Use of functionalized and non-functionalized ECM, ECM fragments, peptides and bioactive components to generate cell-adhesive 3D printed objects

Abstract

Embodiments of the disclosure relate to bioinks and bioink compositions. These bioinks can be 3D printed into hydrogels. The printed hydrogels can support the attachment, proliferation and spreading of primary cells and induced pluripotent stem cells. Compounds in the bioink can be modified, such as by chemical synthesis means, to incorporate chemical functionalities. The incorporation of chemical functionalities can allow the incorporation of modified materials as components in the bioink. The modification can allow chemical conjugation of desired components. The desired components can maintain their cell-interacting properties that aid in cell attachment and proliferation. Such incorporation can allow tuning of the mechanical properties of the bioprinted object without interfering with cell adhesion.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 185,293, filed May 6, 2021, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] background Compositions including hydrogels can be used to form objects for use in biocompatible construction. These objects can be formed using three-dimensional (3D) printing techniques. Cells can be attached for practical applications such as synthetic organs. Summary of the Invention

[0003] Abstract Embodiments of the disclosure relate to bioinks and bioink compositions. These bioinks can be 3D printed into hydrogels. The printed hydrogels can support attachment, proliferation, and spreading of primary cells and induced pluripotent stem cells. Compounds in the bioink can be modified, such as by chemical synthesis means, to incorporate chemical functionalities. Incorporation of chemical functionalities can allow incorporation of the modified materials as components in the bioink. The modifications can allow chemical conjugation of desired components. The desired components can maintain their cell-interactive properties that aid in cell attachment and proliferation. Such incorporation can allow tuning of the mechanical properties of the bioprinted object without interfering with cell adhesion.

[0004] Disclosed herein are objects formed, for example, by casting, flood curing, photocuring, photopylmerization, layer by layer printing, or extrusion of objects using materials disclosed herein. The object may be an organ tissue replacement. The object may be any other object of commercial value. Embodiments of this disclosure relate to 3D printed objects and methods of forming them.

[0005] The embodiments of the present disclosure relate to systems and methods for modifying extracellular matrix (ECM) to improve cell attachment and interaction with the resulting framework. Disclosed herein are the resulting framework and methods for cell attachment and interaction, and objects produced therewith. As shown herein, extracellular matrix (ECM), such as type I collagen, gelatin, elastin, and fibronectin, can be functionalized with groups such as methacrylate groups to allow incorporation into photocrosslinkable hydrogels. Incorporation of the extracellular matrix can allow cell attachment and interaction within the hydrogel, making the hydrogel biocompatible.

[0006] Embodiments of the disclosure relate to compositions comprising crosslinked (meth)acrylated extracellular matrix (ECM) material and non-(meth)acrylated ECM material. The compositions can have a ratio of (meth)acrylated ECM material to non-(meth)acrylated ECM material of about 5:1 to about 1:5 (e.g., about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4 or about 1:5). The (meth)acrylated ECM material can have a degree of (meth)acrylation of about 5 to about 95 percent (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95%). The ECM material may be selected from collagen, gelatin, elastin, and fibronectin. The ECM material may be collagen type I. The (meth)acrylated ECM material may include mono- or di-(meth)acrylated ECM or ECM-like materials.

[0007] The ECM or ECM-like material can be selected from RGD, KQAGDV, YIGSR, REDV, IKVAV, RNIAEIIKDI, KHIFSDDSSE, VPGIG, FHRRIKA, KRSR, APGL, VRN, AAAAAAAAA, GGLGPAGGK, GVPGI, LPETG(G)n, and IEGR. The ECM or ECM-like material can be a sequence that is sensitive to a protease. The protease may be selected from Arg-C proteinase, Asp-N endopeptidase, BNPS-skatole, caspase 1-10, chymotrypsin high specificity (C-terminal to [FYW], not before P), chymotrypsin low specificity (C-terminal to [FYWML], not before P), clostripain (clostridiopeptidase B), CNBr, enterokinase, factor Xa, formate, glutamyl endopeptidase, granzyme B, hydroxylamine, iodosobenzoate, LysC, neutrophil elastase, NTCB (2-nitro-5-thiocyanobenzoate), pepsin, proline endopeptidase, proteinase K, Staphylococcal peptidase I, thermolysin, thrombin, and trypsin.

[0008] The composition may further comprise a polymeric material. The polymeric material may be hydrophilic. The polymeric material may comprise one or more of acrylamide, poly(N-isopropylacrylamide), 2-hydroxyethyl methacrylate, poly(2-hydroxyethyl methacrylate), triethylene glycol dimethacrylate, tetra(ethylene glycol) dimethacrylte, N,N'-methylene biacrylamide, or amine end functionalized 4-arm poly(ethylene glycol). The polymeric material may be polymerized poly(ethylene glycol) di(meth)acrylate, poly(hydroxyethyl)(methacrylate), poly N-hydroxyl acrylamide 3-hydroxypropyl acrylate, or hydroxybutyl acrylate. The polymeric material may have a weight average molecular weight (M) of about 400 to about 20,000. w The polymeric material may be a poly(ethylene glycol) di(meth)acrylate monomer having an M of about 2000 to about 4000. w The polymeric material may be a mixture of any of the compositions described above. The polymeric material may be present in an amount of about 5 to about 50 wt.% (e.g., about 5 wt.%, about 10 wt.%, about 15 wt.%, about 20 wt.%, about 25 wt.%, about 30 wt.%, about 35 wt.%, about 40 wt.%, about 45 wt.%, or about 50 wt.%) of the composition. In some embodiments, the polymeric material can be a polymerized poly(ethylene glycol) di(meth)acrylate monomer present in an amount of about 5 to about 50 wt.% (e.g., about 5 wt.%, about 10 wt.%, about 15 wt.%, about 20 wt.%, about 25 wt.%, about 30 wt.%, about 35 wt.%, about 40 wt.%, about 45 wt.% or about 50 wt.%) of the composition.

[0009] The composition can support the attachment, proliferation and spreading of primary cells and / or induced pluripotent stem cells. The composition can be a molded or 3D printed hydrogel article. The composition can be a molded or 3D hydrogel printed photocrosslinked article. The molded or 3D printed hydrogel article can be a three-dimensional article of an organ or part of an organ. The organ can be an organ of a mammal, such as an adult human.

[0010] An embodiment of the disclosure may relate to a method of making a three-dimensional article, comprising depositing a layer of a printable composition on a surface to obtain a deposited layer, irradiating the deposited layer, and repeating the depositing and irradiating steps until the deposited layer forms a three-dimensional article. The printable composition may, for example, comprise a (meth)acrylated extracellular matrix (ECM) material, a non-(meth)acryalted ECM material, or a mixture of (meth)acrylated and non-(meth)acrylated ECM, and also a photoinitiator. The ECM material may comprise one or more of collagen, gelatin, elastin, and fibronectin.

[0011] The printable composition may also include poly(ethylene glycol) di(meth)acrylate monomers. The printable composition may include mono- or di(meth)acrylated ECM or ECM-like materials. The ECM or ECM-like materials may include RGD, PHSRN(GGGERCG)GGRGDSPY, GCREKKRKRLQVQLSIRT, GCREKKTLQPVYEYMVGV, GCREISAFLGIPFAEPPMGPRRFLPPEPKKP, GCRDGPQGWGQDRCG, GCRDVPMSMRGGDRCG, GFOGER, KQAGDV, YIGSR, REDV, IKVAV, RNIAEIIKDI, KHIFSDDSSE, VPGIG, FHRRIKA, KRSR, APGL, VRN, AAAAAAAAA, GGLGPAGGK, GVPGI, LPETG(G)n, and IEGR. The ECM or ECM-like materials may include sequences that are sensitive to proteases. In some embodiments, the protease can be selected from Arg-C proteinase, Asp-N endopeptidase, BNPS-skatole, caspase 1-10, chymotrypsin high specificity (C-terminal to [FYW], not before P), chymotrypsin low specificity (C-terminal to [FYWML], not before P), clostripain (clostridiopeptidase B), CNBr, enterokinase, factor Xa, formate, glutamyl endopeptidase, granzyme B, hydroxylamine, iodosobenzoate, LysC, neutrophil elastase, NTCB (2-nitro-5-thiocyanobenzoate), pepsin, proline endopeptidase, proteinase K, Staphylococcal peptidase I, thermolysin, thrombin, and trypsin.

[0012] The ratio of (meth)acrylated ECM material to non-(meth)acrylated ECM material can be about 5:1 to about 1:5 (e.g., about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4 or about 1:5). The (meth)acrylated ECM material can have a degree of (meth)acrylation of about 5 to about 95 percent (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95%). 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, and riboflavin, or mixtures thereof.

[0013] An embodiment of the disclosure may include a printable composition, where the one or more additives include a polymer, a photoactive dye, a natural extracellular matrix, a photoinitiator, a peptide, an amino acid, a growth factor, a modified extracellular matrix, an extracellular matrix fragment, or a mixture thereof. The photoactive dye may be a UV dye having an absorbance spectrum between 300 nm and 420 nm. The photoactive dye may have a wavelength range of 300 nm to 400 nm. The photoactive dye may be non-cytotoxic. The photoactive dye may include a benzyne ring in its molecular structure. The photoactive dye may be a quinolone yellow, a UV dye, or a dye having a similar molecular structure thereto. The photoactive dye may be a UV 386A dye.

[0014] The embodiments also include a printed scaffold. The printed scaffold may be non-cytotoxic upon leaching out monomers into a buffer in which the scaffold is placed. The embodiments may include compositions used to print the scaffolds described above. The embodiments may include a formed three-dimensional article. The three-dimensional article may replicate an organ or a portion of an organ.

[0015] Embodiments also include printable compositions that include a protic solvent. The protic solvent may include water, polyethylene glycol, a glycol diacrylate derivative, or a mixture thereof.

[0016] Embodiments of the disclosure also relate to uses of functionalized and non-functionalized extracellular matrices, matrix fragments, peptides and bioactive components formed by the disclosed methods. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 shows a schematic diagram of the reaction between collagen and methacrylate anhydride to form methacrylated collagen. [Diagram 2] Figure 2A shows images and Figure 2B shows graphs of cell spreading, density, and % cell coverage of lung fibroblasts cultured for 7 days on different surfaces: (i) glass (control), (ii) bioink containing collagen with 50% DOF, (iii) bioink containing 90% DOF, and (iv) bioink containing collagen with 0% DOF and 90% DOF (1:2 ratio). Additional explanation is provided in Example 1. [Diagram 3] Figure 3A shows images and Figure 3B shows graphs of cell spreading, density, and % cell coverage of pulmonary artery endothelial cells cultured for 1 day on different surfaces: (i) glass (control), (ii) 9% PEGDA hybrid and 9% PEGDA. Asterisks (*) indicate p-values ​​less than 0.05. Additional explanations are provided in Example 2. [Figure 4] 4 shows the attachment, proliferation and spreading of pulmonary smooth muscle cell cultures on 3D printed disks composed of PEGDA MW 3400, 95% DOF collagen and 0% DOF collagen (2:1 ratio) over the course of 7 days. Additional description is provided in Example 3. [Figure 5-1]5A-5C show cell adhesion to different 3D printed objects made using different bioinks and with different ratios of functionalized to non-functionalized collagen. Additional explanation is provided in Example 4. [Figure 5-2] 5A-5C show cell adhesion to different 3D printed objects made using different bioinks and with different ratios of functionalized to non-functionalized collagen. Additional explanation is provided in Example 4. [Figure 6] 6 shows cell attachment and proliferation of lung fibroblasts onto 3D printed objects made with bioinks containing functionalized and non-functionalized collagen supports over a 7 day period. Additional description is provided in Example 5. [Figure 7-1] 7A-7B show the effect of HEAA content on cell adhesion properties. Further explanation is provided in Example 6. [Figure 7-2] 7A-7B show the effect of HEAA content on cell adhesion properties. Further explanation is provided in Example 6. [Figure 8-1] 8A-8E show the effect of CollMA DOF on cell adhesion and proliferation (>90%, ~50% (~50%), Hybrid (50% non-MA, 50% HM)). Further explanation is provided in Example 7. [Figure 8-2] 8A-8E show the effect of CollMA DOF on cell adhesion and proliferation (>90%, -50%, Hybrid (50% non-MA, 50% HM)). Further explanation is provided in Example 7. [Figure 8-3] 8A-8E show the effect of CollMA DOF on cell adhesion and proliferation (>90%, -50%, Hybrid (50% non-MA, 50% HM)). Further explanation is provided in Example 7. [Figure 8-4] 8A-8E show the effect of CollMA DOF on cell adhesion and proliferation (>90%, -50%, Hybrid (50% non-MA, 50% HM)). Further explanation is provided in Example 7. [Figure 8-5] 8A-8E show the effect of CollMA DOF on cell adhesion and proliferation (>90%, -50%, Hybrid (50% non-MA, 50% HM)). Further explanation is provided in Example 7. [Figure 9] 9 shows a comparison of cell density, cell spreading, and cell coverage of PAEC cells. Additional explanation is provided in Example 8. [Figure 10-1] 10A-10D show the differences in cell spreading, cell density and cell coverage on 5% PEGDA 3D printed substrates with different degrees of functionalization. Further explanation is provided in Example 10. [Figure 10-2] 10A-10D show the differences in cell spreading, cell density and cell coverage on 5% PEGDA 3D printed substrates with different degrees of functionalization. Further explanation is provided in Example 10. [Figure 10-3] 10A-10D show the differences in cell spreading, cell density and cell coverage on 5% PEGDA 3D printed substrates with different degrees of functionalization. Further explanation is provided in Example 10. [Figure 10-4] 10A-10D show the differences in cell spreading, cell density and cell coverage on 5% PEGDA 3D printed substrates with different degrees of functionalization. Further explanation is provided in Example 10. [Figure 11-1]11A-11D show cell coverage, cell spreading and cell density characteristics of cells "inserted" on different 3D printed discs: (FIG. 11A) Image of 3D printed disc on platform, (FIG. 11B) Day 1 analysis of percent area coverage, cell spreading and cell density for AC42 1 mm disc, AC42 3 mm disc, elution control and glass control, (FIG. 11C) Day 4 analysis of percent area coverage, cell spreading and cell density for AC42 1 mm disc, AC42 3 mm disc, elution control and glass control, (FIG. 11D) Day 7 analysis of percent area coverage, cell spreading and cell density for AC42 1 mm disc, AC42 3 mm disc, elution control and glass control. [Figure 11-2] 11A-11D show cell coverage, cell spreading and cell density characteristics of cells "inserted" on different 3D printed discs: (FIG. 11A) Image of 3D printed disc on platform, (FIG. 11B) Day 1 analysis of percent area coverage, cell spreading and cell density for AC42 1 mm disc, AC42 3 mm disc, elution control and glass control, (FIG. 11C) Day 4 analysis of percent area coverage, cell spreading and cell density for AC42 1 mm disc, AC42 3 mm disc, elution control and glass control, (FIG. 11D) Day 7 analysis of percent area coverage, cell spreading and cell density for AC42 1 mm disc, AC42 3 mm disc, elution control and glass control. [Figure 12-1] 12A-12B show cell coverage, cell spreading and cell density characteristics of cells on different 3D printed discs. (A) Percent area coverage, cell spreading and cell density comparison of AC42 1 mm discs, 3 mm discs seeded with LFN, PAEC or SAEC seeds (seed), elution control and glass control. (B) Percent area coverage, cell spreading and cell density comparison of AC42 1 mm discs and 3 mm discs seeded with LFN, PAEC or SAEC seeds. [Figure 12-2]12A-12B show cell coverage, cell spreading and cell density characteristics of cells on different 3D printed discs. (A) Percent area coverage, cell spreading and cell density comparison for AC42 1 mm discs, 3 mm discs seeded with LFN, PAEC or SAEC seeds, elution control and glass control. (B) Percent area coverage, cell spreading and cell density comparison for AC42 1 mm discs and 3 mm discs seeded with LFN, PAEC or SAEC seeds. [Figure 13] FIG. 13 illustrates certain embodiments of biologically active peptides that can be incorporated into the bio-inks of the present disclosure. [Figure 14-1] 14A-14C show cell coverage, cell spreading and cell density characteristics of cells on different 3D printed discs. Additional details are provided in Example 13. [Figure 14-2] 14A-14C show cell coverage, cell spreading and cell density characteristics of cells on different 3D printed discs. Additional details are provided in Example 13. [Figure 15-1] 15A-15B show an embodiment in which peptides enhance the bioactivity of the bioink to promote cell attachment. Additional details are disclosed in Example 14. [Figure 15-2] 15A-15B show an embodiment in which peptides enhance the bioactivity of the bioink to promote cell attachment. Additional details are disclosed in Example 14. [Figure 16] 16A-16B show an embodiment in which peptides enhance the bioactivity of the bioink to promote cell attachment. Additional details are disclosed in Example 14. [Figure 17] 17A-17B show an embodiment in which peptides enhance the bioactivity of the bioink to promote cell attachment. Additional details are disclosed in Example 14. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Detailed Description As used herein, "3D printing" refers to any technique used to create a three-dimensional object using a digital model of the object. Exemplary 3D printing techniques include selective laser sintering (SLS), fused deposition modeling (FDM), 3D inkjet printing, digital light processing (DLP), and stereolithography.

[0019] As used herein, "printable ink" and "printable composition" refer to any composition that can be used to form an object using 3D printing technology. A "bio-ink" is a printable ink that forms a material with one or more desired biocompatible properties. For example, the bio-ink can contain one or more materials that promote adhesion and proliferation of a desired cell type. The printed object can support the attachment, proliferation and spreading of primary cells and induced pluripotent stem cells. In some cases, the bio-ink can be formed into a hydrogel. Compounds in the bio-ink can be selected or modified, such as by chemical synthesis means, to incorporate chemical functionality. The chemical functionality can allow incorporation of the modified material as a component in the bio-ink. The modification can allow chemical conjugation of a desired component. The desired component can maintain its cell-interacting properties. Such incorporation can allow tuning of the mechanical properties of the printed object without interfering with cell adhesion.

[0020] As used herein, "extracellular matrix" and "ECM" refer to natural and synthetic ECM, as well as one or more materials that make up the ECM. For example, ECM can refer to naturally occurring ECM or ECM made using synthetic techniques. ECM can also refer to one or more materials that make up naturally occurring ECM, such as collagen (natural or synthetic). In some cases, "ECM material" is used to refer to a specific material. ECM can be made using various techniques, including 3D printing. ECM can be made using hydrogel materials.

[0021] As used herein, "extracellular matrix" and "ECM" refer to natural and synthetic ECM, as well as one or more materials that make up the ECM. For example, ECM can refer to naturally occurring ECM or ECM made using synthetic techniques. ECM can also refer to one or more materials that make up naturally occurring ECM, such as collagen, either natural or synthetic. In some cases, "ECM material" is used to refer to a particular material. ECM can be made using a variety of techniques, including 3D printing. ECM can be made using hydrogel materials. ECM matrix materials, such as type I collagen, gelatin, elastin, and fibronectin, can be functionalized with methacrylate groups to allow incorporation into photocrosslinkable hydrogels. Incorporation of ECM materials into other materials and objects, such as 3D printed materials, can increase biocompatibility and allow cell attachment and interaction within the materials and objects. The degree to which a material allows cell attachment can vary depending on the amount of ECM material, the availability of binding sites on or within the material, the surface charge of the material, the polarity of the material, and the mechanical properties of the material.

[0022] This application incorporates by reference each of the following documents in their entirety: No. 63 / 185,300, filed May 6, 2021, entitled "CONTROLLING THE SIZE OF 3D PRINTING HYDROGEL OBJECTS USING HDROPHILIC MONOMERS, HYDROPHOBIC MONOMERS, AND CROSSLINKERS," as well as a U.S. non-provisional application and / or PCT application of the same title filed May 6, 2022; (b) U.S. Provisional Application No. 63 / 185,302, filed May 6, 2021, entitled "MODIFIED 3D-PRINTED OBJECTS AND THEIR USES," as well as a U.S. non-provisional application and / or PCT application of the same title filed May 6, 2022; (c) U.S. Provisional Application No. 63 / 185,305, filed May 6, 2021, entitled "PHOTOCURABLE No. 63 / 185,299, filed May 6, 2021, entitled "ADDITIVE MANUFACTURING OF HYDROGEL TUBE FOR BIOMEDICAL APPLICATIONS," and a U.S. non-provisional application and / or PCT application of the same title, filed May 6, 2022; (d) U.S. Provisional Application No. 63 / 185,299, filed May 6, 2021, entitled "ADDITIVE MANUFACTURING OF HYDROGEL TUBE FOR BIOMEDICAL APPLICATIONS," and a U.S. non-provisional application and / or PCT application of the same title, filed May 6, 2022; and (e) U.S. Provisional Application No. 63 / 185,298, filed May 6, 2021, entitled "MICROPHYSIOLOGICAL 3-D PRINTING AND ITS APPLICATIONS," and a U.S. non-provisional application and / or PCT application of the same title, filed May 6, 2022.

[0023] The ECM can be functionalized with methacrylate groups by substituting lysine residues on the amine groups with, for example, methacrylate anhydride (MAA). The degree of (meth)acrylation of the ECM can be defined by the percentage of available amine groups that are modified with AA or MAA. A higher degree of (meth)acrylation correlates with more amine groups modified with AA or MAA, resulting in fewer free amine groups.

[0024] The degree of functionalization may be varied to achieve a particular degree or type of functionalization. Hybrid forms of functionalized and non-functionalized components may be used. In some embodiments, collagen may be modified to form (meth)acrylated collagen. Hybrid combinations may be used, which may refer to hydrogels containing both methacrylated and non-methacrylated collagen. 3D printed objects made of poly(ethylene glycol) diacrylate containing (meth)acrylated and non-(meth)acrylated collagen may support the attachment, spreading and / or proliferation of lung-derived cells, including, for example, fibroblasts, endothelial cells, and smooth muscle cells.

[0025] Instead of or in addition to collagen, various biomaterials can be used. These biomaterials can include type IV collagen, fibronectin, gelatin, type III collagen, short peptides (e.g. RGD), fragments of ECM proteins, proteoglycans, glycosaminoglycans, hyaluronic acid or any other ECM from any species. The available binding sites on these ECMs can be varied through functionalization or modification with different chemical groups. These chemical groups can bind to amine groups or other groups for which cells have an affinity.

[0026] The ECM compositions can be formulated for use in casting, extrusion or 3D printing applications, such as for use in bioinks to form the compositions. In some embodiments, these compositions increase cell adhesion, proliferation, spreading and migration to 3D printed materials, including hydrogels. These 3D printed materials can be used for biological, biomedical, medical device and / or health care applications. These printed materials can be used for diagnostic devices. These printed hydrogels can be useful for applications requiring surfaces that require tight control of binding sites of biological components to the surface.

[0027] Certain embodiments of the disclosure relate to compositions comprising crosslinked (meth)acrylated and non-(meth)acrylated ECM materials. The compositions can have a ratio of (meth)acrylated ECM material to non-(meth)acrylated ECM material of about 5:1 to about 1:5 (e.g., about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, or about 1:5). The (meth)acrylated ECM material can have a degree of (meth)acrylation of about 5 to about 95 percent (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%). The ECM material may be selected from collagen, gelatin, elastin, and fibronectin. The ECM material may be collagen type I. The (meth)acrylated ECM material may include mono- or di-(meth)acrylated ECM or ECM-like materials.

[0028] The ECM can include one or more peptides. Non-limiting examples of suitable peptides include RGD, PHSRN(GGGERCG)GGRGDSPY, GCREKKRKRLQVQLSIRT, GCREKKTLQPVYEYMVGV, GCREISAFLGIPFAEPPMGPRRFLPPEPKKP, GCRDGPQGWGQDRCG, GCRDVPMSMRGGDRCG, GFOGER, KQAGDV, YIGSR, REDV, IKVAV, RNIAEIIKDI, KHIFSDDSSE, VPGIG, FHRRIKA, KRSR, APGL, VRN, AAAAAAAAA, GGLGPAGGK, GVPGI, LPETG(G)n, and IEGR. Suitable peptides also include peptide materials that mimic the properties of native ECM, including integrin binding, syndecan binding, ECM deposition, and / or MMP-dependent remodeling. The peptide may be present in the printable composition in an amount of about 0.5 mM to about 5 mM (e.g., about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, or about 5 mM). In some cases, the ECM material may contain about 0.5 mM peptide to about 10 mM peptide. In other cases, the ECM material may contain about 5 mM peptide to about 20 mM peptide. In other cases, the ECM material may contain about 10 mM peptide to about 100 mM peptide. The ECM or ECM-like material may be a protease-sensitive amino acid sequence.The protease may be selected from Arg-C proteinase, Asp-N endopeptidase, BNPS-skatole, caspase 1-10, chymotrypsin high specificity (C-terminal to [FYW], not before P), chymotrypsin low specificity (C-terminal to [FYWML], not before P), clostripain (clostridiopeptidase B), CNBr, enterokinase, factor Xa, formate, glutamyl endopeptidase, granzyme B, hydroxylamine, iodosobenzoate, LysC, neutrophil elastase, NTCB (2-nitro-5-thiocyanobenzoate), pepsin, proline endopeptidase, proteinase K, Staphylococcal peptidase I, thermolysin, thrombin, and trypsin.

[0029] The composition may further comprise a polymeric material. The polymeric material may be hydrophilic. The polymeric material may be acrylamide, poly(N-isopropylacrylamide), 2-hydroxyethyl methacrylate, poly(2-hydroxyethyl methacrylate), triethylene glycol dimethacrylate, tetra(ethylene glycol) dimethacrylate, N,N'-methylene biacrylamide, or amine end functionalized 4-arm poly(ethylene glycol). The polymeric material may be polymerized poly(ethylene glycol) di(meth)acrylate, poly(hydroxyethyl)(methacrylate), poly N-hydroxyl acrylamide 3-hydroxypropyl acrylate, or hydroxybutyl acrylate. The polymeric material may have a weight average molecular weight (M) of about 400 to about 20,000. w The polymeric material may be a poly(ethylene glycol) di(meth)acrylate monomer having an M of about 2000 to about 4000. wThe polymeric material may be a mixture of any of the compositions described above. The polymeric material may be present in an amount of about 5-50 wt.% (e.g., about 5 wt.%, about 10 wt.%, about 15 wt.%, about 20 wt.%, about 25 wt.%, about 30 wt.%, about 35 wt.%, about 40 wt.%, about 45 wt.%, or about 50 wt.%) of the composition. In some embodiments, the polymeric material can be a polymerized poly(ethylene glycol) di(meth)acrylate monomer present in an amount of about 5 to about 50 wt.% (e.g., about 5 wt.%, about 10 wt.%, about 15 wt.%, about 20 wt.%, about 25 wt.%, about 30 wt.%, about 35 wt.%, about 40 wt.%, about 45 wt.% or about 50 wt.%) of the composition.

[0030] Printable Composition An embodiment of the disclosure may relate to a method of making a three-dimensional article, comprising depositing a layer of a printable composition onto a surface to obtain a deposited layer, irradiating the deposited layer, and repeating the depositing and irradiating steps until the deposited layer forms a three-dimensional article. The printable composition may include a (meth)acrylated extracellular matrix (ECM) material, a non-(meth)acrylated ECM material, and a photoinitiator. The ECM material may include collagen, gelatin, elastin, and fibronectin.

[0031] The printable composition may include poly(ethylene glycol) di(meth)acrylate monomers. The printable composition may include mono- or di(meth)acrylated ECM or ECM-like materials. The ECM or ECM-like materials may include one or more of RGD, PHSRN(GGGERCG)GGRGDSPY, GCREKKRKRLQVQLSIRT, GCREKKTLQPVYEYMVGV, GCREISAFLGIPFAEPPMGPRRFLPPEPKKP, GCRDGPQGWGQDRCG, GCRDVPMSMRGGDRCG, GFOGER KQAGDV, YIGSR, REDV, IKVAV, RNIAEIIKDI, KHIFSDDSSE, VPGIG, FHRRIKA, KRSR, APGL, VRN, AAAAAAAAA, GGLGPAGGK, GVPGI, LPETG(G)n, and IEGR. Suitable peptides also include peptide materials that mimic the properties of native ECM, including integrin binding, syndecan binding, ECM deposition and / or MMP-dependent remodeling. The peptides can be present in the printable composition in an amount of about 0.5 mM to about 5 mM (e.g., about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, or about 5 mM). The ECM or ECM-like material can include a sequence that is susceptible to a protease. The protease may be selected from Arg-C proteinase, Asp-N endopeptidase, BNPS-skatole, caspase 1-10, chymotrypsin high specificity (C-terminal to [FYW], not before P), chymotrypsin low specificity (C-terminal to [FYWML], not before P), clostripain (clostridiopeptidase B), CNBr, enterokinase, factor Xa, formate, glutamyl endopeptidase, granzyme B, hydroxylamine, iodosobenzoate, LysC, neutrophil elastase, NTCB (2-nitro-5-thiocyanobenzoate), pepsin, proline endopeptidase, proteinase K, Staphylococcal peptidase I, thermolysin, thrombin, and trypsin.

[0032] The ratio of (meth)acrylated ECM material to non-(meth)acrylated ECM material can be about 5:1 to about 1:5 (e.g., about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4 or about 1:5). The (meth)acrylated ECM material can have a degree of (meth)acrylation of about 5 to about 95 percent (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95%). 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, and riboflavin, or mixtures thereof.

[0033] An embodiment of the disclosure may include a printable composition, where the one or more additives include a polymer, a photoactive dye, a natural extracellular matrix, a photoinitiator, a peptide, an amino acid, a growth factor, a modified extracellular matrix, an extracellular matrix fragment, or a mixture thereof. The photoactive dye may be a UV dye having an absorbance spectrum between 300 nm and 420 nm. The photoactive dye may have a wavelength range of 300 nm to 400 nm. The photoactive dye may be non-cytotoxic. The photoactive dye may include a benzyne ring in its molecular structure. The photoactive dye may be a quinolone yellow, a UV dye, or a dye having a similar molecular structure thereto. The photoactive dye may be a UV 386A dye.

[0034] The printable compositions described herein can be used to create scaffolds using 3D printing. The printed scaffolds can be formulated, for example, using appropriately formulated bioinks that are non-cytotoxic when placed in an appropriate buffer. The bioinks selected to form the scaffolds can be selected depending on the desired biocompatibility of the resulting scaffold. For example, the scaffolds can support the attachment, growth and proliferation of selected cell types, such as lung-derived cells.

[0035] The printable composition can include a protic solvent. The protic solvent can include water, polyethylene glycol, glycol diacrylate derivatives, or mixtures thereof. In some cases, the printable composition can include a buffer solution of HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) or PBS (phosphate buffered saline). The polyethylene glycol diacrylate can vary from about 1 wt.% to about 20 wt.%. Non-limiting examples of polyethylene glycol diacrylate include polyethylene (olyehylene) diacrylate 3400 (PEGDA 3400), polyethylene diacrylate 6000 (PEGDA 6000), polyethylene diacrylate 575 (PEGDA 575), and mixtures thereof. The printable composition can include about 1 wt.% to about 20 wt.% HPA or HBA. The printable material can also include about 0.5 wt.% to about 3 wt.% N-hydroxyethylacrylamide (HEAA). The printable material can also contain about 0.5 wt.% to about 10 wt.% (meth)acrylated ECM material and / or non-(meth)acrylated ECM material. Additionally, the printable material can contain about 0.5 wt.% to about 3 wt.% PEG-acrylate CGRGDS, e.g., PEG 3400 It may also contain the acrylate CGRGDS. The printable material may contain from about 0.5% to about 5% of a photoinitiator and from about 0.1% to about 5% of a light absorber dye.

[0036] The composition can support the attachment, proliferation and spreading of primary cells and / or induced pluripotent stem cells. The composition can be a molded or 3D printed hydrogel article. The composition can be a molded or 3D hydrogel printed photocrosslinked article. The molded or 3D printed hydrogel article can be a three-dimensional article of an organ. The organ can be a mammalian organ.

[0037] The printable compositions described herein can be formed into three-dimensional objects that mimic or replicate an organ or a portion of an organ. For example, the printable compositions described herein can be formed into structures that mimic or replicate the architecture of a lung, for example, using 3D printing technology. The printable compositions can be used to form a scaffold for cell adhesion and growth, resulting in a structure that has one or more desired properties of an organ, such as a structure that can perform the gas exchange function of the lung. These objects can include hydrogels. The organ or portion of an organ can be a human lung in a preferred embodiment. The shape of the 3D object is not particularly limited and can be a tube shape, or can be substantially the same shape, size, and / or have the same relative dimensions as an organ or a fragment of an organ.

[0038] In some embodiments, the object formed from the printable composition is substantially the same shape, size, and / or has 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 additional embodiments, the shape of the blood vessel comprises a pulmonary artery, renal artery, coronary artery, peripheral artery, pulmonary vein, or renal vein. In certain embodiments, the structure comprises a hemodialysis graft. Other embodiments include where the structure is substantially the shape of a lung lobe, a lung, a pulmonary airway tree, a pulmonary vasculature, or a combination thereof. In some embodiments, the reinforcement includes maintaining air flow or blood (or fluid) flow through the structure when external pressure is applied to the structure.

[0039] Embodiments of this disclosure relate to the use of functionalized and non-functionalized extracellular matrices, matrix fragments, peptides and bioactive components formed by the disclosed methods.

[0040] 3D composition An embodiment of the disclosure may relate to a method of making a three-dimensional article, the method comprising depositing a layer of a printable composition onto a surface to obtain a deposited layer, irradiating the deposited layer, and repeating the depositing and irradiating steps until the deposited layer forms the three-dimensional article. The printable composition may include a hydrogel material, a modified or unmodified extracellular matrix (ECM) material, and a photoinitiator.

[0041] The three-dimensional (3D) hydrogel structure is not particularly limited, and can be, for example, a composite structure made of one or more different polymerized monomers. The hydrogel material that can be used in the present invention can be known to those skilled in the art, as can the method of making it. For example, the hydrogel described in Calo et al., European Polymer Journal Volume 65, April 2015, Pages 252-267 can be used. In some embodiments, the hydrogel structure comprises polymerized (meth)acrylate and / or (meth)acrylamide hydrogel. 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), polylactic acid, polyglycol, In some embodiments, the M of the hydrogel polymer includes polymers comprising poly(methacrylic acid), polyvinyl alcohol, polyanhydrides, such as 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), sodium phenyl-2,4,6-trimethylbenzoylphosphinate (NAP), and combinations thereof. 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 58 00 Da, 5900 Da, 6000 Da, 6100 Da, 6200 Da, 6300 Da, 6400 Da, 6500 Da, 7000 Da, 7500 Da, 8000 Da, 8500 Da, 9000 Da, 9500 Da, 10000 Da, 15000 Da, or 20000 Da. In some embodiments, the hydrogel polymer comprises NAP as a major component and further comprises one or more peptides and / or collagens as described herein, i.e., functionalized with PEGDA.

[0042] In some embodiments, the hydrogel comprises a crosslinked polymer. In some embodiments, the polymer is crosslinked by 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% based on the percentage of crosslinkable moieties in the polymer. The crosslinked moieties may, for example, comprise (meth)acrylate groups.

[0043] Embodiments of the disclosure relate to the use of functionalized and non-functionalized extracellular matrices, matrix fragments, peptides, and bioactive components formed by the disclosed methods. In some cases, these extracellular matrices can be formed from materials including collagen, gelatin, elastin, and / or fibronectin. In some cases, the compounds, such as collagen, can be reacted with methacrylate anhydrides to form methacrylated compounds, such as methacrylated collagen, as shown in FIG. 1.

[0044] As disclosed herein, the degree of collagen functionalization (DOF) in the hydrogel can vary, for example, in the ratio of (meth)acrylated collagen to non-(meth)acrylated collagen. In some cases, the collagen can be hybrid or contain both functionalized and non-functionalized components. For example, hybrid can refer to a hydrogel containing both (meth)acrylated and non-(meth)acrylated collagen. 3D printed objects made of poly(ethylene glycol) diacrylate containing (meth)acrylated and non-(meth)acrylated collagen can support the attachment, spreading and / or proliferation of lung-derived fibroblasts, endothelial cells and smooth muscle cells.

[0045] As disclosed herein, alternative biomaterials may be used in place of collagen. These biomaterials may include type IV collagen, fibronectin, gelatin, type III collagen, short peptides (e.g., RGD), fragments of ECM proteins, proteoglycans, glycosaminoglycans, hyaluronic acid, or any other extracellular matrix from any species.

[0046] The printable compositions may be modified to enhance cell attachment and / or mechanical properties. These printable compositions or inks may have unique mechanical properties or reactivity orthogonal to acrylate reactivity. Components of the inks or printable compositions may include poly(ethylene glycol) di(meth)acrylate monomers. The printable compositions may include mono- or di(meth)acrylated ECM or ECM-like materials. The ECM or ECM-like material may include one or more of RGD, PHSRN(GGGERCG)GGRGDSPY, GCREKKRKRLQVQLSIRT, GCREKKTLQPVYEYMVGV, GCREISAFLGIPFAEPPMGPRRFLPPEPKKP, GCRDGPQGWGQDRCG, GCRDVPMSMRGGDRCG, GFOGER, KQAGDV, YIGSR, REDV, IKVAV, RNIAEIIKDI, KHIFSDDSSE, VPGIG, FHRRIKA, KRSR, APGL, VRN, AAAAAAAAA, GGLGPAGGK, GVPGI, LPETG(G)n, and IEGR. Suitable peptides also include peptide materials that mimic properties of native ECM, including integrin binding, syndecan binding, ECM deposition and / or MMP-dependent remodeling. The peptides may be present in the printable composition in an amount of about 0.5 mM to about 5 mM (e.g., about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, or about 5 mM). Alternatively, or in addition, the 3D printed object may be further surface-modified with one or more peptides. Surface modification may be accomplished by reacting unreacted (meth)acrylate moieties in the 3D printed object with one or more peptides by contacting the unreacted moieties with a solution containing one or more peptides. It should be understood that this disclosure includes surface modification with other ECM or ECM-like materials disclosed herein, similar to that described above for peptides.The ECM or ECM-like material can include sequences susceptible to a protease. The protease can be selected from Arg-C proteinase, Asp-N endopeptidase, BNPS-skatole, caspase 1-10, chymotrypsin high specificity (C-terminal to [FYW], not before P), chymotrypsin low specificity (C-terminal to [FYWML], not before P), clostripain (clostridiopeptidase B), CNBr, enterokinase, factor Xa, formate, glutamyl endopeptidase, granzyme B, hydroxylamine, iodosobenzoate, LysC, neutrophil elastase, NTCB (2-nitro-5-thiocyanobenzoate), pepsin, proline endopeptidase, proteinase K, Staphylococcal peptidase I, thermolysin, thrombin, and trypsin.

[0047] The available binding sites for the extracellular matrix can be varied through functionalization or modification with different chemical groups. These chemical groups can bind to amine groups or other groups for which cells have an affinity. Below are examples of mono- and di(meth)acrylates with bioactive moieties R1 that can be used to create ECM or ECM / ECM-like printable compositions or inks.

[0048] [ka] R can be hydrogen or a methyl group. R1 can be any of the following: RGD Fibronectin, Vitronectin Cell Adhesion PHSRNKRGD Fibronectin cell adhesion GCREKKRKRLQVQLSIRT laminin cell adhesion GCREKKTLQPVYEYMVGV Peptide with affinity to fibronectin GCREISAFLGIPFAEPPMGPRRFLPPEPKKP A peptide having affinity for Col IV, and LMNGCRDGPQGWGQDRCG Cytolytic peptide GCRDVPMSMRGGDRCG Cytolytic peptide KQAGDV smooth muscle cell adhesion YIGSR laminin B1 cell adhesion REDV fibronectin endothelial cell adhesion IKVAV Laminin Neurite Outgrowth RNIAEIIKDI Laminin B2 Neurite Outgrowth KHIFSDDSSE Neural cell adhesion molecule Astrocyte adhesion VPGIG elastin enhances the elastic modulus of artificial ECM FHRRIKA heparin-binding domain improves osteoblast mineralization KRSR heparin-binding domain regulates osteoblast adhesion APGL collagenase-mediated degradation VRN plasmin-mediated degradation AAAAAAAAA Elastase-mediated degradation GGLGPAGGK Protease sensitive peptide GVPGI Elastin associated with mechanical stability LPETG(G) n Sortase-mediated ligation IEGR protease susceptibility ECM components or protease digests of ECM components Monosaccharides, oligosaccharides, polysaccharides (hyaluronic acid, heparan, aggrecan, chondroitin, sialic acid) Redox reactivity Sulfhydryl (-SH) Disulfide (SS) R1 can also be any of the following sequences susceptible to the following proteases, where [P4...P2'] is the generally accepted nomenclature for designating protease cleavage site sequences:

[0049] [Table 1-1]

[0050] [Table 1-2]

[0051] The ratio of modified ECM material to unmodified ECM material can be optimized based on the application. For example, the ratio of modified ECM material to unmodified ECM material can be optimized based on parameters such as material properties and desired cell attachment. In some embodiments, the ratio of modified ECM material to unmodified ECM material can be about 5:1 to about 1:5 (e.g., about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4 or about 1:5). The modified ECM material can have a degree of modification of about 5 to about 95 percent (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95%). In some cases, the modification may be (meth)acrylation of the ECM. In these embodiments, the ratio of (meth)acrylated ECM material to non-(meth)acrylated ECM material may be about 5:1 to about 1:5 (e.g., about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, or about 1:5). The (meth)acrylated ECM material may have a degree of (meth)acrylation of about 5 to about 95 percent (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%).

[0052] The composition may include a photoinitiator. The photoinitiator is not particularly limited. The photoinitiator may be a photoactive dye. The photoactive dye may be a UV dye having an absorbance spectrum between 100 and 420 nm. The UV dye may have an absorbance spectrum between 300 nm and 420 nm. The photoactive dye may have a wavelength range of 300 nm to 400 nm. The photoactive dye may be non-cytotoxic. The photoactive dye may include a benzyne ring in the molecular structure. The photoactive dye may be quinolone yellow, a UV dye, or a dye having a molecular structure similar thereto. The photoactive dye may be a UV 386A dye.

[0053] The photoinitiator may include, for example, benzophenone, phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide (BAPO), 2-hydroxy-2-methyl-l-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, lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate (LAP), and ethyl(2,4,6-trimethylbenzoyl)phenylphosphinic acid, sodium phenyl-2,4,6-trimethylbenzoylphosphinate (NAP), trimethylbenzoyl-based photoinitiators, diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (TPO nanoparticles) Irgacure class photoinitiators, ruthenium, and riboflavin, or mixtures thereof.

[0054] method In certain embodiments, hydrogel scaffolds, objects, and / or methods of making are disclosed.Objects can be formed by 3D printing.The compositions and materials listed above can be used as ink in 3D printers to form 3D printed objects.

[0055] Those skilled in the art will understand printing methods known in the art, non-limiting examples include selective laser sintering (SLS), fused deposition modeling (FDM), 3D inkjet printing, digital light processing (DLP) and stereolithography. In fused deposition modeling (FDM), ink is deposited by an extrusion head along a tool path defined by a CAD file. The material is deposited in fine layers of 25 μm thickness, and the part is built up, layer by layer, from the bottom up. Some 3D printers based on fused deposition modeling are equipped with dual print nozzle heads that can extrude two different materials, one of which is a building material and the other is a support material such as a pillar. The support material can be washed with water.

[0056] 3D inkjet printing is effectively optimized for speed, low cost, high accuracy and ease of use, making it suitable for visualization from the conceptual stage of engineering design to early-stage functional testing. In inkjet printing, complex 3D objects are produced from ink compositions by ejection followed by UV / Vis light. In the inkjet printing process, photocurable inks can be ejected through several nozzles onto a building platform in a pattern defined by a CAD file.

[0057] Among the 3D printing techniques, an effective technique is the digital light processing (DLP) method or stereolithography (SLA). In a 3D printer using the DLP or SLA method, the ink material is layered on a vat or spread on a sheet, and a predetermined area or surface of the ink is exposed to ultraviolet-visible (UV / Vis) light controlled by a digital micromirror device or a rotating mirror. In the DLP method, additional layers 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 the line of a radiation beam. Other methods of 3D printing can be found in 3D Printing Techniques and Processes by Michael Degnan, Dec 2017, Cavendish Square Publishing, LLC, the disclosure of which is incorporated herein by reference.

[0058] In some embodiments, once the 3D printed object is formed, cells are deposited onto it. EXAMPLES

[0059] The following examples describe certain aspects of some embodiments of the present disclosure to illustrate and provide explanations to one of ordinary skill in the art. The examples are not to be construed as limiting the disclosure, as they merely provide certain methods useful in understanding and practicing some embodiments of the disclosure.

[0060] printing All samples used in the examples were prepared using 3D inkjet printing or digital light processing (DLP) methods, where the components of the bioink were mixed and then the 3D objects were printed using, for example, a Labfab inverted digital light projection (DLP) 3D printing system.

[0061] Bioink components were sourced from commercial sources when available. Biologically active peptides were synthesized by performing a Michael-type reaction to link the peptide to one or more (meth)acrylate monomers or polymers.

[0062] Cell adhesion studies The following cell adhesion test was performed according to at least part of the following protocol. First, 3D printed disks were obtained and, if made of fully non-methacrylated collagen, the disks were subjected to a cross-linking treatment with sterile 1M NaHCO3. The disks were washed twice with DPBS++ for at least 30 minutes. The disks were then placed in 5x Anti-Anti solution (100x antibiotic-antimycotic diluted in DPBS--) overnight. The 5x Anti-anti was then replaced with two PBS washes for at least 30 minutes each. Using an optical 96-well plate, three wells were filled with 200uL of wash solution each. The average 384nm absorbance of the wash solution was obtained from these three wells using a SpectraMax i3x (or equivalent). The 384nm absorbance was less than 0.1, indicating that the traces of dye / PI were at acceptable levels.

[0063] The disks are then transferred to wells with 500uL of LFN GM (Lung Fibroblast Growth Medium) and a given number of cells are added to the solution. After a given time, 10% formalin + 0.1% TritonX100 is added, 300uL / well to the control and 500uL / well to the wells containing 3D disks, and incubated at room temperature for 15 minutes. The fixative is then aspirated into a waste container and the samples are washed with DPBS-- (3 x 5 minute washes). The disks are then stained with a solution containing 1:20,000 SytoxOrange and 3:400 Phalloidin 488. After a final DPBS wash, the disks are evaluated for cell attachment.

[0064] Example 1 Lung fibroblasts were cultured for 7 days on glass (control), bioink containing collagen with 50% DOF, bioink containing collagen with 90% DOF, and bioink containing collagen with 0% DOF and collagen with 90% DOF (1:2 ratio).

[0065] Bioinks were formulated according to the procedure described above. Bioinks containing collagen with different degrees of functionalization were then 3D printed into disks. Lung fibroblasts were deposited on each sample according to the procedure described above. Fibroblasts were cultured for 7 days on glass (control), printed disks from bioinks containing collagen at 50% DOF, printed disks from bioinks containing collagen at 90% DOF, and printed disks from hybrid bioink formulations containing a hybrid of collagen at 0% DOF and collagen at 90% DOF (1:2 ratio). Images were taken as shown in Figure 2(A). Graphs of cell spreading, density, and % of cell coverage were plotted for each sample as shown in the graphs of cell spreading, density, and % of cell coverage in Figure 2(B).

[0066] Example 2 Pulmonary artery endothelial cells were cultured for 1 day on glass, printed discs from 9% PEGDA hybrid, and printed discs from 9% PEGDA.

[0067] Bioinks were formulated according to the procedure above. Bioinks containing PEGDA and PEGDA hybrid were 3D printed into disks. Pulmonary artery endothelial cells were deposited on each sample according to the procedure above. Pulmonary artery endothelial cells were then cultured on glass (control), PEGDA and PEGDA hybrid disks for 1 day. Images were taken as shown in Figure 3(A). Graphs of cell spreading, density and % of cell coverage were plotted for each sample as shown in the graphs of cell spreading, density and % of cell coverage in Figure 3(B).

[0068] Example 3 The ability of pulmonary smooth muscle cells to attach and proliferate on 3D printed discs containing PEGDA MW3400, collagen with 95% DOF, and collagen with 0% DOF (2:1 ratio) was investigated.

[0069] Bioink was formulated according to the method described above. The bioink was 3D printed to form a 3D printed disk containing PEGDA MW 3400, collagen at 95% DOF and collagen at 0% DOF (2:1 ratio). Lung smooth cells were deposited on the disk according to the method described above. The cells were allowed to attach, proliferate and spread across the disk over the course of 7 days. Images of the disks at 2, 5 and 7 days were taken as shown in FIG. 4.

[0070] Example 4 Number of cell adhesion properties on 3D printed objects made from bioinks with different ratios of functionalized to non-functionalized collagen. Bioinks C201, C202 and C203 were formed with the components in Table 2 below. These bioinks were 3D printed according to the method described above.

[0071] [Table 2]

[0072] After printing, cells were deposited on each object according to the method described above. Cell adhesion was measured for each sample C201, C202, and C203 and plotted on Figure 5A. Figure 5B shows images of the samples.

[0073] As shown in graph 5C, sample C201 showed significantly greater cell adhesion versus C202 and C203.

[0074] Example 5 3D printed bioinks containing functionalized and non-functionalized collagen were examined to assess their support for lung fibroblast attachment and proliferation on a daily basis.

[0075] Hybrid bioinks containing 30 DOF hybrid collagen (a mixture of 0 DOF (non-methaycrylated collagen) and 90 DOF (methaycrylated collagen)) were formed by adding LAP as a solid (0.5–2 wt.%) to an aqueous solution containing PEGDA 3400 (5–14 wt.%) and HPA (8–17 wt.%), and the resulting solution was speed mixed until clear. Dye (0.12%) was added and the solution was ensured to be between pH 2–3. Next, 30 DOF hybrid collagen (40–55 wt.%) was added and the solution was stirred until homogenous. The degree of functionalization was 30%. The bioinks were 3D printed. Lung cells were deposited on the samples following the method described above. The amount of cell attachment and proliferation was imaged on days 1, 4 and 7 and is shown in Figure 6.

[0076] Example 6 The effect of HEAA (%) content on cell adhesion properties was investigated. 3D printed discs as illustrated in Figure 7 were obtained according to the method described above and cells were deposited on the discs according to the method described above.

[0077] The effect of HEAA (%) content on cell adhesion can be seen in Figure 7. Figure 7A shows the ratio of components in the bioink. Samples containing 5%, 10% and 20% HEAA were tested and cell adhesion is shown in Figure 7B.

[0078] Example 7 Effect of CollMA DOF on cell adhesion and proliferation (>90%, ~50%, hybrid (50% non-MA, 50% HM)). Samples were formed by 3D printing bioinks containing the following sample compositions: Sample 1 Composition PEGDA3.4k(3~10wt.%);LAP(0.3~1.0wt.%);UV386A;ColMA(90 DOF) Sample 2 Composition PEGDA3.4k(3~10wt.%);LAP(0.3~1.0wt.%);UV386A;ColMA(50 DOF) Sample 3 Composition PEGDA3.4k (3-10wt.%); LAP (0.3-1.0wt.%); UV386A; ColMA hybrid (0 DOF+90 DOF)

[0079] Rheology was measured and the results are shown in the graph in Figure 8A.

[0080] The bioink was 3D printed using a Labfab inverted digital light projection (DLP) 3D printing system. Fibroblast cells were deposited and incubated as per the procedure above. Figure 8E shows the graphs of cell spreading, cell density and percent cell coverage by day. Cell spreading, cell density and percent coverage were calculated as per the procedure above on days 1, 4 and 7 as shown in Figure 8B-D. Graphs of cell spreading, cell density and percent cell coverage between samples are graphed in Figure 8E. As shown, cell spreading improved on day 7 for the hybrid and 50% DOF inks. Cells proliferated actively on the 50% DOF ink compared to the 90% DOF and hybrid inks over the course of 7 days. One model for this is that non-methacrylated collagen may be eluted from the hybrid gel.

[0081] Example 8 Comparison of cell density, cell spreading and cell coverage of PAEC cells.

[0082] PAEC cells were cultured at 10,500 / cm 2 The cells were seeded at a dose of 100 μg / ml and cultured for 1 day. The samples were compared to glass slides. As shown in Figure 9, the cell density, cell spreading and cell coverage were compared between the samples and the control.

[0083] Example 9 Samples with compositions like samples 1-3 above were produced using five different batches of collagen. For each of these batches, a sample was formed by mixing the ink components in water and then 3D printing the ink into a disk. Each sample contained 5000 cells / cm. 2 (PAEC) were seeded on the glass plates. In addition, PARC was seeded on the glass controls. Cell spreading, cell density, and percent cell coverage were assessed on days 1, 4, and 7.

[0084] Example 10 LFN on 5% PEGDA with Col I at x% DOF

[0085] Samples of 5% PEGDA and type I collagen with different degrees of functionalization were produced. Samples included a glass control, 50% degree of type I collagen functionalization, 90% type I collagen functionalization, and a hybrid sample. The samples were formed by mixing the ink components in water and then 3D printing the ink into a disk. Each sample was seeded with cells in a similar manner as described above.

[0086] As shown in Figure 10A, cell spreading, cell density, percent cell coverage and images were compared between each sample on day 1. As shown in Figure 10B, cell spreading, cell density, percent cell coverage and images were compared between each sample on day 4. As shown in Figure 10C, cell spreading, cell density, percent cell coverage and images were compared between each sample on day 7. As shown in Figure 10D, cell spreading, cell density and percent cell coverage were graphed for each sample by day.

[0087] Example 11 This study evaluated the biocompatibility of AC42 (a bioink with 6-12 wt.% HPA; 6-12 wt.% PEGDA3.4k, various methacrylated collagens, LAP, and UV dyes). The bioink was printed on a LabFab printer to form 3 mm and 1 mm disks for three time points of testing (1, 4, and 7 days). These disks were glued onto a platform. An image of the disks prior to seeding with cells is shown in Figure 11A.

[0088] The disks were crosslinked in 1M NaHCO3 in 24-well plates for 10 min. Following crosslinking, the disks were transferred to a Petri dish on a shaker plate at speed 60 for PBS++ washes (2 times, at least 10 min each). The disks were transferred to a well plate for overnight 5x washes.

[0089] LFN cells were seeded on each disk. 10K cells were added to each well to seed each sample disk. 20K cells per well were added to seed the control. As shown in FIG. 11B, cell spreading, cell density, percent cell coverage and images were compared between each sample on day 1. The cells spread well (became very confluent) on day 1. As shown in FIG. 11C, cell spreading, cell density, percent cell coverage and images were compared between each sample on day 4. The cells were completely confluent on day 4. As shown in FIG. 11D, cell spreading, cell density, percent cell coverage and images were compared between each sample on day 7. The cells were overlapping each other on day 7. The disks had high cell attachment, but some were detached from the confluent cell sheet on days 4 and 7, which may reduce the cell density for the LFN test.

[0090] Example 12 Figures 12A-12B show percent area coverage, cell spreading comparison, and cell density comparison for AC42 1 mm disks, 3 mm disks, elution control, and glass control seeded with LFN, PAEC, or SAEC seeds. AC42 bioink was printed on a LabFab printer to form 3 mm and 1 mm disks for three time points tested (1, 4, 7 days). The disks were glued to a platform. An image of the disks is shown in Figure 12A.

[0091] The disks were crosslinked in 1M NaHCO3 in 24-well plates for 10 min. Following crosslinking, the disks were transferred to a Petri dish on a shaker plate at speed 60 for PBS++ washes (2 times, at least 10 min each). The disks were transferred to a well plate for overnight 5x washes. LFN, PAEC or SAEC cells were seeded onto each disk.

[0092] Figure 12A shows the percent area coverage, cell spreading comparison, and cell density comparison for AC42 1 mm disks, 3 mm disks seeded with LFN, PAEC, or SAEC seeds, elution control, and glass control on day 1. Figure 12B shows the percent area coverage, cell spreading comparison, and cell density comparison for AC42 1 mm disks and 3 mm disks seeded with LFN, PAEC, or SAEC seeds on day 1.

[0093] Example 13 2-5wt.% HPA or HBA, 1-5wt.% PEGDA575, 3-8wt.% PEGDA6000, 1-6mM PEG 3400 Discs were printed from a bioink containing the acrylate CGRGDS, LAP, UV386A and water.

[0094] The PAEC and SAEC coverage was examined on days 1 and 4. Figure 14A shows the results of the hybridization of 2-5 wt.% HPA, 1-2 wt.% PEGDA575, 3-8 wt.% PEGDA6000, 1-6 mM PEG 3400Figure 14B shows cell coverage on disks containing the acrylates CGRGDS, LAP, and UV386A. Figure 14B shows cell coverage on disks containing 2-5 wt.% HPA, 3-4 wt.% PEGDA575, 3-8 wt.% PEGDA6000, 1-6 mM PEG 3400 Figure 14C shows the cell coverage on disks containing the acrylates CGRGDS, LAP, UV386A, 2-5 wt.% HBA, 3-4 wt.% PEGDA575, 3-8 wt.% PEGDA6000, 1-6 mM PEG 3400 The cell coverage on discs containing the acrylates CGRGDS, LAP, and UV386A is shown.

[0095] Example 14 Discs printed from inks with the following components were surface modified with PHSRNKRGDS (0.5–1 mM) and AG73 (0.3–0.7 mM) by reacting unreacted acrylate groups after printing with peptides in solution.

[0096] [Table 3]

[0097] As shown in Figures 16A-16B, fibroblast growth / attachment was examined on days 1 and 4. As shown in Figures 17A-17B, SAEC growth / attachment was examined on days 1 and 4.

[0098] As used herein, the singular terms "a," "an," and "the" may include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to an object may include a plurality of objects unless the context clearly dictates otherwise.

[0099] As used herein, the terms "substantially" and "about" are used to describe and explain small variations.When used in conjunction with an event or situation, the term can refer to the exact occurrence of the event or situation, as well as the approximate occurrence of the event or situation.When used in conjunction with a numerical value, the term can refer to a range of variation of less than or equal to ±10% of the numerical value, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to ±0.1%, or less than or equal to ±0.05%. When referring to a first numerical value being "substantially" or "about" the same as a second numerical value, the term can refer to the first numerical value being within a range of variation of less than or equal to ±10% of the second numerical value, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to ±0.1%, or less than or equal to ±0.05%.

[0100] In addition, amounts, ratios and other numerical values ​​may be expressed in a range format herein. It should be understood that such range formats are used for convenience and brevity, and should be understood to be flexible, including not only 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 within the range of about 1 to about 200 should be understood to include not only the explicitly recited limits of about 1 and about 200, but also 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.

[0101] Although the present disclosure has been described with reference to certain embodiments thereof, it should be understood by those skilled in the art that various changes can be made and equivalents can be substituted without departing from the true spirit and scope of the present disclosure as defined by the appended claims. In addition, many modifications can be made to adapt a particular situation, material, composition of matter, method, operation(s) to the objective, spirit and scope of the present disclosure. All such modifications are intended to be within the scope of the appended claims. In particular, certain methods are described with reference to certain operations performed in a particular order, but it is understood that these operations may be combined, sub-divided or rearranged to form equivalent methods without departing from the teachings of the present disclosure. Thus, unless specifically indicated herein, the order and grouping of operations are not limitations of the present disclosure.

Claims

1. 1. A composition comprising a crosslinked (meth)acrylated extracellular matrix (ECM) material and a non-(meth)acrylated ECM material, A composition, wherein the (meth)acrylated ECM material comprises collagen and has a degree of (meth)acrylation of about 45 to about 95 percent.

2. The composition of claim 1, wherein the ratio of (meth)acrylated ECM material to non-(meth)acrylated ECM material is from about 5:1 to about 1:

5.

3. The composition of claim 1 or 2, wherein the ECM material is type I collagen.

4. the (meth)acrylated ECM material comprises a mono- or di-(meth)acrylated ECM or ECM-like material; Optionally, the ECM or ECM-like material comprises a sequence sensitive to a protease; 2. The composition of claim 1, further optionally wherein the protease is selected from Arg-C proteinase, Asp-N endopeptidase, BNPS-skatole, caspase 1-10, chymotrypsin high specificity (C-terminal to [FYW], not before P), chymotrypsin low specificity (C-terminal to [FYWML], not before P), 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.

5. The composition of claim 1, wherein the composition further comprises polymerized poly(ethylene glycol) di(meth)acrylate, poly(hydroxyethyl)(methacrylate), poly N-hydroxylacrylamide, 3-hydroxypropyl acrylate, hydroxybutyl acrylate.

6. The poly(ethylene glycol) di(meth)acrylate monomer has a weight average molecular weight (M ｗ ) or has a M w of about 2000 to about 4000; and / or 6. The composition of claim 5, wherein the polymerized poly(ethylene glycol) di(meth)acrylate monomer is present in an amount of about 5 to about 50 wt. % of the composition.

7. The composition of claim 1 , wherein the composition supports the attachment, proliferation and spreading of primary cells and / or induced pluripotent stem cells.

8. 10. The composition of claim 1, wherein the composition is an optionally photocrosslinked, molded or 3D printed hydrogel article.

9. 10. The molded or 3D printed hydrogel article of claim 8, wherein the article is a three-dimensional article of an organ, the organ being a mammalian organ.

10. 1. A method of making a three-dimensional article, comprising: depositing a layer of a printable composition on a surface to obtain a deposited layer; irradiating the deposited layer; and repeating the depositing and irradiating steps until the deposited layers form a three-dimensional article. Including, the printable composition comprises a (meth)acrylated extracellular matrix (ECM) material, a non-(meth)acrylated ECM material, and a photoinitiator; The method, wherein the (meth)acrylated ECM material comprises collagen and has a degree of (meth)acrylation of about 45 to about 95 percent.

11. The method of claim 10 , wherein the printable composition further comprises a poly(ethylene glycol) di(meth)acrylate monomer.

12. the printable composition further comprises a mono- or di-(meth)acrylated ECM or ECM-like material; Optionally, the ECM or ECM-like material comprises a sequence sensitive to a protease; 12. The method of claim 10 or 11, further optionally wherein the protease is selected from Arg-C proteinase, Asp-N endopeptidase, BNPS-skatole, caspase 1-10, chymotrypsin high specificity (C-terminal to [FYW], not before P), chymotrypsin low specificity (C-terminal to [FYWML], not before P), clostripain (clostridiopeptidase B), CNBr, enterokinase, factor Xa, formate, glutamyl endopeptidase, granzyme B, hydroxylamine, iodosobenzoate, LysC, neutrophil elastase, NTCB (2-nitro-5-thiocyanobenzoate), pepsin, proline endopeptidase, proteinase K, Staphylococcal peptidase I, thermolysin, thrombin, and trypsin.

13. and / or the ratio of (meth)acrylated ECM material to non-(meth)acrylated ECM material is from about 5:1 to about 1:5; and / or The photoinitiator comprises 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, and riboflavin, or mixtures thereof; and / or the printable composition further comprises one or more additives including a polymer, a UV386A dye, a native extracellular matrix, a photoinitiator, a peptide, an amino acid, a growth factor, a modified extracellular matrix, an extracellular matrix fragment, or a mixture thereof; and / or 11. The method of claim 10, wherein the printable composition comprises any UV dye having an absorbance spectrum between 300 nm and 420 nm, which is not cytotoxic and has a benzyne ring composition in its molecular structure, or the printed scaffold is not cytotoxic upon elution of monomers into a buffer in which the scaffold is placed.

14. The method of claim 10 , wherein the printable composition further comprises a protic solvent, optionally wherein the protic solvent comprises water, polyethylene glycol, a glycol diacrylate derivative, or a mixture thereof.

15. The method of claim 10 , wherein the three-dimensional article replicates an organ or a portion of an organ.

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