How to Print Hydrogel Scaffolds

Abstract

A method of printing a hydrogel scaffold is provided that includes providing a container containing an ink and a liquid immiscible with the ink, exposing the ink to light from a light source to form a portion of a hydrogel scaffold, and exposing the ink to light from the light source one or more additional times to produce one or more additional portions of a hydrogel scaffold.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 271,670, filed October 25, 2021, which is incorporated by reference in its entirety. [Background technology]

[0002] Compositions including hydrogels may be used to form objects used for biocompatible construction. These objects may be formed using three-dimensional (3D) printing techniques.

[0003] In traditional 3D printing, full containers of ink are used, even when printing large items. Reasons for this include the excess ink providing buoyancy to prevent deformation of the printed object and help maintain adhesion to the build platform. In addition, the ink helps prevent dehydration and shrinkage of the printed hydrogel, which could potentially cause detachment of the build platform. However, there are many drawbacks to using large amounts of ink. For example, ink is expensive. In addition, sourcing materials for a 3L container can have a long lead time between formulation development and large-scale printing.

[0004] In some cases, ink can be reused, but this also has drawbacks. Reusing ink can introduce unwanted variability that can be difficult to quantify. In addition, reusing ink increases the likelihood that a single batch of ink will need to be shared among many people and projects. In addition, reusing ink often requires formulating additional ink and adding it to the reused ink to create the required print volume. This introduces less than optimal interdependencies. These interdependencies add complexity when planning a project.

[0005] It is against this background that the need arose to develop the embodiments described herein. Summary of the Invention

[0006] To overcome this problem, certain embodiments herein use less ink with filler material. The advantage of this is that less ink is used and subsequently costs are saved. In addition, the ink can be made fresh for each print. This eliminates batch variations caused by material print history (which has variables including number of prints, material age, variation in ingredients sourced, and who makes the additional ink). In addition, formulation ingredients can be sourced in smaller volumes.

[0007] An additional benefit is that inks do not have to be shared between different printing runs, removing interdependencies. This shortens the time between new ink development and large-format printing. Large-format printing can be done with almost any formulation, not just reusable inks. In addition, one-off printing can be done at any time without worrying about disposing of residual ink. There is no need for long-term planning to think about how to use reused materials. This can speed up the printing work cycle model.

[0008] The embodiments disclosed herein include a process for printing large, hydrogel-based objects, such as hydrogel scaffolds, with an inverted digital light projection (DLP) 3D printing system. During printing, the hydrogel object may be submerged in liquid for the entire printing time. To reduce the amount of ink required, the excess ink can be replaced with an alternative liquid. The alternative liquid is immiscible with the ink. The alternative liquid provides buoyancy to the 3D printed scaffold and prevents dehydration during printing, allowing for successful printing.

[0009] An embodiment of the present disclosure relates to a method of printing a hydrogel scaffold, the method including providing a container containing an ink and a liquid immiscible with the ink, exposing the ink to light from a light source to form a portion of the hydrogel scaffold, and exposing the ink to light from a light source one or more additional times to produce one or more additional portions of the hydrogel scaffold.

[0010] The hydrogel scaffold may remain immersed in the ink-immiscible liquid throughout the method. In some embodiments, the ink-immiscible liquid is selected from one or more hydrophobic materials. In some embodiments, the hydrophobic material may include an oil having a viscosity of at least 5 cP or an organic solvent having a boiling point above 100° C. In some embodiments, the viscosity of the oil may be at least 10, 15, 20, or 50 cP. In some embodiments, the boiling point of the oil may be above 120° C., 150° C., or 200° C. In some embodiments, the ink-immiscible liquid is mineral oil, butyl acetate, a mixture of butyl ether and mineral oil, petroleum ether (and liquids having similar chemical properties to butyl ether, butyl acetate, petroleum ether, or densities of the immiscible liquid), and mineral oil compositions, where the composition of petroleum ether is between 25% (w / w) and 50% (w / w). In some embodiments, the ink comprises a poly(ethylene glycol) di-(meth)acrylate monomer. In some embodiments, the weight average molecular weight (M w ) is from about 400 to about 20,000. In some embodiments, the M of the poly(ethylene glycol) di-(meth)acrylate monomer is w is approximately 2,000 to 7,000.

[0011] In some embodiments, the ink comprises a photoinitiator. The photoinitiator may include lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), sodium phenyl-2,4,6-trimethylbenzoylphosphinate (NAP), trimethylbenzoyl-based photoinitiators, diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (TPO nanoparticles), Irgacure class photoinitiators, ruthenium, riboflavin, or mixtures thereof. The ink may include DI water. The ink may include about 50% to about 90% DI water. The ink may further include proteins, peptides, and / or extracellular matrix materials.

[0012] An embodiment of the present disclosure may relate to a method of reducing an amount of ink used during a digital light projection printing process to form a hydrogel scaffold, the method may include providing a container containing an ink and a liquid immiscible with the ink, exposing the ink to light from a light source to form a portion of the hydrogel scaffold, and exposing the ink to light from a light source one or more additional times to produce one or more additional portions of the hydrogel scaffold, wherein the amount of ink used to form the hydrogel scaffold is at least 50% less than the amount of ink required to form the same hydrogel scaffold in the absence of the liquid immiscible with the ink.

[0013] The amount of ink used to form the hydrogel scaffold may be at least 50% less than the amount of ink required to form the same hydrogel scaffold in the absence of the ink-immiscible liquid. The ink-immiscible liquid may be selected from one or more hydrophobic substances. The one or more hydrophobic substances may include an oil having a viscosity of at least 5 cP or an organic solvent having a boiling point above 100° C. The ink-immiscible liquid may be a mineral oil, butyl acetate, a mixture of butyl ether and mineral oil, petroleum ether or a mineral oil composition. The petroleum ether composition may be 25% (w / w) to 50% (w / w).

[0014] The ink may include a poly(ethylene glycol) di-(meth)acrylate monomer. The weight average molecular weight (M w ) may be from about 400 to about 20,000. The M of the poly(ethylene glycol) di-(meth)acrylate monomer w may be about 2000 to about 7000. The ink may include a photoinitiator. The photoinitiator may include lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), sodium phenyl-2,4,6-trimethylbenzoylphosphinate (NAP), trimethylbenzoyl-based photoinitiators, diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (TPO nanoparticles) Irgacure-based photoinitiators, ruthenium, riboflavin, or mixtures thereof. The ink may include DI water. The ink may include about 50% to about 90% DI water. The ink may further include proteins, peptides, and / or extracellular matrix materials.

[0015] Embodiments of the present disclosure relate to hydrogel scaffolds formed by the disclosed methods. [Brief description of the drawings]

[0016] [Figure 1]FIG. 1 illustrates an embodiment of a two-phase printing process set-up using a blue immiscible liquid and a yellow ink. [Diagram 2] FIG. 2(a) shows an embodiment of an ink composition with over 85% DI water. FIG. 2(b) is an image showing an embodiment of a printed object printed in mineral oil as the second phase. FIG. 2(c) shows an embodiment of a hydrogel printed outside of the ink resulting in failure. FIG. 2(d) shows the amount of ink used for one-phase and two-phase printing embodiments, showing over 80% savings in ink consumption. [Diagram 3] FIG. 3 shows the mechanical properties modulus, stress, and strain of embodiments of printed ink without collagen (control), printed ink with collagen (AC42), and dual-phase printed bioink with collagen and mineral oil (AC42 mineral oil). [Figure 4] FIG. 4 (A-E) illustrates an embodiment of two-phase printing of models using a mineral oil second phase. [Diagram 5] 5(A-B) show an embodiment of two-phase printing of a Fischer bottle structure (5A is a digital model). [Figure 6] FIG. 6 (A-B) illustrates an embodiment of two-phase printing of small cubic structures. [Figure 7] 7(A-B) show an embodiment of two-phase printing of shaped lobe structures. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] definition As used herein, "3D printing" refers to any technique used to manufacture three-dimensional objects using a digital model of that object by building up layers using photopolymerizable inks.

[0018] As used herein, "printable ink" and "printable composition" refer to any composition that can be used to form an object using 3D printing technology. An "ink" is a printable ink that forms a material with one or more desired mechanical, swelling, and / or biocompatible properties. A bioink (also called "bio ink" and "bio-ink") is a type of ink that includes biological components, whether natural or synthetic, and / or is designed for biocompatibility. The biological components may include peptides. The ink may contain one or more materials that facilitate the attachment and growth of a desired cell type. The printed object may support the attachment, growth, and spreading of primary cells and induced pluripotent stem cells. In some examples, the ink can be formed into a hydrogel.

[0019] Printing / Ink Reduction Process Disclosed herein are processes for printing large, hydrogel-based objects with an inverted digital light projection (DLP) 3D printing system, methods for reducing the amount of ink used during the digital light projection printing process, and hydrogel scaffolds formed by these processes. The hydrogel object may be comprised of an ink. The ink may be water-soluble and photocurable. The ink may contain certain components, such as proteins, peptides, and extracellular matrices. The ink may evaporate during the long printing process. Of course, "light" is used broadly and may include electromagnetic radiation that can cause a polymerization reaction (with or without a photoinitiator) within the ink.

[0020] During printing, the hydrogel object may be submerged in liquid for the entire printing time. This submersion can prevent dehydration and provide buoyancy. A large volume of ink is required to keep the hydrogel submerged throughout the printing time. Manufacturing large quantities of ink cost-effectively can be a challenge. The systems and methods for two-phase printing disclosed herein can reduce the amount of ink required to successfully print large objects by as much as 80%.

[0021] Accordingly, some embodiments are methods of printing a hydrogel scaffold, the method including providing a container including an ink and a liquid immiscible with the ink, exposing the ink to light from a light source to form a portion of the hydrogel scaffold, and exposing the ink to light from a light source one or more additional times to produce one or more additional portions of the hydrogel scaffold.

[0022] Further disclosed herein is a method of reducing the amount of ink required by replacing excess ink with an alternative liquid. The alternative liquid may be immiscible with the ink. The alternative liquid may provide buoyancy to the 3d printed scaffold and allow for successful printing by preventing dehydration during printing. In some embodiments, the liquid may be a hydrophobic liquid, such as an oil. In some embodiments, the liquid may be an alcohol. In some embodiments, the liquid may be an amphipathic liquid.

[0023] Accordingly, some embodiments may include a method of reducing an amount of ink used during a digital light projection printing process to form a hydrogel scaffold, comprising providing a container including ink and a liquid immiscible with the ink, exposing the ink to light from a light source to form a portion of the hydrogel scaffold, and exposing the ink to light from a light source one or more additional times to produce one or more additional portions of the hydrogel scaffold, wherein the amount of ink used to form the hydrogel scaffold is at least 50% less (at least 50%, 55%, 60%, 65%, 70%, 75% less, or ranges therebetween) than an amount of ink required to form the same hydrogel scaffold in the absence of the liquid immiscible with the ink.

[0024] In some embodiments, the liquid immiscible with the ink may be selected from one or more hydrophobic materials. For example, in some embodiments, the immiscible liquid is selected from mineral oil, butyl acetate, petroleum ether, and mixtures thereof. In some embodiments, the mixture comprises about 25% (w / w) to about 50% (w / w) petroleum ether (e.g., about 25%, 30%, 35%, 40%, 45%, or 50% (w / w) petroleum ether). In some embodiments, the mixture comprises about 25% (w / w) to about 50% (w / w) butyl acetate (e.g., about 25, 30, 35, 40, 45, or 50% (w / w) petroleum ether). In some embodiments, the mixture comprises, for example, about 50% (w / w) to about 90% (w / w) mineral oil (e.g., about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% (w / w), or any range therebetween). In some embodiments, the one or more hydrophobic materials comprise an oil having a viscosity at 25° C. of at least 5 cP (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 cP, or any range therebetween) and / or an organic solvent having a boiling point at STP of greater than 100° C. (e.g., greater than 105, 110, 120, 130, 140, 150, 160, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650° C., or any range therebetween).

[0025] The ink of this embodiment is not particularly limited. In some embodiments, the ink comprises a poly(ethylene glycol) di-(meth)acrylate monomer. In some embodiments, the weight average molecular weight (M w) is about 400 to about 20,000 (e.g., about 400, 500, 100, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 10500, 1 1000, 11500, 12000, 12500, 13000, 13500, 14000, 14500, 15000, 15500, 16000, 16500, 17000, 17500, 18000, 18500, 19000, 19500, or 20000, or a range therebetween).

[0026] In some embodiments, the ink comprises hydroxy C 1-2 Alkyl (meth)acrylates, Poly(alkylene oxide) alkyl ether (meth)acrylates, N-hydroxy C 1-2Alkyl(meth)acrylamides, poly(ethylene glycol) methyl ether acrylate (PEGMEA), poly(ethylene glycol) methyl ether methacrylate, poly(propylene glycol) methyl ether acrylate, poly(propylene glycol) methyl ether methacrylate, hydroxyethyl acrylate (HEA), N-hydroxyethyl acrylamide (HEAA), hydroxyethyl methacrylate, hydroxypropyl acrylate (HPA 3-hydroxypropyl acrylate and / or 2-hydroxypropyl acrylate), hydroxypropyl methacrylate, hydroxybutyl acrylate (HBA), hydroxybutyl methacrylate, poly(alkylene oxide) di(meth)acrylate, diethylene glycol di(meth)acrylate, tetraethylene glycol di(meth)acrylate, N,N'-methylenebis(acrylamide), (poly)lactic acid di(meth)acrylate, (poly)glycolic acid di(meth)acrylate, (poly)lactic acid-coglycolic acid di(meth)acrylate acrylate, (poly)caprolactone di(meth)acrylate, (poly)dioxanone di(meth)acrylate, (poly)fumarate di(meth)acrylate, (carboxy)(methyl)cellulose di(meth)acrylate, hyaluronic acid di(meth)acrylate, heparan sulfate di(meth)acrylate, dextran di(meth)acrylate, alginate di(meth)acrylate, pectin di(meth)acrylate, tri-acrylate, or collagen di(meth)acrylate, or mixtures thereof.

[0027] In some embodiments, the ink further comprises a photoinitiator. The photoinitiator is not particularly limited, and any suitable photoinitiator can be used. Examples of suitable photoinitiators include lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), sodium phenyl-2,4,6-trimethylbenzoylphosphinate (NAP), trimethylbenzoyl-based photoinitiators, diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (TPO nanoparticles), Irgacure-type photoinitiators, ruthenium, riboflavin, or mixtures thereof.

[0028] In some embodiments, the ink further comprises a solvent, such as water. In some embodiments, the water is deionized. In certain embodiments, the ink comprises about 50% to about 90% DI water (e.g., about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% DI water, or any range therebetween).

[0029] In some embodiments, the ink may further comprise a protein, peptide, and / or extracellular matrix material. In some embodiments, the peptide is selected from the group consisting of RGD, KQAGDV, YIGSR, REDV, IKVAV, RNIAEIIKDI, KHIFSDDSSE, VPGIG, FHRRIKA, KRSR, APGL, VRN, AAAAAAAAA, GGLGPAGGK, GVPGI, LPETG(G)n, IEGR, and combinations thereof. Other examples of optional additional components include ECM or ECM-like materials, such as amino acid sequences that are sensitive to proteases. The protease may be selected from Arg-C proteinase, Asp-N endopeptidase, BNPS-skatole, caspase 1-10, chymotrypsin-high specificity (not before P, C-terminal to [FYW]), chymotrypsin-low specificity (not before P, C-terminal to [FYWML]), clostripain (clostridiopeptidase B), CNBr, enterokinase, factor Xa, formate, glutamyl endopeptidase, granzyme B, hydroxylamine, iodosobenzoic acid, LysC, neutrophil elastase, NTCB (2-nitro-5-thiocyanobenzoic acid), pepsin, proline-endopeptidase, proteinase K, staphylococcal peptidase I, thermolysin, thrombin, and trypsin.

[0030] In some embodiments, the hydrogel scaffold is of a shape and size similar to a human organ. In some embodiments, the hydrogel scaffold remains immersed or submerged (or partially immersed) in the ink-immiscible liquid during the method. In some embodiments, the hydrogel scaffold is submerged in a container. In some embodiments, the hydrogel scaffold is submerged in a container. In some embodiments, the method further includes adding an ink-immiscible liquid to replace at least a portion of the ink consumed or otherwise lost during the printing. In some embodiments, the ink-immiscible liquid is positioned in the container to prevent evaporation of the ink.

[0031] Printable Composition (Hydrogel Scaffold or Structure) The printable compositions (hydrogel scaffolds or structures) described herein can be formed into three-dimensional objects that mimic or replicate an organ or part of an organ. For example, in some embodiments, the printable compositions described herein can be formed into structures that mimic or replicate the architecture of a lung, e.g., by using 3D printing techniques. The printable compositions can be used to form scaffolds for cell attachment and growth, resulting in a structure that has one or more desired properties of an organ, e.g., a structure that can perform the gas exchange function of the lung. These objects can include hydrogels. The organ or part of an organ can be a human lung in a preferred embodiment.

[0032] In some embodiments, the 3D shape of the hydrogel structure is substantially the same shape, size, and / or the same relative dimensions as an organ or organ fragment. In certain embodiments, the organ or organ fragment comprises a blood vessel, trachea, bronchus, esophagus, ureter, renal tubule, bile duct, renal duct, bile duct, hepatic duct, nerve conduit, CSF shunt, lung, kidney, heart, liver, spleen, brain, gallbladder, stomach, pancreas, bladder, lymphatic vessel, skeletal bone, cartilage, skin, intestine, muscle, larynx, or pharynx. In further embodiments, the vascular shape comprises a pulmonary artery, renal artery, coronary artery, peripheral artery, pulmonary vein, or renal vein. In certain embodiments, the structure comprises a hemodialysis graft. In other embodiments, the structure comprises substantially the shape of a lung lobe, a lung, a pulmonary airway, pulmonary vasculature, or a combination thereof, where the structure is located. In some embodiments, the reinforcement includes maintaining air flow or blood (or liquid) flow through the structure when an external pressure is applied to the structure.

[0033] The three-dimensional (3D) hydrogel structure is not particularly limited and may be, for example, a composite structure made of one or more different polymerized monomers. Hydrogel materials that may be used in the present invention are known to those skilled in the art, as are methods for producing the same. For example, the hydrogels described in Calo et al., European Polymer Journal Volume 65, April 2015, Pages 252-267 may be used. In some embodiments, the hydrogel structure comprises polymerized (meth)acrylate and / or (meth)acrylamide hydrogels. In some embodiments, the structure is selected from the group consisting of polymerized poly(ethylene glycol) di(meth)acrylate, polymerized poly(ethylene glycol) di(meth)acrylamide, polymerized poly(ethylene glycol) (meth)acrylate / (methacrylamide), poly(ethylene glycol)-block-poly(ε-caprolactone), polycaprolactone, polyvinyl alcohol, gelatin, methylcellulose, hydroxyethyl methylcellulose, hydroxypropyl methylcellulose, polyethylene oxide, polyacrylamide, polyacrylic acid, polymethacrylic acid, salts of polyacrylic acid, salts of polymethacrylic acid, poly(2-hydroxyethyl methacrylate), ... The hydrogel polymer may comprise polymers including poly(methacrylic acid) anhydride, poly(acrylic acid) anhydride, polysebacic acid anhydride, collagen, poly(hyaluronic acid), hyaluronic acid-containing polymers and copolymers, polypeptides, dextran, dextran sulfate, chitosan, chitin, agarose gel, fibrin gel, soy-derived hydrogels, alginate-based hydrogels, poly(sodium alginate), hydroxypropyl acrylate (HPA), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and combinations thereof. In some embodiments, the M of the hydrogel polymer may be selected from the group consisting of hydroxypropyl acrylate (HPA), hydroxypropyl acrylate (HPA), hydroxypropyl acrylate (HAP ... wApproximately 400Da, 500Da, 600Da, 700Da, 800Da, 900Da, 1000Da, 1100Da, 1200Da, 1300Da, 1400Da, 1500Da, 1600Da, 1700Da, 1800Da, 1900Da, 2000Da, 2100Da, 22 00Da, 2300Da, 2400Da, 2500Da, 2600Da, 2700Da, 2800Da, 2900Da, 3000Da, 3100Da, 3200Da, 3300Da, 3400Da, 3500Da, 3600Da, 3700Da, 3800Da, 3900Da, 40 00Da, 4100Da, 4200Da, 4300Da, 4400Da, 4500Da, 4600Da, 4700Da, 4800Da, 4900Da, 5000Da, 5100Da, 5200Da, 5300Da, 5400Da, 5500Da, 5600Da, 5700Da, 5 800Da, 5900Da, 6000Da, 6100Da, 6200Da, 6300Da, 6400Da, 6500Da, 7000Da, 7500Da, 8000Da, 8500Da, 9000Da, 9500Da, 10000Da, 15000Da, or 20000Da.

[0034] In some embodiments, the hydrogel comprises a crosslinked polymer. In some embodiments, the polymer is about 0% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% crosslinked, based on the percentage of crosslinkable moieties in the polymer. The crosslinkable moieties may include, for example, (meth)acrylate groups.

[0035] The curable ink is not particularly limited. In some embodiments, the ink is the same or similar in composition to the monomers used in the three-dimensional (3D) hydrogel structure. In some embodiments, the curable ink is a photocurable ink, for example an ink that can be photocured in the UV spectrum range of 100-400 nm. Contemplated inks include combinations that contain photoinitiators and / or dyes that react and absorb light at 100-400 nm. Photoinitiators may include, for example, benzophenone, phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide (BAPO), 2-hydroxy-2-methyl-1-phenyl-propan-1-one, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone), 2,2'-azobis[2-methyl-n-(2-hydroxyethyl)propionamide], 2,2'-dimethoxy-2-phenylacetophenone, diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate (LAP), sodium phenyl-2,4,6-trimethylbenzoylphosphinate (NAP), and ethyl(2,4,6-trimethylbenzoyl)phenylphosphinate.

[0036] Among the 3D printing techniques, an efficient technique is the digital light process (DLP) method or stereolithography (SLA). In a 3D printer using DLP or SLA, the ink material is layered on a container or spread on a sheet, and a predetermined area or surface of the ink is exposed to ultraviolet-visible (UV / Vis) light. This light is controlled by a digital micro-mirror device or a rotating mirror. In the DLP method, additional parts are repeatedly or successively laid down, and each layer is cured until the desired 3D article is formed. The SLA method differs from the DLP method in that the ink is solidified by a radiation beam line. Other 3D printing methods can be found in 3D Printing Techniques and Processes by Michael Degnan, Dec 2017, Cavendish Square Publishing, LLC, which is incorporated herein by reference.

[0037] During printing, the hydrogel object may be submerged in liquid for the entire printing time. Such submersion can prevent dehydration and provide buoyancy. A large volume of ink is required to keep the hydrogel submerged throughout the printing time. Manufacturing large quantities of ink cost-effectively can be a challenge. The systems and methods for two-phase printing disclosed herein can reduce the amount of ink required to successfully print large objects by as much as 80%. FIG. 1 illustrates an embodiment of a two-phase printing process setup using a blue immiscible liquid and a yellow ink.

[0038] FIG. 2 illustrates one embodiment of the disclosure. FIG. 2(a) illustrates the composition of an example ink. The ink contains more than 85% DI water, which makes the ink immiscible with hydrophobic liquids, such as mineral oil. FIG. 2(b) illustrates an image of a printed object printed in mineral oil as the second phase. In contrast, FIG. 2(c) illustrates a hydrogel printed outside of the ink, resulting in a collapse. FIG. 2(d) illustrates the amount of ink used for one-phase and two-phase printing, showing a savings of more than 80% in ink consumption.

[0039] The following examples describe specific aspects of some embodiments of the present disclosure to illustrate and explain to one of ordinary skill in the art, and should not be construed as limiting the present disclosure, as the examples merely provide specific methods useful in understanding and practicing some embodiments of the present disclosure.

[0040] Example 1 FIG. 3 shows mechanical properties of dog-bone shaped samples formed by one-phase or two-phase printing. The graph shows modulus, stress, and strain of three different samples. The first sample shows the printed ink without collagen (control) using one-phase printing. The second sample shows the printed ink with collagen (AC42) using one-phase printing. The third sample shows the ink with collagen using two-phase printing utilizing mineral oil (AC42 mineral oil) for the second phase. The printed dog-bone structures were used to complete the analysis of tensile testing material properties between the ink and the samples printed inside a vat filled with ink covered with mineral oil. Uniaxial tensile testing was used to measure mechanical properties such as break stress, break strain, and tensile modulus. The control samples were tested for certain printing properties including certain exposure times and printer output. The dog-bones were printed with the dimensions shown in FIG. 3. The tensile material properties were measured by connecting the dog-bones to an Instron machine. As can be seen from FIG. 3, the modulus, stress, and strain of the dog-bones formed using mineral oil and a lower amount of ink had acceptable modulus, stress, and strain when compared to the dog-bones formed from a container of ink alone.

[0041] Example 2 The ink was poured into a vat and mineral oil was added to the vat, as shown in Figures 4A and B. After printing, the object was transferred, as shown in Figures 4C and D. An image of the printed object is shown in Figure E.

[0042] A flat printing platform was mounted on a digital light projection (DLP) 3d printing system. 600 ml of 504N containing PEGDA, LAP, and UV386A® (UV dye produced by QCR Solutions Corp.) was produced. Figure 4A shows the 3-D printer settings. After the printer was lowered, 100 ml / min of mineral oil was added to the top of the ink. Figure 4B shows a close-up of the interface between the ink and the mineral oil.

[0043] During printing, a 10-part base was printed using a printing time of 35 seconds per 100 μm layer. The body was printed at a rate of 7 seconds per 20 μm layer. 1.8-2.7 mW / cm 2 Or 2.0~2.5mW / cm 2 or 2.1-2.3mW / cm 2 , e.g. 2.22 mW / cm 2 Then, a 200-400 watt or 250-350 watt or 275-325 watt, e.g., 300W LED was used to complete the light cure.

[0044] As the model was printed, the printed model rose through the ink and into the mineral oil. Figure 4C shows a successfully printed object formed from the 3-D printing ink. Figure 4D is a close-up of the small amount of ink left after 3-D printing. Figure 4E shows the completed object after 3-D printing, coated in mineral oil.

[0045] Example 3 Figure 5 shows a 3D printed ventilation Fischer bottle model printed using an ink containing PEGDA, LAP, and UV dye. After the printer was lowered, 50 ml / min of mineral oil was added to the top of the ink.

[0046] During printing, a print time of 25 seconds per 100 μm layer was used to print the base, which consisted of 10 parts. The body was printed at a speed of 4 seconds per 50 μm layer. 2.5-4.5 mW / cm 2 Or 3.0~4.0mW / cm 2 or 3.25~3.75mW / cm 2 , e.g. 3.5 mW / cm 2 Then, a 300-600 watt or 350-550 watt or 400-500 watt or 425-475 watt, e.g., 450W LED was used to complete the light cure.

[0047] Figure 5A shows a model of the Fisher bottle structure. Figure 5B shows the Fisher bottle structure during printing in a large vat DLP printer in two phases, ink and oil.

[0048] Example 4 Example 4 shows the printing of a large hydrogel structure of a leaf-like model "Object 3". A cubic cutout at the G3-G4 junction was modeled as shown in FIG. 6A. The diameter of the main inlet was approximately 400 μm. The diameter of the main outlet was approximately 350 μm. The diameter of the vascular network was approximately 200 μm. The dimensions of the cube were 3 cm3.

[0049] For 3D printing, a DLP printer was used. A plastic build platform was used to avoid unwanted reactions. Prior to 3D printing, a 300 nm thick titanium layer was sputter-coated onto the plastic build platform. The titanium layer was coated with silane.

[0050] An ink containing PEGDA, LAP, and UV dye was produced. The ink was loaded into a container. The container shown in FIG. 6B consisted of a black bath, a blue gasket, an AF2400 sheet (not shown), and a chrome metal clamp. It was found that contact of the ink with the metal container resulted in coloring of the ink. To prevent contact of the dye with the metal container, a PDMS layer was added to the black surface of the container shown in FIG. 6B. The PDMS was prepared by mixing Sylgard 184 PDMS base with a curing agent in a 10:1 ratio, speed mixing for 2 minutes, and then partially curing the mixture at 80° C. for 30 minutes until the PDMS mixture was partially cured and more viscous. Approximately 30 grams of PDMS was deposited on the black surface of the container in FIG. 4. The container was cured at 80° C. for 3-4 hours before use.

[0051] The cubes were 3-D printed using a 250 watt DLP printer with a build layer thickness of 20 μm. In some embodiments, an ink formulation may be added to the container before printing, and an immiscible liquid, such as oil or alcohol, may be added to cover the leaves as they are 3-D printed. Additional immiscible liquid may be added as needed to keep the leaves submerged. An example of a molded 3-D cube is shown in the photograph of FIG. 6C.

[0052] For 3D printing, a NextDent printer was used. A plastic build platform was used to avoid unwanted reactions. Prior to 3D printing, a 300 nm thick titanium layer was sputter-coated onto the plastic build platform. To improve adhesion of the hydrogel to the printing platform, the titanium layer was coated with silane methacrylate.

[0053] An ink was made from 503N combined with 1% LAP and 0.1% UV386A® pigment (UV pigment produced by QCR Solutions Corp.). The ink was loaded into a container. The container shown in FIG. 6B consisted of a black bath, a blue gasket, an AF2400 sheet (not shown), and a chrome metal clamp. It was found that contact of the ink with the metal container would result in coloring of the ink. To prevent contact of the pigment with the metal container, a PDMS layer was added to the black surface of the container shown in FIG. 6B. The PDMS was prepared by mixing Sylgard 184 PDMS base with a curing agent in a 10:1 ratio, speed mixing for 2 minutes, and then partially curing the mixture at 80° C. for 30 minutes until the PDMS mixture was partially cured and more viscous. Approximately 30 grams of PDMS was deposited on the black surface of the container in FIG. 4. The container was cured at 80° C. for 3-4 hours before use.

[0054] The lobe structures were 3-D printed using a DLP printer with a build layer thickness of 20 μm and 250 mW irradiance [please also confirm that the above is accurate for the lobe structures as well as the cubic structures].

[0055] In some embodiments, an ink formulation may be added to the container prior to printing, and an immiscible liquid, such as oil or alcohol, may be added to cover the leaves as they are 3-D printed. Additional immiscible liquid may be added as needed to keep the leaves submerged. An example of a molded 3-D cube is shown in the photograph of FIG. 6C.

[0056] MicroCt images were taken from the molded leaf structures as shown in Figure 7B.

[0057] As used herein, the singular terms "a," "an," and "the" can include the plural unless the context clearly dictates otherwise. Thus, for example, when referring to an object, this can include a plurality of objects unless the context clearly dictates otherwise.

[0058] As used herein, the terms "substantially" and "about" are used to describe and take into account small variations. When used in the context of an event or circumstance, these terms can refer to instances where the event or circumstance occurs exactly as well as instances where the event or circumstance occurs very similarly. When used in the context of a numerical value, these terms can refer to a variation range of ±10% or less of the numerical value, for example, ±5% or less, ±4% or less, ±3% or less, ±2% or less, ±1% or less, ±0.5% or less, ±0.1% or less, or ±0.05% or less. When referring to a first numerical value that is "substantially" or "about" the same as a second numerical value, these terms can mean that the first numerical value is within a range of variation of the second numerical value by ±10% or less, for example, ±5% or less, ±4% or less, ±3% or less, ±2% or less, ±1% or less, ±0.5% or less, ±0.1% or less, or ±0.05% or less.

[0059] In addition, amounts, ratios, and other numerical values ​​may be defined herein in a range format. It will be appreciated that such range formats are used for convenience and brevity, and should be understood to include the numerical values ​​explicitly specified as the limits of the range, but also to include all individual numerical values ​​or subranges subsumed within the range, as if each numerical value and subrange were explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly specified limits of about 1 and about 200, but also to include the individual ratios, such as about 2, about 3, and about 4, and subranges, such as about 10 to about 50, about 20 to about 100, etc.

[0060] Although the disclosure has been described with reference to specific embodiments thereof, it will be apparent to those skilled in the art that various modifications can be made and equivalents can be substituted without departing from the true spirit and scope of the disclosure as defined by the appended claims. In addition, numerous modifications may be made to adapt particular conditions, materials, compositions of matter, methods, and operations to the objective, spirit, and scope of the disclosure. All such modifications are intended to be within the scope of the appended claims. Although a particular method has been specifically described with reference to certain operations performed in a particular order, it will be understood that these operations can be combined, further divided, or reordered to form equivalent methods without departing from the teachings of the disclosure. Thus, unless specifically indicated herein, the order and grouping of operations is not intended to limit the disclosure.

Claims

1. A method for printing a hydrogel scaffold, Prepare a container containing ink and a liquid that is immiscible with the ink. To replace at least a portion of the ink consumed or otherwise lost during printing, a liquid that is immiscible with the ink is added. To form a portion of the hydrogel scaffold, light from a light source is shone onto the ink, and To produce one or more further portions of the hydrogel scaffold, light from a light source is applied one or more times. A method for printing hydrogel scaffolds, including the following.

2. The method according to claim 1, wherein the liquid immiscible with the ink comprises an oil having a viscosity of at least 5 cP, or an organic solvent having a boiling point above 100°C.

3. The method according to claim 1, wherein the liquid that is immiscible with the ink is selected from the group consisting of mineral oil, butyl acetate, a mixture of butyl ether and mineral oil, and a mixture of petroleum ether and mineral oil.

4. The method according to claim 1, wherein the ink comprises poly(ethylene glycol) di(meth)acrylate.

5. The method according to claim 4, wherein the weight-average molecular weight (Mw) of the poly(ethylene glycol) di(meth)acrylate is about 400 to about 20,000.

6. The method according to claim 4, wherein the ink further comprises at least one photoinitiator selected from the group consisting of lithium phenyl-2,4,6-trimethylbenzoyl phosphinate (LAP), trimethylbenzoyl-based photoinitiators, diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide (TPO nanoparticles) Irgacure class photoinitiators, ruthenium, riboflavin, and mixtures thereof.

7. The method according to claim 4, wherein the ink further comprises about 50% to about 90% DI water.

8. The method according to claim 4, wherein the ink further comprises a protein, a peptide, and / or an extracellular matrix material.

9. The method according to claim 1, wherein the hydrogel scaffold is human-scale.

10. The method according to claim 1, wherein the printed portion of the hydrogel scaffold is immersed in a liquid that is immiscible with the ink while exposed to light.

11. A hydrogel scaffold formed by the method described in Claim 1.

12. A method for reducing the amount of ink used in a digital light projection printing process for forming a hydrogel scaffold, Prepare a container containing ink and a liquid that is immiscible with the ink. To form a portion of the hydrogel scaffold, light from a light source is shone onto the ink, and To produce one or more further portions of the hydrogel scaffold, light from a light source is applied one or more times. This includes, The amount of ink used to form the hydrogel scaffold is at least 50% less than the amount of ink required to form the same hydrogel scaffold when no liquid immiscible with the ink is present, and The liquid that is immiscible with the ink is a mixture of butyl ether and mineral oil containing 25% (w / w) to 50% (w / w) petroleum ether. How to reduce the amount of ink used.

13. The method according to claim 12, wherein the hydrogel scaffold is submerged in the container.

14. The method according to claim 13, wherein the hydrogel scaffold is completely submerged in the ink or a liquid that is immiscible with the ink.

15. The method according to claim 12, wherein the liquid that is immiscible with the ink is hydrophobic.

16. The method according to claim 12, wherein the ink comprises poly(ethylene glycol) di(meth)acrylate.

17. The method according to claim 16, wherein the ink further comprises a photoinitiator.

18. The method according to claim 16, wherein the ink further comprises about 50% to about 90% DI water.

19. A method for printing a hydrogel scaffold, Prepare a container containing ink and a liquid that is immiscible with the ink. To form a portion of the hydrogel scaffold, light from a light source is shone onto the ink, and To produce one or more further portions of the hydrogel scaffold, light from a light source is applied one or more times. This includes, To prevent the evaporation of the ink, a liquid that is immiscible with the ink is positioned on the upper surface of the ink in the container. How to print hydrogel scaffolds.

20. The method according to claim 19, wherein the hydrogel scaffold is submerged in the container.

21. The method according to claim 20, wherein the hydrogel scaffold is completely submerged in the ink or a liquid that is immiscible with the ink.

22. The method according to claim 19, further comprising adding a liquid that is immiscible with the ink to replace at least a portion of the ink that is consumed or otherwise lost during printing.

23. The method according to claim 19, wherein a liquid that is immiscible with the ink is positioned in the container in order to prevent the evaporation of the ink.

24. The method according to claim 19, wherein the liquid that is immiscible with the ink is hydrophobic.

25. The method according to claim 19, wherein the liquid immiscible with the ink comprises an oil having a viscosity of at least 5 cP, or an organic solvent having a boiling point above 100°C.

26. The method according to claim 19, wherein the ink comprises a poly(ethylene glycol) di(meth)acrylate monomer, optionally having a weight-average molecular weight (Mw) of the poly(ethylene glycol) di(meth)acrylate monomer of about 400 to about 20,000, and optionally having a Mw of about 2,000 to about 7,000.

27. ​​The method according to claim 19, wherein the ink further comprises at least one photoinitiator, optionally the photoinitiator being selected from the group consisting of lithium phenyl-2,4,6-trimethylbenzoyl phosphine (LAP), trimethylbenzoyl-based photoinitiators, diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide (TPO nanoparticles) Irgacure photoinitiators, ruthenium, riboflavin, and mixtures thereof.

28. The method according to claim 19, wherein the ink contains DI water, and optionally the ink contains about 50% to about 90% DI water (w / w).

29. The method according to claim 19, wherein the ink comprises a protein, a peptide, and / or an extracellular matrix material.

30. The method according to claim 19, wherein the hydrogel scaffold is human-scale.

31. The method according to claim 19, wherein the printed portion of the hydrogel scaffold is immersed in a liquid that is immiscible with the ink while exposed to light.

32. The method according to claim 19 as a digital light projection printing process, The amount of ink used to form the hydrogel scaffold is at least 50% less than the amount of ink required to form the same hydrogel scaffold when no liquid immiscible with the ink is present. method.

33. The method according to claim 19, wherein the liquid immiscible with the ink is a mixture of petroleum ether and mineral oil.

34. The method according to claim 33, wherein the mixture of petroleum ether and mineral oil contains 25% (w / w) to 50% (w / w) petroleum ether.

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