Composite biomaterial

Abstract

To provide an improved scaffold biomaterial.SOLUTION: Provided is a composite scaffold biomaterial including two or more scaffold biomaterial subunits, each including a decellularized plant or fungal tissue from which cellular materials and nucleic acids of the tissue are removed, the decellularized plant or fungal tissue having a 3-dimensional porous structure, the two or more scaffold biomaterial subunits being assembled into the composite scaffold biomaterial and held together via gel casting using a hydrogel glue; via complementary interlocking geometry of the two or more scaffold biomaterial subunits; via guided assembly based biolithography (GAB); via chemical cross-linking; or any combinations thereof. Methods for producing such a scaffold biomaterial, as well as methods and uses thereof, are also provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates generally to scaffold biomaterials, and more particularly to composite scaffold biomaterials comprising decellularized plant and / or fungal tissue, such as composite scaffold biomaterials comprising two or more scaffold biomaterial subunits. [Background technology]

[0002] Biological tissues are complex structures consisting of a collection of different cell types. Within the body, various cell types can interact to perform specialized functions. Cell and extracellular matrix organization are often directly related to function; as a result, defects in cell, tissue, and / or organ function can result from both biochemical and physical defects ranging from genetic disorders to physical injuries. The biochemical and physical environment within the body can vary within, at the interfaces between, and between different tissue types and organs. Therefore, it is difficult to recreate or approximate the natural in vivo environment of cells.

[0003] Much research has been conducted to produce biomimetic constructs. Various approaches, including synthetic biology, regenerative medicine, transplantation, templated, and scaffolding, have been proposed as potential routes to recapture or mimic the native environment. However, recreating the cellular microenvironment has been challenging. Because tissues are themselves complex composites, simple materials typically do not allow for the biochemical and physical complexity to mimic the natural environment. Therefore, extensive research has been devoted to composite materials developed from one or more of the above-listed approaches to biomaterial production.

[0004] An attractive approach for biomaterial design is decellularization, which replaces damaged tissue with a scaffold material composed of animal ECM proteins but devoid of cells. This concept is based on the idea that damaged tissue can be replaced with a scaffold of the same tissue or organ; the scaffold can be repopulated with healthy cells, and native tissue or organ function can be restored. Typically, such organ decellularization approaches have had several difficulties, including dependency on donor tissue, compatibility issues, and practical limitations.

[0005] Alternatively, synthetic approaches may offer several other biomaterial production methods, including 3D printing, casting, and electrospinning, that have been developed to create custom structures that attempt to circumvent certain shortcomings of organ decellularization approaches. However, the development of synthetic scaffold biomaterials that mimic animal tissues and / or conditions has proven challenging in this field, particularly because it is difficult to provide bio-like complexity.

[0006] Nevertheless, scaffolds that can be tailored to mimic in vivo tissues or conditions are highly sought after, especially in the fields of, for example, tissue regeneration and / or repair, bone engineering, and wound healing.

[0007] Additionally, due to sustainability and environmental concerns associated with global meat consumption, cultured and / or meat substitute foods are highly sought after by the industry, however, creating the mouthfeel and / or taste experience of traditional meat foods has proven to be a formidable challenge facing the industry. Summary of the Invention [Problem to be solved by the invention]

[0008] Alternative, additional and / or improved scaffold biomaterials are desirable. [Means for solving the problem]

[0009] Provided herein is a composite scaffold biomaterial comprising two or more scaffold biomaterial subunits, each comprising decellularized plant or fungal tissue, wherein the decellularized plant or fungal tissue has been removed of cellular material and nucleic acids, and wherein the decellularized plant or fungal tissue has a three-dimensional porous structure, wherein the two or more scaffold biomaterial subunits are assembled into the composite scaffold biomaterial, for example, by gel casting using a hydrogel adhesive, by complementary interlocking geometry of the two or more scaffold biomaterial subunits, by guided assembly based biolithography (GAB), by chemical crosslinking, or any combination thereof.

[0003] A composite scaffold biomaterial is provided in which the biomaterial is held together or bound by a matrix of structural elements. Methods for producing such a scaffold biomaterial, as well as methods and uses thereof, are also provided. The scaffold biomaterial can be produced for a variety of different applications, including, but not limited to, medical applications and / or applications in the food industry as plant-based meat substitutes.

[0010] In certain embodiments, the scaffold biomaterials described herein may comprise two or more scaffold biomaterial subunits. By combining two or more subunits, various benefits can be achieved. For example, the selection of individual subunits and their properties can significantly increase the complexity of the resulting scaffold biomaterial after subunit assembly, and the properties of the resulting scaffold biomaterial (overall, at specific sites or regions, or both) can be tailored or customized to suit specific applications, as needed, by appropriately designing and selecting the subunits and the techniques for assembling and connecting them. Similarly, by combining two or more subunits, larger constructs can be prepared without encountering the problems associated with decellularizing larger structures and / or without being limited by the dimensions imposed by plant and / or fungal source materials. Described herein are various subunits that have been developed, as well as various assembly techniques and techniques for imparting structural integrity and tenability to the resulting structures.

[0011] In one embodiment, the present specification provides 1. A composite scaffold biomaterial comprising two or more scaffold biomaterial subunits each comprising decellularized plant or fungal tissue, wherein the decellularized plant or fungal tissue has been stripped of its cellular material and nucleic acids, and the decellularized plant or fungal tissue comprises a three-dimensional porous structure. Composite scaffold biomaterials are provided in which two or more scaffold biomaterial subunits are assembled into a composite scaffold biomaterial and held together by gel casting using a hydrogel adhesive, by complementary / interlocking shapes of the two or more scaffold biomaterial subunits, by guided assembly-based biolithography (GAB), by chemical crosslinking, or any combination thereof.

[0012] In another embodiment of the composite scaffold biomaterial, two or more scaffold biomaterial subunits may be assembled into a composite scaffold biomaterial and held together by gel casting using a hydrogel adhesive.

[0013] In another embodiment of any of the one or more composite scaffold biomaterials described above, the hydrogel adhesive may comprise gelatin, collagen, agarose, hyaluronic acid, alginate, fibrin, fibronectin, agar, PEG, PVA, or any combination thereof.

[0014] In yet another embodiment of any of the one or more composite scaffold biomaterials described above, two or more scaffold biomaterial subunits may act as a scaffold for a hydrogel adhesive to form around.

[0015] In yet another embodiment of any of the one or more composite scaffold biomaterials described above, at least a portion of the two or more scaffold biomaterial subunits may be coated with a hydrogel adhesive.

[0016] In another embodiment of any of the one or more composite scaffold biomaterials described above, the hydrogel adhesive may be cured or solidified by temperature change, crosslinking, or a combination thereof.

[0017] In yet another embodiment of any of the one or more composite scaffold biomaterials described above, the hydrogel adhesive may comprise gelatin that may be crosslinked with glutaraldehyde and sodium borohydride reduction.

[0018] In yet another embodiment of any of the one or more composite scaffold biomaterials described above, the hydrogel adhesive may further comprise one or more agents, such as a therapeutic agent, a signaling molecule, a growth factor, a metabolite, an ECM protein or component, or any combination thereof.

[0019] In another embodiment of any of the one or more composite scaffold biomaterials described above, two or more scaffold biomaterial subunits may be assembled into the composite scaffold biomaterial and held together by complementary interlocking geometries of the two or more scaffold biomaterial subunits.

[0020] In yet another embodiment of any of the one or more composite scaffold biomaterials described above, the complementary interlocking geometries of the two or more scaffold biomaterial subunits may comprise peg-and-hole friction-fit interlocking geometries.

[0021] In yet another embodiment of any of the one or more composite scaffold biomaterials described above, at least one of the scaffold biomaterial subunits may comprise or be seeded with a first cell type. In another embodiment of any of the one or more composite scaffold biomaterials described above, at least one other scaffold biomaterial subunit may comprise or be seeded with a second cell type. In yet another embodiment of any of the one or more composite scaffold biomaterials described above, the first and second cell types may be contact-inhibited or contact-inhibited. In yet another embodiment of any of the one or more composite scaffold biomaterials described above, the composite scaffold biomaterial may comprise an interface between adjacent scaffold biomaterial subunits that can mimic a tissue interface, such as a bone-fibroblast tissue interface. In another embodiment of any of the one or more composite scaffold biomaterials described above, the scaffold biomaterial may comprise ECM deposition at at least one interface between adjacent scaffold biomaterial subunits.

[0022] In yet another embodiment of any of the one or more composite scaffold biomaterials described above, the scaffold biomaterial may comprise an effective Young's modulus that varies based on the direction of an applied force.

[0023] In yet another embodiment of any of the one or more composite scaffold biomaterials described above, the two or more scaffold biomaterial subunits may be further held together by gel casting using a hydrogel adhesive, by guided assembly-based biolithography (GAB), by chemical crosslinking, or any combination thereof.

[0024] In another embodiment of any of the one or more composite scaffold biomaterials described above, two or more scaffold biomaterial subunits may be assembled into the composite scaffold biomaterial and held together by guided assembly-based biolithography (GAB).

[0025] In another embodiment of any of the one or more composite scaffold biomaterials described above, the two or more scaffold biomaterial subunits may comprise at least one subunit comprising a plant- or fungal-derived biomaterial and at least one subunit comprising bacterial cellulose.

[0026] In yet another embodiment of any of the one or more composite scaffold biomaterials described above, bacterial cellulose can be grown on plant or fungal derived biomaterials by guided assembly-based biolithography (GAB).

[0027] In yet another embodiment of any of the one or more composite scaffold biomaterials described above, two or more scaffold biomaterial subunits may be assembled into the composite scaffold biomaterial and may be held together by chemical crosslinks.

[0028] In yet another embodiment of any of the one or more composite scaffold biomaterials described above, at least a portion of two or more of the scaffold biomaterial subunits may be modified to include carboxymethyl and / or hydroxyethyl cellulose functional groups that can be crosslinked together with citric acid and heat to hold the composite scaffold biomaterial together.

[0029] In another embodiment of any of the one or more composite scaffold biomaterials described above, the composite scaffold biomaterial may further comprise one or more agents, such as a therapeutic agent, a signaling molecule, a growth factor, a metabolite, an ECM protein or component, or any combination thereof.

[0030] In yet another embodiment of any of the one or more composite scaffold biomaterials described above, the decellularized plant or fungal tissue may be cellulose-based, hemicellulose-based, chitin-based, chitosan-based, pectin-based, lignin-based, lignan-based, or any combination thereof.

[0031] In yet another embodiment of any of the one or more composite scaffold biomaterials described above, the composite scaffold biomaterial may comprise at least two scaffold biomaterial subunits that may be structurally distinct from one another. In yet another embodiment of any of the one or more composite scaffold biomaterials described above, the at least two structurally distinct scaffold biomaterial subunits may be derived from different plant or fungal sources, may be derived from different parts of the same plant or fungal source, may be derived from two different species of plant or fungal source, may exhibit different Young's modulus properties, may comprise different cell types, may comprise different hydrogels, or any combination thereof.

[0032] In another embodiment, the present specification provides a crosslinked matrix, such as a crosslinked cellulose matrix, and One or more channels formed in the crosslinked matrix by removing a temporary space filler from the crosslinked matrix. Hydrogels, such as cellulose-based hydrogels, are provided.

[0033] In another embodiment of the hydrogel, the crosslinked matrix may be prepared from decellularized plant or fungal tissue, from which cellular material and nucleic acids have been removed, and which may comprise a three-dimensional porous structure.

[0034] In another embodiment of any of the one or more hydrogels described above, the temporary space-filling material may comprise a temporary gel, such as an alginate hydrogel.

[0035] In another embodiment of any of the one or more hydrogels described above, the matrix may be crosslinked around the fugitive space filler, and the fugitive space filler may then be removed from the crosslinked matrix, thereby forming one or more channels.

[0036] In another embodiment of any of the one or more hydrogels above, the transient space-filling agent is The oxidative stress can be removed by dissolving, heating, changing salt concentration, decomposition, or any combination thereof.

[0037] In yet another embodiment of any of the one or more hydrogels described above, a transient space-filling material may be disposed within the matrix to template a network of channels within the crosslinked matrix.

[0038] In another embodiment of any of the one or more hydrogels described above, the temporary space-filling material may be disposed within the matrix by deposition via 3D printing.

[0039] In another embodiment of any of the one or more hydrogels described above, the hydrogel may further comprise one or more agents, such as a therapeutic agent, a signaling molecule, a growth factor, a metabolite, an ECM protein or component, or a combination thereof.

[0040] In yet another embodiment of any of the one or more hydrogels described above, the matrix can be prepared from decellularized plant or fungal tissue that is cellulose-based, hemicellulose-based, chitin-based, chitosan-based, pectin-based, lignin-based, lignan-based, or any combination thereof.

[0041] In another embodiment of any of the one or more hydrogels described above, the crosslinked matrix may comprise at least two structurally different celluloses.

[0042] In yet another embodiment of any of the one or more hydrogels described above, the at least two structurally different celluloses may be derived from different plant or fungal sources, may be derived from different parts of the same plant or fungal source, may be derived from two different species of plant or fungal source, may exhibit different Young's modulus properties, may comprise different cell types, may comprise different hydrogels, or any combination thereof.

[0043] In another embodiment, the present specification provides 1. A scaffold biomaterial comprising decellularized plant or fungal tissue, wherein the decellularized plant or fungal tissue has been removed of cellular material and nucleic acids of the tissue, and the decellularized plant or fungal tissue comprises a three-dimensional porous structure; At least a portion of the decellularized plant or fungal tissue is functionalized with, complexed with, or covalently bound to, one or more agents, such as therapeutic agents, signaling molecules, growth factors, metabolites, ECM proteins or components, linkers for subsequent cross-linking or binding to any of these agents, or any combination thereof; A scaffold biomaterial is provided.

[0044] In another embodiment of the above scaffold biomaterials, the decellularized plant or fungal tissue may be modified with linkers such as succinyl linkers that can be used for cross-linking or covalently binding to one or more drugs, or the decellularized plant or fungal tissue may be modified with carboxymethyl and / or hydroxyethyl cellulose functional groups that can be used for covalently binding to one or more drugs via citric acid and heat-based coupling.

[0045] In another embodiment, the present disclosure provides a method for tissue repair or regeneration; in an implant; for culturing one or more cell types in vitro or in vivo; for mimicking in vivo tissues or tissue interfaces; for bone tissue engineering; for bone repair or regeneration; for fluid or Use of any of the above composite scaffold biomaterials, hydrogels, or scaffold biomaterials, or any combination thereof, for transporting fluids; for mimicking a tissue interface; for wound healing; for delivering agents such as therapeutic agents; signaling molecules, growth factors, metabolites, ECM proteins or components, or any combination thereof; or any combination thereof is provided.

[0046] In another embodiment, a method is provided for repairing or regenerating tissue; for providing an implant; for culturing one or more cell types; for mimicking in vivo tissue or tissue interfaces; for bone tissue engineering; for repairing or regenerating bone; for transporting fluids or liquids; for mimicking tissue interfaces; for wound healing; for delivering agents such as therapeutic agents, signaling molecules, growth factors, metabolites, ECM proteins or components, or any combination thereof; or any combination thereof in a subject in need thereof, comprising: Providing any of the composite scaffold biomaterials, hydrogels, or scaffold biomaterials described above, or any combination thereof; and introducing the composite scaffold biomaterial, hydrogel, or scaffold biomaterial, or any combination thereof, into a subject at a site in need thereof; A method comprising:

[0047] In another embodiment, provided herein is a method for inducing cell alignment, comprising: providing a decellularized scaffold biomaterial comprising one or more channels or grooves; Seeding the decellularized scaffold biomaterial with cells; and Culturing the cells on the decellularized scaffold biomaterial, thereby aligning the cells along one or more channels or grooves. A method is provided which includes:

[0048] In another embodiment of the above method, the decellularized scaffold biomaterial may comprise decellularized celery tissue.

[0049] In another embodiment of one or more of the above methods, the cells may comprise muscle cells or precursors thereof. In another embodiment, the cells may comprise myoblasts. In another embodiment, the cells may comprise C2C12 myoblasts.

[0050] In yet another embodiment, provided herein is a method for preparing a hydrogel having one or more channels, such as a cellulose-based hydrogel having one or more channels, comprising: providing a first crosslinkable material or gel, such as a natural or modified cellulose, chitin, lignin, lignan, hemicellulose, or pectin-based material or gel; providing a transient space-filling material; generating a three-dimensional structure comprising a first crosslinkable material or gel having the fugitive space-filling material dispersed therein, and crosslinking the first crosslinkable material or gel during or after generation of the three-dimensional structure, such that the fugitive space-filling material becomes a template for one or more channels in the first crosslinkable material or gel; and Removing the transient space-filling material from the three-dimensional structure to provide a hydrogel having one or more channels. A method is provided which includes:

[0051] In another embodiment, provided herein is a method for producing a scaffold biomaterial, comprising: providing plant or fungal tissue; Extracting one or more structures from plant or fungal tissue; and Preparing a scaffold biomaterial from one or more extracted structures. A method is provided which includes:

[0052] In another embodiment of the above method, the extracting step may comprise a liquid-based extraction to isolate one or more structures from the plant or fungal tissue.

[0053] In yet another embodiment of any of the one or more methods above, the extracting step may include maceration using at least one of treatment with a salt solution, treatment with a base solution, treatment with an acid solution, or treatment with an acid and peroxide solution.

[0054] In yet another embodiment of any one or more of the above methods, the salt solution can comprise a salt concentration of about 0.5 M to 3 M. Of course, the salt concentration can be adjusted depending on the particular desired application. For example, if the structures extracted from plant or fungal tissue include vascular bundles or microchannels (see below), a higher salt concentration (e.g., about 1 to about 3 M) may be preferred.

[0055] In another embodiment of any of the one or more methods above, the salt solution may include a NaCl solution or a LiCl solution.

[0056] In another embodiment of any one or more of the above methods, the base solution can include a base concentration of about 0.5 M to 3 M. Of course, the base concentration can be adjusted depending on the particular desired application.

[0057] In yet another embodiment of any of the one or more methods above, the base solution may comprise a NaOH solution.

[0058] In another embodiment of any one or more of the above methods, the acid and peroxide solution can comprise an acid-to-peroxide ratio of about 3:1 to about 1:3, or any ratio value therebetween (which may be rounded to the nearest 0.1), or any subrange spanning any two of these ratios. By way of example, in certain embodiments, an acid-to-peroxide ratio of about 3:1, 1:1, or 1:3 can be used. By way of example, in certain embodiments, the acid and peroxide solution can comprise acetic acid as the acid and hydrogen peroxide as the peroxide, and can be provided in an acid-to-peroxide ratio of about 3:1 (i.e., 13.05 M acetic acid, 2.45 M peroxide), 1:1 (i.e., 8.7 M acetic acid, 4.9 M peroxide), or 1:3 (i.e., 4.35 M acetic acid, 7.35 M peroxide). Of course, the acid and peroxide concentrations can be adjusted depending on the particular desired application.

[0059] In another embodiment of any of the one or more methods above, the acid and peroxide solution may include acetic acid and hydrogen peroxide.

[0060] In yet another embodiment of any of the one or more methods above, the acid and peroxide solution may comprise a ratio (by volume) of glacial acetic acid to 30% hydrogen peroxide of 3:1 (containing 13.05 M acetic acid and 2.45 M hydrogen peroxide) to 1:3 (containing 4.35 M acetic acid and 7.35 M hydrogen peroxide).

[0061] In yet another embodiment of any of the one or more methods above, the extracting step may include heating the plant or fungal tissue in a salt solution, a base solution, or an acid and peroxide solution.

[0062] In another embodiment of any of the one or more methods above, the extracting step comprises salting The method may further include mechanically agitating, e.g., stirring, the plant or fungal tissue in the liquid, base solution, or acid and peroxide solution.

[0063] In yet another embodiment of any of the one or more methods above, the one or more structures derived from plant or fungal tissue may include three-dimensional structures such as thixosa or pulp (i.e., fleshy material) structures, microchannels such as xylem and / or phloem (e.g., microchannel-type structures in vascular plants / materials), or any combination thereof, or one or more structures derived from other such structures.

[0064] In yet another embodiment of any of the one or more methods above, the thixosa or pulp structure may comprise an extended 3D structure (which may be composed of any one or more of cellulose, hemicellulose, pectin, or lignin, etc., and typically the extended 3D structure may comprise a lignocellulosic structure), a single structural cell or a group of structural cells derived from the extended 3D structure, or any combination thereof. In certain embodiments, the extended 3D structure may be cellulose-based.

[0065] In another embodiment of any of the one or more methods above, the extracting step may further comprise centrifuging.

[0066] In yet another embodiment of any of the one or more methods above, centrifugation can separate extended 3D structures and / or microchannels, such as xylem and / or phloem, from single structural cells or groups of structural cells derived from the extended 3D structures.

[0067] In yet another embodiment of any of the one or more methods above, the extracting step may further comprise centrifuging the extended 3D structure to separate single structural cells or groups of structural cells derived from the extended 3D structure.

[0068] In another embodiment of any one or more of the methods above, centrifugation can produce an upper band or pellet comprising a single structural cell or group of structural cells derived from the extended 3D structure.

[0069] In yet another embodiment of any of the one or more methods above, the one or more structures derived from the plant or fungal tissue may comprise a single structural cell or group of structural cells derived from the extended 3D structure that are localized in an upper band or pellet.

[0070] In another embodiment of any of the one or more methods above, the extracting step may further comprise washing the one or more structures from the plant or fungal tissue.

[0071] In yet another embodiment of any of the one or more methods above, preparing a scaffold biomaterial from the one or more extracted structures may include mixing, agitating, or physically manipulating the extracted structures to remove any residual undesired plant tissue material; washing the one or more extracted structures derived from plant or fungal tissue; or both.

[0072] In yet another embodiment of any one or more of the methods above, the plant or fungal tissue may comprise decellularized plant or fungal tissue, from which cellular material and nucleic acids of the tissue have been removed.

[0073] In another embodiment of any of the one or more methods above, the method may further comprise the step of decellularizing the plant or fungal tissue prior to the extracting step. Alternatively, the conditions of the extracting step can be selected such that decellularization or partial decellularization occurs during the extracting step.

[0074] In yet another embodiment of any of the one or more methods above, the method may further comprise the step of decellularizing the one or more structures extracted from the plant or fungal tissue.

[0075] In yet another embodiment of any of the one or more methods above, the method may further comprise decellularizing the plant or fungal tissue, or decellularizing one or more structures derived from the plant or fungal tissue, or both.

[0076] In another embodiment of any of the one or more methods above, preparing a scaffold biomaterial from the one or more extracted structures may include decellularizing the one or more extracted structures.

[0077] In certain embodiments of any of the one or more methods described above, a comminution step may be performed on the plant or fungal tissue prior to extraction and / or on one or more extracted structures after extraction.

[0078] In yet another embodiment of any of the one or more methods above, preparing a scaffold biomaterial from the one or more extracted structures may include grinding the one or more extracted structures.

[0079] In certain embodiments, the material to be milled, particularly for milling to produce small particles, may first be freeze-dried to remove moisture to prevent moisture-induced aggregation during milling.

[0080] In yet another embodiment of any of the one or more methods above, preparing a scaffold biomaterial from the one or more extracted structures may include incorporating the one or more extracted structures into a matrix; or gluing or attaching the extracted structures and / or scaffold biomaterial together; drying or freeze-drying the one or more extracted structures or scaffold biomaterial; seeding or culturing cells, such as animal cells, on the extracted structures and / or scaffold biomaterial; joining two or more extracted structures and / or scaffold biomaterials by layering, stacking, or other complementary / interlocking configurations; or any combination thereof.

[0081] In another embodiment of any one or more of the methods above, the matrix may comprise a hydrogel to form a composite hydrogel.

[0082] In yet another embodiment of any one or more of the methods above, the matrix or adhesive comprises an alginate matrix.

[0083] In another embodiment, provided herein is a scaffold biomaterial produced by any one or more of the methods described above.

[0084] In another embodiment, provided herein is a scaffold biomaterial comprising one or more structures extracted from plant or fungal tissue.

[0085] In another embodiment of the above scaffold biomaterial, the one or more structures are extracted from plant or fungal tissue by liquid-based extraction.

[0086] In yet another embodiment of any of the one or more scaffold biomaterials described above, the one or more structures can be extracted from plant or fungal tissue by treatment with a salt solution, treatment with a base solution, treatment with an acid solution, or treatment with an acid and peroxide solution.

[0087] In yet another embodiment of any of the one or more scaffold biomaterials described above, the one or more structures derived from plant or fungal tissue may comprise three-dimensional structures such as one or more structures derived from thipus or pulp structures, microchannels such as xylem and / or phloem, or any combination thereof.

[0088] In another embodiment of any of the one or more scaffold biomaterials described above, the thixosa or pulp structure may comprise an extended 3D structure (which may be composed of any one or more of cellulose, hemicellulose, pectin, or lignin, and typically the extended 3D structure may include a lignocellulosic structure), a single structural cell or a group of structural cells derived from the extended 3D structure, or any combination thereof. In certain embodiments, the extended 3D structure may be cellulose-based.

[0089] In yet another embodiment of any of the one or more scaffold biomaterials described above, the one or more structures may comprise a single structural cell or group of structural cells derived from the expanded 3D structure by centrifugation.

[0090] In yet another embodiment of any one or more scaffold biomaterials described above, the scaffold biomaterial may be a decellularized scaffold biomaterial that is devoid of cellular material and nucleic acids of plant or fungal tissue.

[0091] In another embodiment of any of the one or more scaffold biomaterials described above, the one or more structures may be milled to reduce particle size.

[0092] In further embodiments of any of the one or more scaffold biomaterials described above, the scaffold biomaterial may comprise a matrix having one or more incorporated structures; a product formed by gluing or attaching extracted structures and / or scaffold biomaterials together; a product formed by drying or freeze-drying one or more extracted structures or scaffold biomaterials; a product formed by seeding or culturing cells, such as animal cells, on or in the extracted structures and / or scaffold biomaterials; a product formed by joining two or more extracted structures and / or scaffold biomaterials by layering, stacking, or other complementary / interlocking configurations; or any combination thereof.

[0093] In yet another embodiment of any of the one or more scaffold biomaterials described above, the matrix may comprise a hydrogel.

[0094] In another embodiment of any of the one or more scaffolding biomaterials described above, the matrix or adhesive may comprise alginate.

[0095] In another embodiment, provided herein is a food product comprising a scaffold biomaterial, wherein the scaffold biomaterial comprises one or more three-dimensional structures derived from or extracted from plant or fungal tissue.

[0096] In another embodiment of the above food product, the one or more three-dimensional structures include one or more structures derived from a thixotropic or pulp structure, a microchannel, or any combination thereof. It can be seen.

[0097] In yet another embodiment of any of the one or more food products above, the microchannels may comprise xylem and / or phloem.

[0098] In yet another embodiment of any of the one or more foods described above, the one or more structures derived from the thixosa or pulp structure may comprise an extended 3D structure (which may be composed of any one or more of cellulose, hemicellulose, pectin, lignin, etc., and typically the extended 3D structure may include a lignocellulosic structure), a single structural cell or a group of structural cells derived from the extended 3D structure, or any combination thereof. In certain embodiments, the extended 3D structure may be cellulose-based.

[0099] In another embodiment of any of the one or more food products described above, the scaffold biomaterial may be decellularized, where the one or more three-dimensional structures are devoid of cellular material and nucleic acids of plant or fungal tissue.

[0100] In yet another embodiment of any of the one or more food products above, the scaffold biomaterial may comprise any of the one or more scaffold biomaterials described above.

[0101] In yet another embodiment of any of the one or more food products described above, the scaffold biomaterial may include a matrix having one or more three-dimensional structures incorporated therein; a product formed by gluing or attaching one or more three-dimensional structures and / or scaffold biomaterials together; a product formed by drying or freeze-drying one or more three-dimensional structures or scaffold biomaterials; a product formed by seeding or culturing cells, such as animal cells, on or in the three-dimensional structures and / or scaffold biomaterials; a product formed by joining two or more extracted structures and / or scaffold biomaterials by layering, stacking, or other complementary / interlocking configurations; or any combination thereof.

[0102] In another embodiment of any of the one or more food products described above, the scaffold biomaterial may comprise a matrix having one or more three-dimensional structures embedded therein; a product formed by adhering or attaching one or more three-dimensional structures and / or scaffold biomaterials together; or any combination thereof.

[0103] In yet another embodiment of any of the one or more food products above, the matrix or adhesive may comprise an alginate.

[0104] In yet another embodiment of any of the one or more food products above, the scaffold biomaterial may comprise two or more different three-dimensional structures derived from or extracted from the same or different plant or fungal tissues.

[0105] In another embodiment of any of the one or more food products described above, the food product may comprise two or more different scaffold biomaterials having different structural or physical properties.

[0106] In yet another embodiment of any of the one or more food products described above, two or more different three-dimensional structures and / or two or more different scaffold biomaterials can be selected to provide the food product with a desired stiffness, mouthfeel, and / or texture.

[0107] In yet another embodiment of any of the one or more food products above, the one or more cells are seeded or cultured on a scaffold biomaterial and / or three-dimensional structure.

[0108] In another embodiment of any of the one or more food products above, the one or more cells comprise animal cells.

[0109] In yet another embodiment of any of the one or more food products described above, the animal cells may include cells of livestock animals, fish, or insects, or other animals of interest.

[0110] In yet other embodiments of any one or more of the food products described above, the animal cells may include, for example, bovine, porcine, fish, elk, chicken, turkey, or avian cells.

[0111] In another embodiment of any of the one or more food products described above, the one or more cells may comprise muscle cells, adipocytes, connective tissue cells (i.e., fibroblasts), cartilage, bone, epithelial, or endothelial cells, or any combination thereof.

[0112] In yet another embodiment of any of the one or more food products described above, the food product may comprise at least a first layer of a scaffold biomaterial and a second layer of a scaffold biomaterial, one of the layers seeded with muscle cells and the other of the layers seeded with fat cells.

[0113] In yet another embodiment of any of the one or more food products described above, the one or more cells may be aligned along one or more channels or grooves in the scaffold biomaterial and / or three-dimensional structure.

[0114] In another embodiment of any of the one or more food products described above, the one or more cells may comprise muscle cells.

[0115] In yet another embodiment of any of the one or more food products above, the food product may comprise one or more three-dimensional structures derived from the thixotropic or pulp structure, and one or more microchannel structures.

[0116] In yet another embodiment of any of the one or more foods described above, the three-dimensional structure derived from the thixosa or pulp structure comprises an extended 3D structure (which may be composed of any one or more of cellulose, hemicellulose, pectin, or lignin, and typically the extended 3D structure may include a lignocellulosic structure), a single structural cell or a group of structural cells derived from the extended 3D structure, or any combination thereof. In certain embodiments, the extended 3D structure may be cellulose-based.

[0117] In another embodiment of any of the one or more food products described above, the microchannel structure may comprise xylem and / or phloem.

[0118] In yet another embodiment of any of the one or more food products above, the xylem and / or phloem is vascular.

[0119] In yet other embodiments of any of the one or more food products above, the scaffold biomaterial may comprise any of the composite scaffold biomaterials described above.

[0120] In another embodiment, provided herein is a method for preparing a food product, comprising: Providing plant or fungal tissue deriving or extracting one or more three-dimensional structures from plant or fungal tissue; and By preparing a scaffold biomaterial from one or more derived or extracted three-dimensional structures, Producing a scaffold biomaterial; and Preparing food from scaffold biomaterials A method is provided which includes:

[0121] In another embodiment of the above method, producing a scaffold biomaterial may comprise performing any one or more of the methods described above.

[0122] In another embodiment of any of the one or more methods above, preparing a food product from a scaffold biomaterial may include one or more of the following: incorporating one or more derived or extracted three-dimensional structures into a matrix; or gluing or attaching the derived or extracted three-dimensional structures and / or scaffold biomaterial together; drying or freeze-drying the derived or extracted three-dimensional structures or scaffold biomaterial; seeding or culturing cells on the derived or extracted three-dimensional structures and / or scaffold biomaterial; or linking two or more derived or extracted three-dimensional structures and / or scaffold biomaterials by layering, stacking, or other complementary / interlocking configurations; or any combination thereof.

[0123] In yet another embodiment of any of the one or more methods above, preparing a food product from the scaffold biomaterial may include seeding or culturing cells on the derived or extracted three-dimensional structure and / or scaffold biomaterial.

[0124] In another embodiment of any of the one or more methods above, the one or more cells may comprise animal cells.

[0125] In further embodiments of any one or more of the methods above, the animal cells may comprise cells of livestock animals, fish, or insects.

[0126] In yet another embodiment of any one or more of the methods above, the animal cells may comprise bovine, porcine, fish, elk, chicken, turkey, or avian cells.

[0127] In another embodiment of any of the one or more methods above, the one or more cells may comprise muscle cells, adipocytes, connective tissue cells (e.g., fibroblasts), cartilage, bone, epithelial, or endothelial cells, or any combination thereof.

[0128] In yet another embodiment of any of the one or more methods above, the method may include producing at least a first layer of scaffold biomaterial and a second layer of scaffold biomaterial, one of the layers seeded with muscle cells and the other of the layers seeded with adipocytes.

[0129] In yet another embodiment of any of the one or more methods above, the one or more cells may be aligned along one or more channels or grooves in the scaffold biomaterial and / or three-dimensional structure.

[0130] In another embodiment of any of the one or more methods above, the one or more cells may comprise muscle cells.

[0131] In yet another embodiment of any of the one or more methods above, the method may comprise producing two or more different scaffold biomaterials having different structural or physical properties; producing one or more scaffold biomaterials comprising two or more different three-dimensional structures derived or extracted from the same or different plant or fungal tissues; or any combination thereof.

[0132] In yet another embodiment of any of the one or more methods above, the two or more different three-dimensional structures and / or two or more different scaffold biomaterials can be selected to provide a desired stiffness, mouthfeel, and / or texture to the food product. (1) 1. A composite scaffold biomaterial comprising two or more scaffold biomaterial subunits each comprising decellularized plant or fungal tissue, wherein the decellularized plant or fungal tissue has been removed from cellular material and nucleic acids of the tissue, and the decellularized plant or fungal tissue comprises a three-dimensional porous structure; The two or more scaffold biomaterial subunits are assembled into the composite scaffold biomaterial and held together by gel casting using a hydrogel adhesive, by the complementary / interlocking shapes of the two or more scaffold biomaterial subunits, by guided assembly-based biolithography (GAB), by chemical crosslinking, or any combination thereof. (2) A composite scaffold biomaterial according to (1) above, wherein two or more scaffold biomaterial subunits are assembled into a composite scaffold biomaterial and held together by gel casting using a hydrogel adhesive. (3) The composite scaffold biomaterial according to (2) above, wherein the hydrogel adhesive comprises gelatin, collagen, agarose, hyaluronic acid, alginate, fibrin, fibronectin, agar, PEG, PVA, or any combination thereof. (4) The composite scaffold biomaterial according to (2) or (3) above, wherein two or more scaffold biomaterial subunits act as a scaffold for the hydrogel adhesive to form around. (5) The composite scaffold biomaterial according to any one of (2) to (4) above, wherein at least a portion of the two or more scaffold biomaterial subunits is coated with a hydrogel adhesive. (6) The composite scaffold biomaterial according to any one of (2) to (5) above, wherein the hydrogel adhesive is cured or solidified by temperature change, crosslinking, or a combination thereof. (7) The composite scaffold biomaterial according to any one of (2) to (6) above, wherein the hydrogel adhesive comprises gelatin cross-linked by glutaraldehyde and sodium borohydride reduction. (8) The composite scaffold biomaterial according to any one of (2) to (7) above, wherein the hydrogel adhesive further comprises one or more drugs, such as a therapeutic agent, a signaling molecule, a growth factor, a metabolite, an ECM protein or component, or any combination thereof. (9) 1. The composite scaffold biomaterial of claim 1, wherein two or more scaffold biomaterial subunits are assembled into a composite scaffold biomaterial and held together by the complementary interlocking shapes of the two or more scaffold biomaterial subunits. (10) The composite scaffold biomaterial according to (9) above, wherein the complementary interlocking shapes of the two or more scaffold biomaterial subunits comprise a peg-and-hole friction-fit interlocking shape. (11) A composite scaffold biomaterial according to (9) or (10) above, wherein at least one of the scaffold biomaterial subunits comprises or is seeded with a first cell type. (12) A composite scaffold biomaterial according to (11) above, wherein at least one other scaffold biomaterial subunit comprises or is seeded with a second cell type. (13) The first and second cell types may be contact-inhibited or not contact-inhibited. 2) A composite scaffold biomaterial according to the present invention. (14) The composite scaffold biomaterial according to (12) or (13) above, comprising an interface between adjacent scaffold biomaterial subunits that mimics a tissue interface, such as a bone-fibroblast tissue interface. (15) A composite scaffold biomaterial according to any one of (11) to (14) above, wherein the scaffold biomaterial comprises ECM deposition at at least one interface between adjacent scaffold biomaterial subunits. (16) The composite scaffold biomaterial according to any one of (9) to (15) above, wherein the scaffold biomaterial comprises an effective Young's modulus that varies depending on the direction of an applied force. (17) The composite scaffold biomaterial according to any one of (9) to (16) above, wherein two or more of the scaffold biomaterial subunits are further held together by gel casting using a hydrogel adhesive, by guided assembly-based biolithography (GAB), by chemical crosslinking, or any combination thereof. (18) The composite scaffold biomaterial described in (1) above, wherein two or more scaffold biomaterial subunits are assembled into the composite scaffold biomaterial and held together by guided assembly-based biolithography (GAB). (19) The composite scaffold biomaterial described in (18) above, wherein the two or more scaffold biomaterial subunits include at least one subunit comprising a plant- or fungal-derived biomaterial and at least one subunit comprising bacterial cellulose. (20) A composite scaffold biomaterial as described in (19) above, wherein bacterial cellulose is grown on a plant or fungal-derived biomaterial by guided assembly-based biolithography (GAB). (twenty one) 10. The composite scaffold biomaterial according to claim 1, wherein two or more scaffold biomaterial subunits are assembled into the composite scaffold biomaterial and held together by chemical crosslinks. (twenty two) 22. The composite scaffold biomaterial of claim 21, wherein at least a portion of two or more of the scaffold biomaterial subunits have been modified to include carboxymethyl and / or hydroxyethyl cellulose functional groups that are crosslinked together with citric acid and heat to hold the composite scaffold biomaterial together. (twenty three) The composite scaffold biomaterial according to any one of (1) to (22) above, further comprising one or more agents, such as therapeutic agents, signaling molecules, growth factors, metabolites, ECM proteins or components, or any combination thereof. (twenty four) The composite scaffold biomaterial according to any one of (1) to (23) above, wherein the decellularized plant or fungal tissue is cellulose-based, hemicellulose-based, chitin-based, chitosan-based, pectin-based, lignin-based, lignan-based, or any combination thereof. (twenty five) The composite scaffold biomaterial according to any one of (1) to (24) above, wherein the two composite scaffold biomaterials comprise at least two scaffold biomaterial subunits that are structurally different from each other. (26) 26. The composite scaffold biomaterial of claim 25, wherein the at least two scaffold biomaterial subunits that are structurally different from each other are derived from different plant or fungal sources, derived from different parts of the same plant or fungal source, derived from two different species of plant or fungal sources, exhibit different Young's modulus properties, comprise different cell types, comprise different hydrogels, or any combination thereof. (27) A hydrogel, such as a cellulose-based hydrogel, a crosslinked matrix, such as a crosslinked cellulose matrix, and One or more channels formed in the crosslinked matrix by removing the fugitive space-filling material from the crosslinked matrix. A hydrogel comprising: (28) 28. The hydrogel of claim 27, wherein the crosslinked matrix is ​​prepared from decellularized plant or fungal tissue, from which cellular material and nucleic acids have been removed, and wherein the decellularized plant or fungal tissue comprises a three-dimensional porous structure. (29) The hydrogel according to (27) or (28) above, wherein the temporary space-filling material comprises a temporary gel such as an alginate hydrogel. (30) The hydrogel according to any one of (27) to (29) above, wherein the matrix is ​​crosslinked around the fugitive space filler, and then the fugitive space filler is removed from the crosslinked matrix, thereby forming one or more channels. (31) The hydrogel according to (30) above, wherein the transient space-filling material is removed by dissolution, heating, a change in salt concentration, degradation, or any combination thereof. (32) The hydrogel according to any one of (27) to (31) above, wherein the transient space-filling material is disposed within the matrix so as to form a template for a network of channels within the crosslinked matrix. (33) The hydrogel according to (32) above, wherein the temporary space-filling material is disposed within the matrix by deposition via 3D printing. (34) The hydrogel according to any one of (27) to (33) above, further comprising one or more drugs, such as a therapeutic agent, a signaling molecule, a growth factor, a metabolite, an ECM protein or component, or any combination thereof. (35) The hydrogel according to any one of (27) to (34) above, wherein the matrix is ​​prepared from decellularized plant or fungal tissue that is cellulose-based, hemicellulose-based, chitin-based, chitosan-based, pectin-based, lignin-based, lignan-based, or any combination thereof. (36) The hydrogel according to any one of (27) to (35) above, wherein the crosslinked matrix comprises at least two structurally different celluloses. (37) 36. The hydrogel of claim 35, wherein the at least two structurally different celluloses are derived from different plant or fungal sources, from different parts of the same plant or fungal source, from two different species of plant or fungal source, exhibit different Young's modulus properties, comprise different cell types, comprise different hydrogels, or any combination thereof. (38) 1. A scaffold biomaterial comprising decellularized plant or fungal tissue, from which cellular material and nucleic acids of said tissue have been removed, said decellularized plant or fungal tissue comprising a three-dimensional porous structure, At least a portion of the decellularized plant or fungal tissue is functionalized with, complexed to, or covalently linked to one or more agents, such as a therapeutic agent, a signaling molecule, a growth factor, a metabolite, an ECM protein or component, a linker for subsequent cross-linking or conjugation with any of these agents, or any combination thereof; The scaffold biomaterial. (39) 31. The scaffold biomaterial according to claim 31, wherein the decellularized plant or fungal tissue is modified with a linker such as a succinyl linker for cross-linking or covalently binding to one or more drugs, or the decellularized plant or fungal tissue is modified with carboxymethyl and / or hydroxyethyl cellulose functional groups for covalently binding to one or more drugs by citric acid and heat-based coupling. (40) Use of a composite scaffold biomaterial according to any one of (1) to (26), a hydrogel according to any one of (27) to (37), or a scaffold biomaterial according to (38) or (39), or any combination thereof, for tissue repair or regeneration; in an implant; for culturing one or more cell types in vitro or in vivo; for mimicking in vivo tissue or tissue interfaces; for bone tissue engineering; for bone repair or regeneration; for transporting fluids or liquids; for mimicking tissue interfaces; for wound healing; for delivering agents such as therapeutic agents, signaling molecules, growth factors, metabolites, ECM proteins or components, or any combination thereof; or any combination thereof. (41) for repairing or regenerating tissue in a subject in need thereof; for providing an implant; for culturing one or more cell types; for mimicking in vivo tissue or tissue interfaces; for bone tissue engineering; for repairing or regenerating bone; for transporting fluids or liquids; for mimicking tissue interfaces; for wound healing; for delivering agents such as therapeutic agents, signaling molecules, growth factors, metabolites, ECM proteins or components, or any combination thereof; or any combination thereof, Providing a composite scaffold biomaterial according to any one of (1) to (26), a hydrogel according to any one of (27) to (37), or a scaffold biomaterial according to (38) or (39), or any combination thereof; and introducing the composite scaffold biomaterial, the hydrogel, or the scaffold biomaterial, or any combination thereof, into the subject at a site in need thereof. The method comprising: (42) 1. A method for inducing cell alignment, comprising: providing a decellularized scaffold biomaterial comprising one or more channels or grooves; seeding the decellularized scaffold biomaterial with cells; and Culturing the cells on the decellularized scaffold biomaterial, thereby aligning the cells along the one or more channels or grooves. The method comprising: (43) The method according to (42) above, wherein the decellularized scaffold biomaterial comprises decellularized celery tissue. (44) The method according to (42) or (43) above, wherein the cells include muscle cells or precursors thereof. (45) The method according to (44) above, wherein the cells comprise myoblasts. (46) The method according to (45) above, wherein the cells comprise C2C12 myoblasts. (47) 1. A method for preparing a hydrogel having one or more channels, such as a cellulose-based hydrogel having one or more channels, comprising: providing a first crosslinkable material or gel, such as a natural or modified cellulose, chitin, lignin, lignan, hemicellulose, or pectin-based material or gel; providing a transient space-filling material; generating a three-dimensional structure comprising the first crosslinkable material or gel having the fugitive space-filling material dispersed therein such that the fugitive space-filling material forms a template for one or more channels in the first crosslinkable material or gel, and crosslinking the first crosslinkable material or gel during or after generating the three-dimensional structure; and removing the transient space-filling material from the three-dimensional structure to provide a hydrogel having the one or more channels. The method comprising: (48) 1. A method for producing a scaffold biomaterial, comprising: providing plant or fungal tissue; extracting one or more structures from said plant or fungal tissue; and preparing the scaffold biomaterial from the one or more extracted structures; The method comprising: (49) 49. The method of claim 48, wherein the extracting step comprises a liquid-based extraction to isolate one or more structures from plant or fungal tissue. (50) 49. The method of claim 48, wherein the extracting step comprises at least one of treatment with a salt solution, treatment with a base solution, treatment with an acid solution, or treatment with an acid and peroxide solution. (51) The method according to (50) above, wherein the salt solution has a salt concentration of about 0.5M to 3M. (52) The method according to (50) or (51) above, wherein the salt solution comprises a NaCl solution or a LiCl solution. (53) The method according to (50) above, wherein the base solution contains a base concentration of about 0.5M to 3M. (54) The method according to (50) or (53) above, wherein the base solution comprises a NaOH solution. (55) 51. The method of claim 50, wherein the acid and peroxide solution comprises an acid to peroxide ratio of about 3:1 to about 1:3. (56) The method according to (50) or (55) above, wherein the acid and peroxide solution comprises acetic acid and hydrogen peroxide. (57) The method according to any one of (50), (55), or (56) above, wherein the acid and peroxide solution comprises glacial acetic acid and 30% hydrogen peroxide in a ratio of 3:1 to 1:3. (58) The method according to any one of (50) to (57) above, wherein the extracting step comprises heating the plant or fungal tissue in a salt solution, a base solution, or an acid and peroxide solution. (59) 59. The method of claim 58, wherein the extracting step further comprises mechanically agitating, e.g., stirring, the plant or fungal tissue in a salt solution, a base solution, or an acid and peroxide solution. (60) The method according to any one of (48) to (60) above, wherein the one or more structures derived from plant or fungal tissue include three-dimensional structures such as one or more structures derived from a thixosa or pulp cellulose structure, microchannels such as xylem and / or phloem, or any combination thereof. (61) 61. The method according to claim 60, wherein the thixosa or pulp cellulose structure comprises an extended 3D structure, a single structural cell or a group of structural cells derived from said extended 3D structure, or any combination thereof. (62) The method according to any one of (48) to (61) above, wherein the extracting step further comprises a step of centrifuging. (63) 63. The method of claim 62, wherein the centrifugation separates the extended 3D structure and / or microchannels, such as xylem and / or phloem, from single structural cells or groups of structural cells derived from the extended 3D structure. (64) The method according to any one of (48) to (61) above, wherein the extracting step further comprises a step of centrifuging the extended 3D structure to separate the extended 3D structure from single structural cells or groups of structural cells derived from the extended 3D structure. (65) 64. The method of claim 64, wherein the centrifugation produces an upper band or pellet containing single structural cells or groups of structural cells derived from the extended 3D structure. (66) The method of (65) above, wherein one or more structures derived from plant or fungal tissue comprise a single structural cell or group of structural cells derived from an extended 3D structure that is localized in an upper band or pellet. (67) The method according to any one of (48) to (66) above, wherein the extracting step further comprises the step of washing one or more structures derived from plant or fungal tissue. (68) 64. The method of any one of claims 48 to 67, wherein preparing a scaffold biomaterial from the one or more extracted structures comprises: mixing, agitating, or physically manipulating the extracted structures to remove any residual undesired plant tissue material; washing the one or more extracted structures derived from plant or fungal tissue; or both. (69) The method according to any one of (48) to (68) above, wherein the plant or fungal tissue comprises decellularized plant or fungal tissue, from which cellular material and nucleic acids have been removed. (70) The method according to any one of (48) to (68) above, further comprising the step of decellularizing the plant or fungal tissue before the extraction step. (71) The method according to any one of (48) to (68) above, further comprising the step of decellularizing one or more structures extracted from plant or fungal tissue. (72) The method according to any one of (48) to (68) above, further comprising a step of decellularizing plant or fungal tissue, or a step of decellularizing one or more structures derived from said plant or fungal tissue, or both. (73) The step of preparing a scaffold biomaterial from one or more extracted structures comprises: The method according to any one of (48) to (68) above, comprising the step of decellularizing the extracted structure. (74) A method according to any one of (48) to (73) above, wherein the step of preparing a scaffold biomaterial from one or more extracted structures comprises a step of pulverizing the one or more extracted structures. (75) The method according to any one of (48) to (74) above, wherein the step of preparing a scaffold biomaterial from one or more extracted structures comprises the steps of incorporating the one or more extracted structures into a matrix; or gluing or attaching the extracted structures and / or scaffold biomaterial together; drying or freeze-drying the one or more extracted structures or scaffold biomaterial; seeding or culturing cells, such as animal cells, on the extracted structures and / or scaffold biomaterial; linking two or more extracted structures and / or scaffold biomaterials by layering, stacking, or other complementary / interlocking configurations; or any combination thereof. (76) The method according to (75) above, wherein the matrix comprises a hydrogel to form a composite hydrogel. (77) The method according to (75) or (76) above, wherein the matrix or adhesive comprises an alginate matrix. (78) A scaffold biomaterial produced by the method according to any one of (48) to (77) above. (79) A scaffold biomaterial comprising one or more structures extracted from plant or fungal tissue. (80) 79. A scaffold biomaterial according to claim 79, wherein the one or more structures are extracted from plant or fungal tissue by liquid-based extraction. (81) The scaffold biomaterial according to (79) or (80), wherein the one or more structures are extracted from plant or fungal tissue by treatment with a salt solution, a base solution, an acid solution, or an acid and peroxide solution. (82) A scaffold biomaterial according to any one of (79) to (81) above, wherein the one or more structures derived from plant or fungal tissue include three-dimensional structures such as one or more structures derived from thixosa or pulp cellulose structures, microchannels such as xylem and / or phloem, or any combination thereof. (83) The scaffold biomaterial according to (82) above, wherein the thixosa or pulp cellulose structure comprises an extended 3D structure, a single structural cell or a group of structural cells derived from said extended 3D structure, or any combination thereof. (84) A scaffold biomaterial as described in (83) above, wherein one or more structures comprise a single structural cell or a group of structural cells obtained from the expanded 3D structure by centrifugation. (85) A scaffold biomaterial according to any one of (79) to (84) above, which is a decellularized scaffold biomaterial lacking cellular material and nucleic acids of plant or fungal tissue. (86) A scaffold biomaterial according to any one of (79) to (85) above, wherein one or more structures have been pulverized to reduce particle size. (87) A scaffold biomaterial according to any of (79) to (86) above, comprising: a matrix having one or more incorporated structures; a product formed by gluing or attaching extracted structures and / or scaffold biomaterials together; a product formed by drying or freeze-drying said one or more extracted structures or scaffold biomaterials; a product formed by seeding or culturing cells, such as animal cells, on said extracted structures and / or scaffold biomaterials; a product formed by joining two or more extracted structures and / or scaffold biomaterials by layering, stacking, or other complementary / interlocking configurations; or any combination thereof. (88) The scaffold biomaterial according to (87) above, wherein the matrix comprises a hydrogel. (89) The scaffold biomaterial according to (87) or (88) above, wherein the matrix or adhesive comprises alginate. (90) 1. A food product comprising a scaffold biomaterial, said scaffold biomaterial comprising one or more three-dimensional structures derived from or extracted from plant or fungal tissue. (91) The food product according to (90), wherein the one or more three-dimensional structures include one or more structures derived from a thia or pulp structure, microchannels, or any combination thereof. (92) The food product according to (91) above, wherein the microchannels comprise xylem and / or phloem. (93) The food product according to (91) or (92), wherein the one or more structures derived from the thixosa or pulp structure comprise an extended 3D structure, a single structural cell or a group of structural cells derived from the extended 3D structure, or any combination thereof. (94) A food product according to any one of (90) to (93) above, wherein the scaffold biomaterial is decellularized and one or more three-dimensional structures are devoid of cellular material and nucleic acids of plant or fungal tissue. (95) The food according to any one of (90) to (94) above, wherein the scaffold biomaterial comprises a scaffold biomaterial defined in any one of (78) to (89) above. (96) A food product according to any of (90) to (95) above, comprising a scaffold biomaterial, a matrix in which one or more three-dimensional structures are incorporated; a product formed by gluing or attaching said one or more three-dimensional structures and / or scaffold biomaterials together; a product formed by drying or freeze-drying said one or more three-dimensional structures or scaffold biomaterials; a product formed by seeding or culturing cells, such as animal cells, on said three-dimensional structures and / or scaffold biomaterials; a product formed by linking two or more extracted structures and / or scaffold biomaterials by layering, stacking, or other complementary / interlocking configurations; or any combination thereof. (97) A food product according to any one of (90) to (96) above, wherein the scaffold biomaterial comprises a matrix having one or more three-dimensional structures incorporated therein; a product formed by adhering or attaching the one or more three-dimensional structures and / or scaffold biomaterial together; or any combination thereof. (98) The food product according to (97) above, wherein the matrix or adhesive comprises alginate. (99) A food product according to any one of (90) to (98) above, wherein the scaffold biomaterial comprises two or more different three-dimensional structures derived from or extracted from the same or different plant or fungal tissues. (100) The food product according to any one of (90) to (99) above, comprising two or more different scaffold biomaterials having different structural or physical properties. (101) The food product according to (99) or (100) above, wherein the two or more different three-dimensional structures and / or the two or more different scaffold biomaterials are selected to provide the food product with a desired stiffness, mouthfeel, and / or texture. (102) The food product according to any one of (90) to (101) above, wherein one or more cells are seeded on or cultured in a scaffold biomaterial and / or a three-dimensional structure. (103) The food product according to (102) above, wherein the one or more cells include animal cells. (104) The food product according to (103) above, wherein the animal cells include cells of livestock animals, fish, or insects. (105) The food product according to (104) above, wherein the animal cells include bovine, porcine, fish, elk, chicken, turkey, or avian cells. (106) The food according to any one of (102) to (105) above, wherein the one or more cells include muscle cells, adipocytes, connective tissue cells, cartilage, bone, epithelial, or endothelial cells, or any combination thereof. (107) The food product according to (106) above, comprising at least a first layer of a scaffold biomaterial and a second layer of a scaffold biomaterial, one of the layers being seeded with muscle cells and the other layer being seeded with fat cells. (108) The food product according to any one of (102) to (107) above, wherein one or more cells are aligned along one or more channels or grooves of the scaffold biomaterial and / or three-dimensional structure. (109) The food product according to (108) above, wherein the one or more cells comprise muscle cells. (110) The food product according to any one of (90) to (109) above, which comprises one or more three-dimensional structures derived from a thixosa or pulp structure, and one or more microchannel structures. (111) The food product according to (110) above, wherein the three-dimensional structure derived from the thixosa or pulp structure comprises an extended 3D structure, a single structural cell or a group of structural cells derived from said extended 3D structure, or any combination thereof. (112) The food product according to (110) or (111) above, wherein the microchannel structure comprises xylem and / or phloem. (113) The food product according to (112) above, wherein the xylem and / or phloem are vascular bundles. (114) The food according to any one of (90) to (113) above, wherein the scaffold biomaterial comprises a composite scaffold biomaterial as defined in 1 above. (115) 1. A method for preparing a food product, comprising: providing plant or fungal tissue; deriving or extracting one or more three-dimensional structures from said plant or fungal tissue; and preparing a scaffold biomaterial from the one or more derived or extracted three-dimensional structures; producing the scaffold biomaterial; and Preparing the food product from the scaffold biomaterial. The method comprising: (116) The method according to (115) above, wherein the step of producing a scaffold biomaterial comprises carrying out a method defined in any one of (48) to (77) above. (117) The method of any one of (115) or (116) above, wherein the step of preparing a food product from a scaffold biomaterial comprises one or more of the following steps: incorporating one or more derived or extracted three-dimensional structures into a matrix; or gluing or attaching the derived or extracted three-dimensional structures and / or scaffold biomaterial together; drying or freeze-drying the one or more derived or extracted three-dimensional structures or scaffold biomaterial; seeding or culturing cells on the derived or extracted three-dimensional structures and / or scaffold biomaterial; linking two or more derived or extracted three-dimensional structures and / or scaffold biomaterials by layering, stacking, or other complementary / interlocking configurations; or any combination thereof. (118) A method according to any one of (115) to (117) above, wherein the step of preparing a food product from the scaffold biomaterial comprises the step of seeding or culturing cells on the derived or extracted three-dimensional structure and / or scaffold biomaterial. (119) The method according to (118) above, wherein the one or more cells comprise animal cells. (120) The method according to (119) above, wherein the animal cells include cells of livestock animals, fish, or insects, or other animals of interest. (121) The method according to (120) above, wherein the animal cells include bovine, porcine, fish, elk, chicken, turkey, or avian cells. (122) The method according to any one of (118) to (121) above, wherein the one or more cells comprise muscle cells, adipocytes, connective tissue cells (i.e., fibroblasts), cartilage, bone, epithelial, or endothelial cells, or any combination thereof. (one two three) The method according to (122) above, comprising the steps of producing at least a first layer of scaffold biomaterial and a second layer of scaffold biomaterial, one of the layers being seeded with muscle cells and the other of the layers being seeded with fat cells. (124) The method according to any one of (118) to (123) above, wherein one or more cells are aligned along one or more channels or grooves of the scaffold biomaterial and / or three-dimensional structure. (125) The method according to (124) above, wherein the one or more cells comprise muscle cells. (126) Producing two or more different scaffold biomaterials having different structural or physical properties; Producing one or more scaffold biomaterials comprising two or more different three-dimensional structures derived or extracted from the same or different plant or fungal tissues; or The method according to any one of (115) to (125) above, including any combination thereof. (127) The method according to (126) above, wherein the two or more different three-dimensional structures and / or two or more different scaffold biomaterials are selected to provide the food with a desired stiffness, mouthfeel, and / or texture. [Brief explanation of the drawings]

[0133] These and other features will be better understood with reference to the following description and accompanying drawings. [Figure 1] Figure 1 shows an example of a contemplated application of the composite material described herein, in which the composite material may be produced in the form of a blood vessel. Blood vessels are complex structures with various layers of tissue (top left and center images). In the example shown below, a ring structure was prepared from apple-derived cellulose (bottom left and right images), which can be layered and coated with a hydrogel such as 1.5% agarose to produce a tube (see also top and bottom center images). This tube may be wrapped with various layers or membranes containing various cell types. Sample material for the membrane may be, for example, decellularized orange mesocarp membrane (see top right image). Combining multiple elements can be used to more closely reproduce complex structures such as blood vessels, with various tissues and cell types organized radially. [Figure 2] Figure 2 shows an interlocking cellulose-based biomaterial. As shown, the blocks can be assembled using a Lego-style peg-and-hole geometry. Tightly interlocking subunits can be assembled / combined to provide a biocompatible intact structure. [Figure 3] Figure 3 shows that cellulose-based materials can be crushed into gels. Different concentrations can have different consistencies (left = low concentration, right = high concentration). Crosslinking with citric acid and heat can be achieved after chemical modification with carboxymethyl and hydroxylethyl groups. Without these groups, crosslinking would not occur. This figure shows the uncrosslinked hydrogel in the absence of chemical functional groups after exposure to heat and citric acid. [Figure 4]Figure 4 shows (A) a 6x2mm longitudinal scaffold; and (B) a cross section of the vascular bundle. (Blue) ground tissue and phloem, (Red) xylem. [Figure 5] Figure 5 shows (A) SEM images of longitudinally sectioned vascular bundles and (B) C2C12 myotube alignment. Scale bar = 100 μm. [Figure 6] Figure 6 shows the chemical functionalization of cellulose with succinic anhydride. (A) Depiction of the covalent attachment of succinic acid to the cellulose chain and the absorption bands in the IR spectrum. (B) IR spectra of the control (gray) and functionalized (black) scaffolds. The strong absorption peaks near the wavelengths predicted for the ester and carboxyl groups indicate successful covalent attachment of succinic anhydride to the cellulose chain. Note the wavelength shift due to neighboring groups. [Figure 7] Figure 7 shows the results of chemical functionalization of cellulose with denatured collagen. IR spectra of control (gray) and functionalized (black) scaffolds. The control scaffold was functionalized with a succinic acid linker molecule but lacked collagen. The absorption peaks corresponding to amide (I and II) bonds indicate successful covalent attachment of collagen to the cellulose chains via the succinic acid linker molecule. [Figure 8] Figure 8 shows images of the artificial vessel and its assembly from ring-like subunits. The ring subunits were stacked and coated with 1.5% agarose to create the artificial vessel. [Figure 9] FIG. 9 shows the results of a fluid transport study in which Congo red staining passed through the blood vessels shown in FIG. [Figure 10] Figure 10 shows images of the bonded composite biomaterial. The biomaterial was bonded with gelatin cross-linked with glutaraldehyde and reduced with sodium borohydride. (A) shows two subunits of an apple-derived cellulose scaffold bonded together. (B) shows that the bonded construct supported cell growth of GFP 3T3 cells. [Figure 11]Figure 11 shows the fabrication, model and product of a Lego-style subunit: (A) shows the CNC cutting, (B) shows a schematic model of the subunit assembly, and (C) shows the assembled composite structure containing two assembled subunits. [Figure 12] Figure 12 shows the results of single cell type migration. (A) shows the combined PEG-and-Hole subunits used. (B) shows the specificity of subunit preloading. (C) shows cell migration of 3T3 GFP fibroblasts 1 week after assembly. (D) shows cell migration of 3T3 GFP fibroblasts 3 weeks after assembly. [Figure 13] Figure 13 shows the results of dual cell type migration. Two cell populations were seeded onto two subunits of the composite (green = GFP 3T3 cells, blue = 3T3 cells stained with Hoechst dye). The subunits were then combined (T=0). [Figure 14] Figure 14 shows cell area coverage in migration assays for single and dual cell type migration. Cell area coverage of adjacent scaffolds measured 7, 14 and 21 days after assembly. [Figure 15] Figure 15 shows the tensile test of the apple bite composite. The combined piece was disassembled by pulling the two subunits apart. The force for separation was recorded. [Figure 16] Figure 16 shows the stress-isolating composite. Compression of decellularized apple and carrot composites in series and parallel configurations results in different Young's moduli. [Figure 17] Figure 17 shows a composite bone-fibroblast structure. Assembled interlocking Lego-block units seeded with differentiated bone MC3T3 E1 subclone 4 cells (top) and GFP3T3 fibroblasts (bottom) are shown. At the top, a mineralized scaffold can be seen. The bottom unit has no calcium deposits. [Figure 18]Figure 18 shows (A) a visual representation of the celery-scaffold preparation. (B) The samples were 6 mm wide and 2.15 ± 0.15 mm thick. "XY" corresponds to the scaffold cut longitudinally through the celery stalk, while "CS" corresponds to the cross section. Approximately 50,000 cells were seeded onto the decellularized scaffold (C) and left on the scaffold for 4.5 hours. VB = vascular bundle. [Figure 19] Figure 19 shows the vascular bundles of celery. (A) Maximum projection of a cross section and (B) 3D reconstruction of a longitudinal section of a vascular bundle. Phloem and ground tissue were stained with calcofluor (green); lignified tissue was stained with propidium iodide (red). SEM images of a cross section (C) and a longitudinal section (D) of a vascular bundle. Scale bar = 100 μm. [Figure 20] Figure 20 shows alignment of myoblasts on decellularized vascular bundles of 10-day-old celery (A. graveolens). (A and E) Phloem and ground tissue: blue (calcofluor); (B and F) Actin filaments: green (phalloidin 488); (C and G) Nucleus and lignified tissue: red (propidium iodide). (A-D) Scale bar = 100 μm. (E-H) Scale bar = 25 μm. [Figure 21] Figure 21 shows (A-D) alignment of myotubes on decellularized vascular bundles from celery (Anetum graveolens). Myotubes: green (myosin heavy chain antibody); nuclei: red (propidium iodide); ground tissue and vascular bundles: blue (calcofluor). Scale bar = 100 μm. Alignment was based on the long axis direction of the myotubes. (Top) The tilted histogram corresponds to the directional output of anisotropic myotubes (B). (E-H) Isotropic conformation of myotubes on ground tissue. (Bottom) The uniform histogram corresponds to the directional output of isotropic myotubes (F). (I) Polar plot of normalized myotube data (black region) (-2.44° ± 3.83°) (0° corresponds to the direction of the vascular bundles in the polar plot). [Figure 22] Figure 22 shows an orthogonal view of phloem stained with calcofluor. Images were taken using a multiphoton microscope. Arrows: flattened areas. Scale bar = 25 μm. [Figure 23]Figure 23 shows Hoechst 33342 staining of decellularized (B and D) and native (A and C) vascular bundles. (C) Nuclei correspond to companion cells of the phloem. (A and B) Scale bar: 100 μm. (C and D) Scale bar: 25 μm. [Figure 24] Figure 24 shows a composite cellulose-based material. Two subunits were glued together with gelatin cross-linked with glutaraldehyde and reduced with sodium borohydride to obtain a composite structure. These structures were biocompatible after reduction and supported the growth of GFP 3T3 fibroblasts. [Figure 25] Figure 25 shows an excised composite vessel. Composite vessels were formed by casting 1.5% agarose around two laminated decellularized apple-derived cellulose rings. Samples were implanted subcutaneously in rats for four weeks. They were then excised and observed to remain intact and show no signs of infection or encapsulation / rejection. [Figure 26] Figure 26 shows (A) decellularized apple thixosa tissue and pore size distribution; and (B) particles obtained from maceration. Particle size is 241 ± 8 μm (mean ± standard error). [Figure 27] FIG. 27 shows images of freshly macerated apple thip tissue stained in 0.1% Congo red. [Figure 28] FIG. 28 shows images of macerated decellularized apple thip tissue stained in 0.1% Congo red. [Figure 29] FIG. 29 shows images of macerated decellularized apple thip tissue in a 1% alginate matrix. [Figure 30] FIG. 30 shows images of macerated elementary particles made from decellularized apple thip tissue in a 1% alginate matrix. [Figure 31] FIG. 31 shows an example of isolated apple cells obtained through maceration of decellularized apple strips combined with 1% alginate to form a composite hydrogel. [Figure 32]Figure 32 shows (A) decellularized pear thipial tissue macerated with a 1:1 mixture of acetic acid and peroxide; and (B) particle size distribution. The particle size is 96.4 ± 1.8 μm (mean ± standard error). [Figure 33] FIG. 33 shows an image of a cellulose scaffold after the reaction in the presence of succinic anhydride was completed, as described in Example 8. [Figure 34] FIG. 34 shows an image of the cellulose scaffold of FIG. 33 after washing, as described in Example 8, has been completed. [Figure 35] FIG. 35 shows the FTIR spectrum of a chemically bonded composite of decellularized scaffold (2AP-DECEL) and succinylated plant-derived cellulose (5AP-AS), as described in Example 8. [Figure 36] FIG. 36 shows CL macerated in dilute acetic acid and peroxide boiled for 15 minutes, as described in Example 9. [Figure 37] Figure 37 shows macerated celery, with only the xylem and phloem fibers visible. [Figure 38] FIG. 38 shows macerated apple solution boiled for 15 minutes in 1:1 glacial acetic acid and 30% hydrogen peroxide, as described in Example 9. [Figure 39] Figure 39 shows decellularized asparagus bundles, celery bundles (left) and apple slices (right) cross-linked with various concentrations of sodium alginate. [Figure 40] Figure 40 shows decellularized apple slices (left) and apple powder (center and right) cross-linked with alginate. The center and right images are of the same apple "meatballs" before and after frying. [Figure 41] 41 shows decellularized pear slices, as described in Example 9. (A) shows an uncooked pear with the celery bundle, (B) shows an uncooked pear without the celery bundle, (C) shows an uncooked pear "burger," (D) shows a cooked pear with the celery bundle (top layer removed), (E) shows a cooked pear without the celery bundle, and (F) shows a cooked pear "burger." [Figure 42] FIG. 42 shows decellularized celery strips (left), decellularized celery strips seeded with muscle cells (center), and decellularized celery strips layered and cross-linked with alginate solution (right). [Figure 43] Figure 43 shows a layering of decellularized celery with alternating layers of muscle cells and fat cells (left), which is then layered and fried in a cast iron dry skillet (right). [Figure 44] Figure 44 shows raw (left) and pan-fried (right) plant-based AA meat-free "burger patty" held together with 1% alginate. [Figure 45] FIG. 45 shows a burger product prepared from the patty of FIG. [Figure 46] Figure 46 shows a comparison of stress / strain curves comparing results from cooked meat with results from cooked alginate-celery "miniburgers." Representative stress / strain curves for individual samples of uncooked (red) and cooked (gray) plant-based meat and real meat are shown. (A) shows the ACL (alginate-celery) plant-based meat, which became softer after cooking. (B) Alternatively, the CCL (cell-cultured celery) plant-based meat shows that their mechanical properties did not change significantly before and after cooking. (C) shows the stress / strain curve for chicken breast (C), which became significantly tougher after cooking; a similar trend was observed for beef (B) steak, where the sample became significantly tougher after cooking (D). [Figure 47] Figure 47 shows the bulk modulus (N=10) from 3 10 cm CL burgers at various concentrations of CL:alginate. DETAILED DESCRIPTION OF THE INVENTION

[0134] Described herein is a composite scaffold biomaterial comprising two or more scaffold biomaterial subunits, each comprising decellularized plant or fungal tissue, from which cellular material and nucleic acids have been removed, and the decellularized plant or fungal tissue has a three-dimensional porous structure, where the two or more scaffold biomaterial subunits are assembled into the composite scaffold biomaterial and held or bonded together, for example, by gel casting using a hydrogel adhesive, by complementary interlocking shapes of the two or more scaffold biomaterial subunits, by guided assembly-based biolithography (GAB), by chemical crosslinking, or any combination thereof. Also provided are methods for producing such scaffold biomaterials, as well as methods and uses thereof. The scaffold biomaterials can be produced for a variety of different applications, including, but not limited to, medical applications and / or applications in the food industry as plant-derived meat substitutes. It will be understood that the embodiments and examples are provided for illustrative purposes intended for those skilled in the art and are not meant to be limiting in any way.

[0135] In one embodiment, there is provided a composite scaffold biomaterial comprising two or more scaffold biomaterial subunits each comprising decellularized plant or fungal tissue, wherein the decellularized plant or fungal tissue has been removed of tissue cellular material and nucleic acids, and wherein the decellularized plant or fungal tissue comprises a three-dimensional porous structure, Two or more scaffold biomaterial subunits are assembled into a composite scaffold biomaterial and held together by gel casting using a hydrogel adhesive, by complementary interlocking shapes of the two or more scaffold biomaterial subunits, by guided assembly-based biolithography (GAB), by chemical crosslinking, or any combination thereof. Provided herein are composite scaffold biomaterials.

[0136] In certain embodiments, the biomaterials described herein may be derived from cell wall architectures and / or vascular structures found in the plant and fungal kingdoms to create 3D scaffolds that may promote cell infiltration, cell proliferation, bone tissue repair and / or bone remodeling, etc. As will be appreciated, the biomaterials described herein can be produced from any suitable part of a plant or fungal organism. The biomaterials may include, for example, substances such as cellulose, chitin, lignin, lignans, hemicellulose, pectin, and / or any other suitable biochemicals / biopolymers naturally found in these organisms.

[0137] As will be understood, unless otherwise indicated, the meanings / definitions of the kingdoms Plantae and Fungi used herein are based on the Cavalier-Smith classification (1998).

[0138] In certain embodiments, the plant or fungal tissue may generally include any suitable plant or fungal tissue or part containing a suitable scaffold structure appropriate for a particular application.

[0139] In certain embodiments of one or more of the scaffold biomaterials described above, the plant or fungal tissue is selected from the group consisting of apple thip (Malus pumila) tissue, fern (Monilophytes) tissue, turnip (Brassica rapa) root tissue, ginkgo biloba (Ginkgo biloba) tissue, and the like. Weave, Horsetail (Equisetum) tissue, Hemerocallis hybrid Leaf tissue, kale (Brassica oleracea) stem tissue, conifer bay Pine (Pseudotsuga menziesii) tissue, cactus fruit (Pi Taya (Plantago major) flesh tissue, Maculata Vinca tissue, Aquatic lotus (Nelumbo nucifera) tissue, Tulip (Tulipagesneriana) petal tissue, Banana (Musa paradisiaca) tissue, Roccoli (Brassica oleracea) stem tissue, maple leaf (Platanine occidentalis (Acer psuedoplatanus) stem tissue, beet (Beta vulgaris) primary root tissue, leek (Allium cepa) tissue, orchid (Orchidaceae) tissue, turnip (Brassica rapa) stem tissue, chive (Allium ampeloprasum) tissue, maple (Acer serrata) Acer (Acer spp.) branch tissue, celery (Apium graveolens) tissue, leek (onion) stem tissue, pine tissue, aloe vera tissue, watermelon (Citrullus lanatus var. lanatus) tissue, creeping jenny (Lysimachia nummularia) tissue, cactae tissue, Lychnis alpina tissue, rhubarb (Tilapia spp.) Rheum rhabarbarum tissue, pumpkin pulp (Cucurbita pepo) tissue Woven fabric, Dracaena (Asparagaceae) stem tissue, Tradescantia virginiana (Tradescantia virginiana) stem tissue, Asparagus (Dutch Asparagus) (Asparagus officinalis) stem tissue, mushroom (fungal) tissue, fennel (wild The tissue may include bellflower (Foeniculum vulgare) tissue, rose (Rosa) tissue, carrot (Daucus carota) tissue, or pear (Pomaceous) tissue. Additional examples of plant and fungal tissues are described in Example 18 of WO 2017 / 136950, entitled "Decellularized Cell Wall Structures from Plants and Fungus and Use Thereof as Scaffold Materials," which is incorporated herein by reference in its entirety.

[0140] In certain embodiments, the decellularized plant or fungal tissue is cellulose-based, chitin-based, chitosan-based, lignin-based, lignan-based, hemicellulose-based, or pectin-based, or any combination thereof. In certain embodiments, the plant or fungal tissue is apple stipule (Apple) tissue, fern (Monilophytes) tissue, turnip (Brassica rapa) root tissue, ginkgo biloba branch tissue, horsetail (Equisetum aestivum) tissue, daylily hybrid leaf tissue, kale (Brassica oleracea) stem tissue, conifer Douglas fir (Pseudotsuga menziesii) tissue, cactus fruit (Pitaya) flesh tissue, maculata vinca tissue, water lotus (Nelumbo nucifera) tissue, tulip (Tulipa gesneriana) petal tissue, Plantago spp. (Banana) tissue, broccoli (Brassica oleracea) stem tissue, maple leaf (Platanus occidentalis) stem tissue, beet (Sugar cane) tissue, or the like. Radish (radish) primary root tissue, leek (Allium sativum) tissue, orchid (Orchidaceae) tissue, turnip (Brassica rapa) stem tissue, chive (Allium sieboldii) tissue, maple (Acer spp.) branch tissue, celery (Honeywort) tissue, leek (Allium sativum) stem tissue, pine tissue, aloe vera tissue, watermelon (Citrullus lanatus var. lanatus) tissue, creeping jenny (Solanum saxifraga) tissue, octopus tissue, Lychnis alpina tissue, rhubarb (Rheum palmatum) tissue, pumpkin flesh (Cucurbita pepo) tissue, Dracaena (Asparagaceae) stem tissue, Tradescantia recutita (Tradescantia recutita) stem tissue, asparagus (Asparagus asiaticus) stem tissue, mushroom (fungus) tissue The present invention may include tissue derived from plant tissue, fennel (Fennel) tissue, rose (Rosa) tissue, carrot (Daucus carota) tissue, or pear (Malus) tissue, or genetically modified tissue produced by direct genomic modification or through selective breeding, or any combination thereof.

[0141] It will also be understood that the cellular material and nucleic acid of plant or fungal tissue may include intracellular contents such as organelles (e.g., chloroplasts, mitochondria), nuclei, nucleic acids, and / or proteins. These may be substantially removed, partially removed, or completely removed from the plant or fungal tissue and / or from the scaffold biomaterial. Nevertheless, it will be recognized that trace amounts of such components may be present in the decellularized plant or fungal tissue described herein. It will also be understood that references herein to decellularized plant or fungal tissue are intended to reflect that such cellular material found in the plant or fungal tissue source has been substantially removed, and this does not exclude the possibility that the decellularized plant or fungal tissue may, in certain embodiments, contain or include subsequently introduced or reintroduced cells, cellular material, and / or nucleic acids of generally any type, e.g., animal or human cells, e.g., bone or bone precursor cells / tissue.

[0142] Various methods can be used to produce the scaffold biomaterials or scaffold biomaterial subunits described herein. By way of example, in certain embodiments of the above-described scaffold biomaterials and / or subunits, decellularized plant or fungal tissue can include plant or fungal tissue(s) decellularized by heat shock, treatment with detergents (e.g., SDS, Triton X, EDA, alkaline treatment, acid, ionic detergents, non-ionic detergents, and zwitterionic detergents), osmotic shock, lyophilization, physical lysis (e.g., hydrostatic pressure), electrical disruption (e.g., non-thermal irreversible electroporation), or enzymatic digestion, or any combination thereof. In certain embodiments, the biomaterials described herein can be obtained from plants and / or fungi by using a decellularization process that can include any of several techniques (individually or in combination), including, but not limited to, heat shock (e.g., rapid thawing), chemical treatment (e.g., detergents), osmotic shock (e.g., distilled water), lyophilization, physical lysis (e.g., pressure treatment), electrical disruption, and / or enzymatic digestion.

[0143] In certain embodiments, decellularized plant or fungal tissue may include plant or fungal tissue that has been decellularized by treatment with a detergent or surfactant. Examples of detergents include sodium dodecyl sulfate (SDS), Triton X, EDA, and alkaline treatment. These detergents may include, but are not limited to, acids, ionic detergents, non-ionic detergents and zwitterionic detergents.

[0144] In yet a further embodiment, the decellularized plant or fungal tissue may include plant or fungal tissue that has been decellularized by treatment with SDS. In yet another embodiment, residual SDS can be removed from the plant or fungal tissue by washing with an aqueous divalent salt solution. The aqueous divalent salt solution can be used to precipitate / displace salt residues, including SDS micelles, from the solution / scaffold, and dH2O, acetic acid, or dimethylsulfoxide (DMSO) treatment, or sonication can be used to remove the salt residues or SDS micelles. In certain embodiments, the divalent salt of the aqueous divalent salt solution can include, for example, MgCl or CaCl.

[0145] In another embodiment, plant or fungal tissue may be decellularized by treatment with an SDS solution of 0.01 to 10%, e.g., about 0.1% to about 1%, or e.g., about 0.1% SDS or about 1% SDS, in a solvent such as water, ethanol, or another suitable organic solvent, and residual SDS may be removed using an aqueous CaCl2 solution at a concentration of about 100 mM, followed by incubation in dH2O. In certain embodiments, the SDS solution may be used to effect decellularization. Concentrations above 0.1% may be advantageous and may involve increased washing to remove residual SDS. In certain embodiments, plant or fungal tissue may be decellularized by treatment with an SDS solution of about 0.1% SDS in water, and residual SDS may be removed using an aqueous CaCl solution at a concentration of about 100 mM, followed by incubation in dH2O.

[0146] Further examples of decellularization protocols that can be adapted to produce decellularized plant or fungal tissue for the scaffold biomaterials described herein can be found in WO 2017 / 136950, entitled "Decellularized Cell Wall Structures from Plants and Fungus and Use Thereof as Scaffold Materials," which is incorporated herein by reference in its entirety.

[0147] In certain embodiments, the scaffold biomaterials described herein may comprise decellularized plant or fungal tissue comprising a pore size of about 100 to about 200 μm, or about 150 to about 200 μm. In certain embodiments, the scaffold biomaterial may comprise a Young's modulus of about 20 to about 200 kPa. In certain embodiments, the decellularized plant or fungal tissue may comprise decellularized apple, such as decellularized apple thipia tissue.

[0148] In certain embodiments of the scaffold biomaterials described herein, the scaffold biomaterials may further comprise living cells, particularly non-native cells, on and / or within the decellularized plant or fungal tissue. In certain embodiments, the living cells may be animal cells. In certain embodiments, the living cells may be mammalian cells. In certain embodiments, the living cells may be human cells.

[0149] In certain embodiments, two or more scaffold biomaterial subunits may be assembled into a composite scaffold biomaterial and held together by gel casting using a hydrogel adhesive.

[0150] As will be appreciated, gel casting can include any suitable gel casting process known to those of skill in the art having skill in the art in connection with the teachings herein. By way of example, in certain embodiments, gel casting can include any suitable process that surrounds all or a portion of a material with a gel. A variety of techniques can be used to cast the gel. By way of example, in certain embodiments, the gel can be cast by dipping, pipetting, coating, or other such techniques.

[0151] In certain embodiments, the hydrogel adhesive may include any suitable hydrogel adhesive, or a mimetic or functional equivalent thereof. In certain embodiments, the hydrogel adhesive may include an adhesive based on one or more polymeric polymers that may be crosslinked and may retain water. In certain embodiments, rather than crosslinking, polymer entanglement may be used, polymer melts may be used, or any combination thereof. In certain embodiments, colloids may be used. Some examples of hydrogel adhesives may include gelatin, collagen, hyaluronic acid, agarose, fibronectin, or other such adhesives.

[0152] In certain embodiments, the hydrogel adhesive may comprise gelatin, collagen, agarose, hyaluronic acid, alginate, fibrin, fibronectin, agar, PEG, PVA, or any combination thereof. In certain embodiments, two or more scaffold biomaterial subunits may act as a scaffold for the hydrogel adhesive to form around. In certain embodiments, at least a portion of two or more scaffold biomaterial subunits may be coated with the hydrogel adhesive. In certain embodiments, the hydrogel The adhesive may be cured or solidified by temperature change, cross-linking, or a combination thereof. In certain embodiments, the hydrogel adhesive may comprise gelatin that has been cross-linked with glutaraldehyde and sodium borohydride reduction. In certain embodiments, the hydrogel adhesive may further comprise one or more agents, such as a therapeutic agent, a signaling molecule, a growth factor, a metabolite, an ECM protein or component, or any combination thereof.

[0153] In certain embodiments, two or more scaffolding biomaterial subunits may be assembled into a composite scaffolding biomaterial and held together by the complementary interlocking shapes of the two or more scaffolding biomaterial subunits.

[0154] In certain embodiments, the complementary interlocking shapes may include any suitable shape or configuration or structural feature of the scaffold biomaterial subunits that allows the scaffold biomaterial subunits to fit together. Typically, each of the scaffold biomaterial subunits may include one or more interlocking shape features that may be complementary to corresponding interlocking shape feature(s) on one or more adjacent scaffold biomaterial subunits to be assembled together. In certain embodiments, the interlocking shapes may include friction-fit type complementarity. In certain embodiments, the complementary interlocking shapes may include corresponding male and female interlocking shapes on adjacent scaffold biomaterial subunits. In certain embodiments, the complementary interlocking shapes may include peg-and-hole (i.e., Lego-style) structures that may allow two adjacent scaffold biomaterial subunits to be compressed or squeezed together, resulting in a friction fit between the peg and hole features of the scaffold biomaterial subunits. Various other complementary interlocking features are also contemplated, such as, for example, keyhole, latch, stud, taper-fit designs (where the fit of adjacent subunits can be modified by adjusting or modifying the taper to provide a loose, tight, or intermediate fit therebetween to suit a particular application), or clip-type designs. In certain embodiments, the complementary / interlocking features can include any suitable shape or geometry that allows the scaffold biomaterial subunits to be positioned relative to one another, for example, such that the subunits contact one another over a large surface area, and the surface texture of the subunits at the contact / interface region, the surface features of the subunits, or any combination thereof, can prevent slippage.

[0155] It is contemplated that in certain embodiments, the complementary interlocking geometries may be designed, adjusted, or modulated to achieve a desired mechanical result. For example, in certain embodiments, a composite biomaterial intended to act as a conduit for drug delivery may be designed to interlock relatively loosely compared to a composite biomaterial intended to act as a larger scaffold.

[0156] In certain embodiments, the complementary interlocking features of the two or more scaffold biomaterial subunits comprise peg-and-hole or other friction-fit interlocking features.

[0157] In certain embodiments, at least one of the scaffold biomaterial subunits may comprise or be seeded with a first cell type. In certain further embodiments, at least one other scaffold biomaterial subunit may comprise or be seeded with a second cell type. In certain embodiments, the first and second cell types may be contact-inhibited or non-contact-inhibited. In certain embodiments, the composite scaffold biomaterial may comprise an interface between adjacent scaffold biomaterial subunits that may mimic a tissue interface, such as a bone-fibroblast tissue interface. In certain embodiments, the first cell type, the second cell type, or both may be animal cells, such as mammalian cells or human cells. In certain embodiments, the scaffold biomaterial may comprise ECM deposition at at least one interface between adjacent scaffold biomaterial subunits. In certain embodiments, the scaffold biomaterial may comprise an effective Young's modulus that may vary based on the direction of an applied force. In certain embodiments, the scaffold biomaterial subunits may comprise an effective Young's modulus that may vary based on the direction of an applied force. Two or more may be further held together by gel casting using a hydrogel adhesive, by guided assembly-based biolithography (GAB), by chemical crosslinking, or any combination thereof.

[0158] In certain embodiments, two or more scaffold biomaterial subunits can be assembled into a composite scaffold biomaterial and held together by guided assembly-based biolithography (GAB).

[0159] As will be appreciated, guided assembly-based biolithography (GAB) can include techniques that use templates to transfer topography to biomaterials. In certain embodiments, the templates can act as guides for, for example, bacterial cellulose growing on a scaffold biomaterial. In certain embodiments, composite biomaterials comprising plant-derived and bacterial-derived cellulose can be prepared, for example, with customizable topography and / or density.

[0160] Certain bacteria produce and excrete cellulose. The crystalline structure of the produced cellulose differs from that of plant-derived cellulose, and the amount and / or arrangement of amorphous and crystalline regions may differ, which can significantly affect the material properties. Therefore, the physical, mechanical, and / or chemical properties of plant and bacterial cellulose may differ. Those skilled in the art, armed with the teachings herein, will recognize the various cellulose-producing bacteria and the types of cellulose that can be obtained therefrom.

[0161] In certain embodiments, the two or more scaffold biomaterial subunits include at least one subunit comprising a plant or fungal derived biomaterial and at least one subunit comprising bacterial cellulose.

[0162] In certain embodiments, bacterial cellulose can be grown on plant or fungal derived biomaterials by guided assembly-based biolithography (GAB).

[0163] In certain embodiments, two or more scaffold biomaterial subunits may be assembled into a composite scaffold biomaterial and held together by chemical crosslinks.

[0164] In certain embodiments, chemical cross-linking can involve the covalent bonding of two separate cellulose chains. Cross-linking sites can depend on the availability and / or location of functional groups, such as hydroxyl groups, as well as on the density of the chains. In one embodiment, cellulose chains can be modified to add linkers, such as succinic acid, and cross-linking can be performed on the free moieties. In certain embodiments, such modifications can be used to introduce cross-linkable end groups for subsequent chemical cross-linking. In certain embodiments, chemical cross-linking can be performed on, for example, cellulose, chitin, lignin, lignans, hemicellulose, pectin, and / or any other suitable biochemicals / biopolymers / structures found naturally in these organisms.

[0165] In certain embodiments, the cellulose structure can be biochemically functionalized based on the intended use of the biomaterial. Such modifications can expand functionality to suit specific uses. For example, cellulose has free hydroxyl groups that can be utilized to conjugate the material with various molecules. Two commonly used classes of reactions for this type of modification are acylation and alkylation reactions. Such reactions can allow hydrocarbon chains of various lengths to be attached to the cellulose structure via the free hydroxyl groups. Varying chain lengths and geometries can be particularly useful when steric hindrance is a factor. The use of larger chains can reduce steric hindrance, and vice versa. Acylation reactions using dicarboxylic acids can provide options for functionalizing biomaterials. Dicarboxylic acids that can be used include: Some classes of dicarboxylic acids may include linear saturated dicarboxylic acids, branched dicarboxylic acids, unsaturated dicarboxylic acids, substituted dicarboxylic acids, and aromatic dicarboxylic acids. In addition to acylation and alkylation reactions, other compounds, such as compounds containing boron, sulfur, nitrogen, and / or phosphorous, may be used to mediate the connection between the functional group and cellulose. Various functional groups may be added to the other end of the chain to provide specific functionality. Such functional groups may include, for example, groups containing hydrocarbons, oxygen, nitrogen, sulfur, phosphorous, boron, and / or halogens. The selection of functional groups may be tailored to the specific intended application. For example, in certain embodiments, if the intended application is to prevent cell growth in a specific area, a sterically nonpolar hydrocarbon functional group may be used; conversely, if the intended application is to promote cell growth, a carboxylic acid may be used to allow extracellular matrix proteins, such as collagen, to bind to cellulose.

[0166] In certain embodiments, chemical modification of cellulose (or other such materials) may allow for control of the chemical and / or physical properties of biomaterials. As a result, biomaterials may, in certain embodiments, be specialized for specific purposes. For example, patterned cell growth can be provided by inhibiting cell growth (temporarily or permanently) in certain regions and promoting it in others. Furthermore, cell type-specific molecules may be introduced into biomaterials through such functionalization methods to promote the proliferation / invasion / differentiation of specific cell types. Functionalization of biomaterials may allow for closer reproduction of biologically relevant microenvironments, which may be relevant for cell function and / or tissue engineering.

[0167] In certain embodiments, at least a portion of two or more of the scaffold biomaterial subunits can be modified to include carboxymethyl and / or hydroxyethyl cellulose functional groups that can be crosslinked together with citric acid and heat to hold the composite scaffold biomaterial together.

[0168] In certain embodiments, two or more scaffold biomaterial subunits may be assembled into a composite scaffold biomaterial and held together by gel casting using a hydrogel adhesive, by complementary interlocking shapes of the two or more scaffold biomaterial subunits, by guided assembly-based biolithography (GAB), by chemical crosslinking, or any combination thereof. It is contemplated that structural integrity, for example, may be further increased through a combination of two or more of these approaches.

[0169] In certain embodiments of any of the composite materials, structures, or composite scaffold biomaterials described herein, the material may further comprise one or more agents, such as a therapeutic agent, a signaling molecule, a growth factor, a metabolite, an ECM protein or component, or any combination thereof.

[0170] In certain embodiments, example growth factors may include those described in the Wikipedia entry for growth factors (https: / / en.wikipedia.org / wiki / Growth_factor), which is incorporated herein by reference. In certain embodiments, the therapeutic agent may include an anticoagulant, an anti-inflammatory and / or an immunosuppressant, or another therapeutic agent of interest, or any combination thereof. In certain embodiments, the ECM protein or component may include, for example, collagen, elastin, fibronectin, laminin, or another ECM protein or component, or any combination thereof.

[0171] In yet another embodiment of any of the materials described herein, the decellularized plant or fungal tissue can be cellulose-based, hemicellulose-based, chitin-based, chitosan-based, pectin-based, lignin-based, lignan-based, or any combination thereof.

[0172] In yet another embodiment of any of the materials described herein, the composite scaffold biomaterial may comprise at least two scaffold biomaterial subunits that are structurally distinct from one another. In certain embodiments, the at least two structurally distinct scaffold biomaterial subunits may be derived from different plant or fungal sources, may be derived from different parts of the same plant or fungal source, may be derived from two different species of plant or fungal source, may exhibit different Young's modulus properties, may comprise different cell types, may comprise different hydrogels, or any combination thereof.

[0173] In yet another embodiment, a hydrogel, such as a cellulose-based hydrogel, includes a crosslinked matrix, such as a crosslinked cellulose matrix; and One or more channels formed in the crosslinked matrix by removing the transient space-filling material from the crosslinked matrix. Provided herein is a hydrogel comprising:

[0174] In certain embodiments, the hydrogel may comprise any suitable hydrogel, or a mimetic or functional equivalent thereof. In certain embodiments, the hydrogel may comprise a gel based on one or more macromolecular polymers, which may be crosslinked and may retain water. In certain embodiments, rather than crosslinking, polymer entanglement may be used, polymer melts may be used, or any combination thereof. In certain embodiments, colloids may be used. In certain embodiments, the hydrogel may comprise a crosslinked matrix comprising cellulose, chitin, lignin, lignans, hemicellulose, pectin, and / or any other suitable biochemical / biopolymer found naturally in plants or fungi, or any combination thereof.

[0175] In certain embodiments, the temporary space filler may comprise a space-filling material that may be used to prevent the surrounding material from occupying a space or region before the surrounding material is placed. After placement of the surrounding material, the temporary space filler may be removed, leaving a gap in the structure. In certain embodiments, the space filler may comprise, for example, a non-crosslinked polymer, a hydrogel, or a colloid.

[0176] In certain embodiments, the crosslinked matrix may be prepared from decellularized plant or fungal tissue, from which cellular material and nucleic acids have been removed, and which comprises a three-dimensional porous structure. In certain embodiments, the temporary space-filling material may comprise a temporary gel, such as an alginate hydrogel.

[0177] In certain embodiments, a matrix may be crosslinked around the fugitive space filler, which may then be removed from the crosslinked matrix, thereby forming one or more channels. In certain embodiments, the fugitive space filler may be removed by dissolving, heating, changing salt concentration, decomposition, or any combination thereof. In certain embodiments, the fugitive space filler may be disposed within the matrix to form a template for a network of channels within the crosslinked matrix. In certain embodiments, the fugitive space filler may be disposed within the matrix by deposition via 3D printing.

[0178] In certain embodiments, the hydrogel may further comprise one or more agents, such as a therapeutic agent, a signaling molecule, a growth factor, a metabolite, an ECM protein or component, or any combination thereof.

[0179] In certain embodiments, the matrix is ​​cellulose-based, hemicellulose-based The crosslinked matrix can be prepared from decellularized plant or fungal tissue that is chitin-based, chitosan-based, pectin-based, lignin-based, lignan-based, or any combination thereof. In certain embodiments, the crosslinked matrix can comprise at least two structurally distinct celluloses. In certain embodiments, the at least two structurally distinct celluloses can be derived from different plant or fungal sources, from different parts of the same plant or fungal source, from two different species of plant or fungal sources, exhibit different Young's modulus properties, comprise different cell types, comprise different hydrogels, or any combination thereof.

[0180] In certain embodiments, a method for preparing a hydrogel having one or more channels, such as a cellulose-based hydrogel having one or more channels, is provided, comprising: providing a first crosslinkable material or gel, such as a natural or modified cellulose, chitin, lignin, lignan, hemicellulose, or pectin-based material or gel; providing a transient space-filling material; generating a three-dimensional structure comprising a first crosslinkable material or gel having the fugitive space-filling material dispersed therein such that the fugitive space-filling material forms a template for one or more channels in the first crosslinkable material or gel, and crosslinking the first crosslinkable material or gel during or after generation of the three-dimensional structure; and Removing the transient space-filling material from the three-dimensional structure to provide a hydrogel having one or more channels. Provided herein is a method comprising:

[0181] In certain embodiments, the first crosslinkable material or gel and the fugitive space filler may be printed in the xy plane, layer by layer, with one or more extruders (e.g., a dual extruder using one extruder for the first crosslinkable material or gel and one extruder for the fugitive space filler) to generate a three-dimensional structure.

[0182] In certain embodiments, the temporary space filler may comprise a gel. In certain embodiments, the temporary space filler may comprise gelatin, and the structure may be cooled to maintain the gelatin in place. In certain embodiments, the first crosslinkable material or gel may comprise cellulose or a derivative thereof, and the cellulose may be crosslinked during or after printing. In certain embodiments, after the formation and crosslinking of the cellulose three-dimensional structure, the temperature may be increased to dissolve the gelatin from the three-dimensional structure. Alternatively, in certain embodiments, the temporary space filler may comprise a calcium-crosslinked alginate gel, and after the formation and crosslinking of the first crosslinkable material or gel three-dimensional structure, the calcium may be exchanged for sodium in wash salts to dissolve the alginate from the three-dimensional structure.

[0183] In yet another embodiment, 1. A scaffold biomaterial comprising decellularized plant or fungal tissue, wherein the decellularized plant or fungal tissue has been removed of cellular material and nucleic acids of the tissue, and the decellularized plant or fungal tissue comprises a three-dimensional porous structure; At least a portion of the decellularized plant or fungal tissue is functionalized with, complexed to, or covalently bound to one or more agents, such as therapeutic agents, signaling molecules, growth factors, metabolites, ECM proteins or components, or any combination thereof; SUMMARY OF THE INVENTION Provided herein are scaffold biomaterials.

[0184] In certain embodiments, the decellularized plant or fungal tissue may be modified with a linker, such as a succinyl linker, that can be used to covalently attach one or more drugs; or Decellularized plant or fungal tissue may be modified with carboxymethyl and / or hydroxyethyl cellulose functional groups that can be used for covalent attachment of one or more drugs via citric acid and heat-based coupling.

[0185] In another embodiment, provided herein is the use of any of the composite scaffold biomaterials, hydrogels or scaffold biomaterials described herein, or any combination thereof, for tissue repair or regeneration; in an implant; for culturing one or more cell types in vitro or in vivo; for mimicking in vivo tissues or tissue interfaces; for bone tissue engineering; for bone repair or regeneration; for transporting fluids or liquids; for mimicking tissue interfaces; for wound healing; for delivering agents such as therapeutic agents, signaling molecules, growth factors, metabolites, ECM proteins or components, or any combination thereof; or any combination thereof.

[0186] In yet another embodiment, a method is provided for repairing or regenerating tissue; for providing an implant; for culturing one or more cell types; for mimicking in vivo tissue or tissue interfaces; for bone tissue engineering; for repairing or regenerating bone; for transporting fluids or liquids; for mimicking tissue interfaces; for wound healing; for delivering agents such as therapeutic agents, signaling molecules, growth factors, metabolites, ECM proteins or components, or any combination thereof; or any combination thereof in a subject in need thereof, comprising: providing a composite scaffold biomaterial described herein, a hydrogel described herein, or a scaffold biomaterial described herein, or any combination thereof; and introducing the composite scaffold biomaterial, hydrogel or scaffold biomaterial, or any combination thereof, into a subject at a site in need thereof. Provided herein is a method comprising:

[0187] In yet another embodiment, there is provided a method for inducing cell alignment, comprising: providing a decellularized scaffold biomaterial comprising one or more channels or grooves; Seeding the decellularized scaffold biomaterial with cells or implanting the decellularized scaffold biomaterial in a position where it will come into contact with the cells; and Culturing the cells on the decellularized scaffold biomaterial, thereby aligning the cells along one or more channels or grooves. Provided herein is a method comprising:

[0188] In certain embodiments, decellularized scaffold biomaterials may contain grooves or channels derived from natural grooves or channels in structures formed from vascular bundles of plant or fungal sources. In certain embodiments, the grooves or channels may typically be about 1 to 100 micrometers in size, or any subrange or value therebetween. In certain embodiments, the structures may be adapted to accommodate mammalian cell types or structures of interest, including, for example, fibroblasts, myofibroblasts, neurons, neuronal structures such as axons, endogenous stem cells, neutrophils, mesenchymal stem cells, satellite cells, myoblasts, myotubes, muscle progenitor cells, chondrocytes, tendon progenitor cells, tenocytes, periodontal ligament stem cells, or any combination thereof.

[0189] In certain embodiments, the decellularized scaffold biomaterial may comprise decellularized celery tissue. In certain embodiments, the cells may comprise muscle cells or precursors thereof. In certain embodiments, the cells may comprise myoblasts. In certain embodiments, the cells may comprise C2C12 myoblasts.

[0190] In certain embodiments, the composite structures described herein provide microchannels that may be suitable for transporting water, nutrients, and / or for inducing directed cell growth. In certain embodiments, the composite structures described herein may be prepared by incorporating a variety of materials with different structural characteristics to provide mechanisms such as stress shielding (i.e., hard and soft materials) and / or soft and hard bone (i.e., trabecular and cortical).

[0191] In certain embodiments, unlike many commercially available biomaterials, the plant / fungal-derived biomaterials described herein can be substantially non-resorbable or poorly resorbable (i.e., they do not substantially disintegrate and are not absorbed by the body). The non-resorbable nature of these scaffolds can provide certain advantages. For example, in certain embodiments, the biomaterials described herein can be resistant to conformational changes and / or retain their intended shape for extended periods of time. In certain embodiments, because they can have a minimal footprint compared to certain other products, they can be considered effectively invisible to the body and may elicit little immune response. When absorbable biomaterials disintegrate, their by-products often trigger adverse immune responses and induce oxidative stress, leading to increased pH in healing tissues (which can be avoided by using non-resorbable biomaterials).

[0192] Indeed, in certain embodiments, the decellularized plant or fungal tissue and / or scaffold biomaterial described herein may further comprise living cells on and / or within the scaffold biomaterial, hi certain embodiments, the living cells may be animal cells, mammalian cells, or human cells.

[0193] In certain embodiments, plant or fungal tissues may be genetically modified, either by direct genomic modification or through selective breeding, to create additional plant or fungal architectures that can be configured to physically mimic the tissue and / or functionally promote desired tissue effects. Those skilled in the art, armed with the teachings herein, will be able to select appropriate scaffold biomaterials to suit a particular application. In certain embodiments, appropriate tissues may be selected for a particular application based on physical characteristics such as size, structure (porous / tubular), stiffness, strength, hardness, and / or ductility, which can be measured and adapted to the particular application.

[0194] Additionally, chemical properties such as reactivity, coordination number, enthalpy of formation, heat of combustion, stability, toxicity, and / or type of bonding may also be considered for selection to suit a particular application, and such characteristics (physical and chemical) may be directly modified before or after decellularization and / or functionalization to respond to a particular application.

[0195] In certain embodiments, the scaffold biomaterial may be derived from the same tissue or part of a plant or fungus, or from different parts or tissues of a plant or fungus. In certain embodiments, the scaffold biomaterial may be derived from the same individual plant or fungus, or from multiple plants or fungi of the same species. In certain embodiments, the scaffold biomaterial may be derived from different species of plants or fungi, such that the scaffold includes structures from more than one species. In certain embodiments, the scaffold biomaterial may be selected to provide specific characteristics. For example, in certain embodiments, a scaffold biomaterial having porosity and / or stiffness falling within a certain range may be selected to mimic natural tissue and / or structure. In certain embodiments, the plant or fungal tissue may include apple or apple thipium tissue, or tissue of another plant or fungus having similar porosity and / or stiffness characteristic(s).

[0196] In certain embodiments, the scaffold biomaterial may be a scaffold biomaterial formed to physically mimic a tissue of a subject and / or to functionally promote a desired tissue effect in a subject. Methods of using such scaffold biomaterials described herein may, in certain embodiments, involve the decellularization of plant or fungal tissue to physically mimic the tissue of a subject. and / or selecting a scaffold biomaterial as described herein configured to functionally promote a desired tissue effect in a subject.

[0197] In certain embodiments, the decellularized plant or fungal tissue and / or scaffold biomaterial described herein may further comprise living cells on and / or within the plant or fungal tissue. In certain embodiments, the living cells may be animal cells, mammalian cells, or human cells. In certain embodiments, the cells may be, for example, cells introduced or seeded within and / or onto the scaffold biomaterial and / or decellularized plant or fungal tissue, or cells that infiltrate into or onto the scaffold biomaterial and / or decellularized plant or fungal tissue after implantation of the scaffold biomaterial and / or decellularized plant or fungal tissue into a living animal or plant subject.

[0198] In one embodiment, there is provided a method for producing a scaffold biomaterial, comprising: providing plant or fungal tissue; Extracting one or more structures from plant or fungal tissue; and Providing a scaffold biomaterial from one or more extracted structures. Provided herein is a method comprising:

[0199] In certain embodiments, extraction of one or more structures from plant or fungal tissue may include one or more manual steps that may be performed to extract or separate the one or more structures of interest from the surrounding plant or fungal tissue. Such manual steps may include cutting, slicing, peeling, and / or other physical separation techniques. As will be appreciated, for large-scale operations, such manual steps may be cumbersome. Therefore, as described herein, the inventors have developed extraction techniques that may, for example, be less cumbersome and / or easily modifiable for scale-up. Thus, in certain embodiments, the extracting step may include a liquid-based extraction to isolate one or more structures from the plant or fungal tissue.

[0200] As will be appreciated, liquid-based extraction can include any suitable process for treating and extracting one or more structures from plant or fungal tissue (which can be either native plant or fungal tissue, or decellularized plant or fungal tissue, or a combination thereof) using a liquid extraction solution. Typically, liquid-based extraction produces a maceration of the plant or fungal tissue, whereby the plant or fungal tissue is disaggregated into tissue / cellular components (including, for example, vascular bundles, lignocellulosic matrix or structures, single cells, and / or other structures of interest, or any combination thereof). In certain embodiments, the liquid extraction solution can include a maceration solution, such as a salt solution, an acid solution, an acid and peroxide solution, or an alkali / base solution. In certain embodiments, two or more treatments or solutions can be used simultaneously or sequentially.

[0201] In certain embodiments, the extracting step may include maceration with at least one of treatment with a salt solution, treatment with a base solution, treatment with an acid solution, or treatment with an acid and peroxide solution.

[0202] In certain embodiments, the extracting step may include at least one treatment with a salt solution. As will be appreciated, the salt solution may generally include any suitable salt, such as any suitable salt capable of osmotic shock and / or disruption of hydrogen bonding and / or polymer crystalline structure to extract intact tissue structures. As will be appreciated, particularly for dietary and / or medical applications, the salt may be selected to be appropriate for the particular application, e.g., where desired, selected to be physiologically occurring, easily washed away, non-harmful, and / or according to various factors related to the particular application. In certain embodiments, the salt may include NaCl, LiCl, CaCl, AlCl, magnesium sulfate, potassium chloride, or calcium chloride, or any combination thereof. In certain embodiments, the salt may include NaCl, LiCl, or any combination thereof. In certain embodiments, the salt may be monovalent, divalent, or trivalent. As will be appreciated, in certain embodiments, the salt may be selected, at least in part, based on the intended application. Using different salts may have, for example, different electrical screening properties, activities, coordination profiles, and solubilities. The salt may be dissolved / mixed in a suitable solvent to form a salt solution. Typically, the solvent may include water, although other solvents or solvent combinations (e.g., a mixture of water and ethanol, etc.) are also contemplated. The salt concentration in the salt solution may be adjusted to suit the particular application of interest, the particular solubility, and / or other factors. Typically, the salt solution can comprise a salt concentration of about 0.1 to 10 M, or any concentration therebetween (which may be rounded to the nearest 0.1), or any subrange spanning between any two of these concentrations. In certain embodiments, the salt concentration can be about 0.5 M to 3 M, or any value therebetween (which may be rounded to the nearest 0.1), or any subrange spanning between any two of these concentrations. By way of example, in certain embodiments, the salt solution can comprise an aqueous solution of NaCl or LiCl having a salt concentration of about 0.5 M to 3 M. As will be appreciated, the salt solution and processing conditions (i.e., heating, agitation) can be adjusted, as desired, to suit the particular application, the desired structure to be extracted, the plant or fungal tissue used, etc.

[0203] In certain embodiments, the salt may include a salt selected from the group consisting of salts having a cation selected from lithium, sodium, potassium, magnesium, calcium, iron, copper, zinc, aluminum, or ammonium, in any suitable combination; and an anion selected from chloride, acetate, carbonate, citrate, fluoride, nitrate, phosphate, sulfate, iodide, or borate. An example of a pharmaceutical salt may include ibuprofenate. Depending on the intended use of the salt and / or product, neutralization and / or washing may be performed, for example, to remove residual salts and other reagents and prevent undesired contamination.

[0204] In certain embodiments, the extracting step may include at least one treatment with a base solution. As will be appreciated, the base solution may generally include any suitable base, such as any suitable base capable of osmotic shock and / or disruption of hydrogen bonds and / or polymer crystalline structure to extract intact tissue structures. As will be appreciated, particularly for food and / or medical applications, the base may be selected to be appropriate for the particular application, e.g., may be selected to be physiologically occurring, easily washed away, non-toxic, and / or may be selected according to various factors related to the particular application. In certain embodiments, the base may include NaOH, KOH, or a combination thereof. In one embodiment, the base may be dissolved / mixed in a suitable solvent to form a base solution. Typically, the solvent may include water, although other solvents or solvent combinations (e.g., a mixture of water and ethanol) are also contemplated. The concentration of the base in the base solution may be adjusted to suit the particular application of interest. Typically, the base solution can comprise a base concentration of about 0.1 to 10 M, or any concentration therebetween (which may be rounded to the nearest 0.1), or any subrange spanning any two of these concentrations. In certain embodiments, the base concentration can be about 0.5 M to 3 M, or any value therebetween (which may be rounded to the nearest 0.1), or any subrange spanning any two of these concentrations. By way of example, in certain embodiments, the base solution can comprise an aqueous solution of NaOH having a concentration of about 0.5 M to 3 M. As will be appreciated, the base solution and processing conditions (i.e., heating, agitation) can be adjusted, as desired, to suit the particular application, the desired structure to be extracted, the plant or fungal tissue used, etc.

[0205] In certain embodiments, the base may comprise a base selected from the group consisting of carbonate, nitrate, phosphate, sulfate, ammonia, sodium hydroxide, calcium hydroxide, magnesium hydroxide, potassium hydroxide, lithium hydroxide, zinc hydroxide, sodium carbonate, sodium bicarbonate, butyllithium, sodium azide, sodium amide, sodium hydride, sodium borohydride, and lithium diisopropylamine. Depending on the intended use of the base and / or the product, neutralization and / or washing may be performed, for example, to remove residual base and other reagents and prevent undesired contamination.

[0206] In certain embodiments, the extracting step may include at least one treatment with an acid solution. As will be appreciated, the acid solution may generally include any suitable acid, such as any suitable acid capable of osmotic shock and / or disruption of hydrogen bonds and / or polymer crystalline structure to extract intact tissue structures. As will be appreciated, particularly for food and / or medical applications, the acid may be selected to be appropriate for the particular application, e.g., may be selected to be physiologically occurring, easily washed away, non-toxic, and / or selected according to various factors related to the particular application. In certain embodiments, the acid solution may include acetic acid, hydrochloric acid (HCl), H2SO4, or a combination thereof. In certain embodiments, the acid may be dissolved / mixed in a suitable solvent to form the acid solution. Typically, the solvent may include water, although other solvents or solvent combinations (e.g., a mixture of water and ethanol) are also contemplated. In certain embodiments, the acid may be used "neat," i.e., without a solvent. For example, acetic acid was used in certain examples described herein. The acid concentration in the acid solution can be adjusted to suit the particular application of interest. Typically, the acid solution can include an acid concentration of about 0.1 to 10 M, or any concentration therebetween (which may be rounded to the nearest 0.1), or any subrange spanning any two of these concentrations. By way of example, in certain embodiments, the acid solution can include glacial acetic acid; acetic acid diluted in water (e.g., 50% diluted acetic acid); or HCl in water, at a concentration of about 4 M or less. As will be appreciated, the acid solution and processing conditions (i.e., heating, agitation) can be adjusted, as desired, to suit the particular application, the desired structure to be extracted, the plant or fungal tissue used, etc.

[0207] In certain embodiments, the acid may comprise an acid selected from the group consisting of acetic acid, boric acid, carbonic acid, hydrochloric acid, citric acid, hydrofluoric acid, nitric acid, oxalic acid, phosphoric acid, sulfuric acid, boron trifluoride, oxalic acid, malonic acid, succinic acid, and malic acid. Additional examples may include dicarboxylic acids, such as linear saturated dicarboxylic acids, branched dicarboxylic acids, unsaturated dicarboxylic acids, substituted dicarboxylic acids, and aromatic dicarboxylic acids. Depending on the intended use of the acid and / or product, neutralization and / or washing may be performed, for example, to remove residual acid and other reagents and prevent undesired contamination.

[0208] In certain embodiments, the extracting step may include at least one treatment with an acid and peroxide solution. As will be appreciated, the acid and peroxide solution may generally include any suitable acid and peroxide, such as any suitable acid and peroxide capable of osmotic shock and / or disruption of hydrogen bonding and / or polymer crystalline structure to extract intact tissue structures. As will be appreciated, particularly for food and / or medical applications, the acid and peroxide may be selected to be appropriate for the particular application, e.g., may be selected to be physiologically occurring, easily washed away, non-toxic, and / or may be selected according to various factors related to the particular application. Typically, the solution may include a suitable acid and a suitable peroxide mixture. In certain embodiments, the acid may include acetic acid, HCl, H2HO4, or any combination thereof; the peroxide may generally include any suitable peroxide, such as hydrogen peroxide. In certain embodiments, the acid and peroxide are dissolved / mixed in a suitable solvent to form the acid and peroxide solution. The solution can form a solution. Typically, the solvent can include water, although other solvents or solvent combinations (e.g., a mixture of water and ethanol) are also contemplated. The concentrations of the acid and peroxide in the solution can be adjusted to suit the particular application of interest. Typically, the solution can include an acid concentration of about 0.1 to 15 M, or any concentration therebetween (which may be rounded to the nearest 0.1), or any subrange between any two of these concentrations; and a peroxide concentration of about 0.1 to 10 M, or any concentration therebetween (which may be rounded to the nearest 0.1), or any subrange between any two of these concentrations. In certain embodiments, the maceration solution may comprise an acid to peroxide ratio of about 3:1 (e.g., a solution containing 13.05 M acetic acid and 2.45 M hydrogen peroxide) to about 1:3 (e.g., a solution containing 4.35 M acetic acid and 7.35 M hydrogen peroxide), or any ratio value therebetween (which may be rounded to the nearest 0.1), or any subrange spanning any two of these ratios. By way of example, in certain embodiments, the acid and peroxide solution may comprise acetic acid and hydrogen peroxide. In certain embodiments, it may comprise a solution of glacial acetic acid and 30% hydrogen peroxide in a 1:1 (by volume) ratio; or a solution of glacial acetic acid and 30% hydrogen peroxide in a 1:1 (as above) ratio diluted 50% in water. As will be appreciated, the acid and peroxide solutions, as well as the processing conditions (i.e., heating, agitation), may be adjusted, as desired, to suit the particular application, the desired structure to be extracted, the plant or fungal tissue being used, etc.

[0209] In certain embodiments, the acid may comprise an acid selected from the group consisting of acetic acid, boric acid, carbonic acid, hydrochloric acid, citric acid, hydrofluoric acid, nitric acid, oxalic acid, phosphoric acid, sulfuric acid, boron trifluoride, oxalic acid, malonic acid, succinic acid, and malic acid. Additional examples may include dicarboxylic acids, such as linear saturated dicarboxylic acids, branched dicarboxylic acids, unsaturated dicarboxylic acids, substituted dicarboxylic acids, and aromatic dicarboxylic acids. Depending on the intended use of the acid and / or product, neutralization and / or washing may be performed, for example, to remove residual acid and other reagents and prevent undesired contamination.

[0210] In certain embodiments, the peroxide may comprise a peroxide selected from the group consisting of hydrogen peroxide, peracids such as peracetic acid, metal peroxides (e.g., lithium peroxide, barium peroxide), and organic peroxides (e.g., dibenzoyl peroxide, benzoyl peroxide, methyl ethyl ketone peroxide). Depending on the intended use of the peroxide and / or product, neutralization and / or washing may be performed, for example, to remove residual peroxide and other reagents to prevent undesired contamination.

[0211] In certain embodiments, the extracting step can include heating the plant or fungal tissue in a salt solution, a base solution, an acid solution, or an acid and peroxide solution. In certain embodiments, heating can include heating to or near boiling, such as heating to a temperature of about 80-100°C, or any temperature value therebetween (which may be rounded to the nearest 0.1), or any subrange spanning any two of these temperatures. As will be appreciated, the processing temperature can be adjusted to suit, for example, the particular application, the solution used (e.g., the processing temperature may be lowered for harsher processing solutions or increased for milder processing solutions), the processing time, the desired structure to be extracted, and the plant or fungal tissue used. In certain embodiments, the processing temperature can vary over the course of the process, for example, starting at a higher temperature and ending at a lower temperature, or vice versa.

[0212] In certain embodiments, treatment can be carried out for at least about 1 minute, at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, or longer, or any time value or subrange therebetween. As will be appreciated, treatment times can vary depending on, for example, the particular application, the solution used (e.g., treatment times may be shortened for harsher treatment solutions or extended for milder treatment solutions), the treatment temperature, the amount of water extracted, and the amount of water present in the solution. It can be tailored to suit the desired structure and plant or fungal tissue used.

[0213] In certain embodiments, example solvents may include solvents selected from the group consisting of polar protic (e.g., propanol, ethanol, methanol, ammonia, acetic acid, water), polar aprotic (e.g., acetone, dimethylformamide, dimethylsulfoxide), organic, inorganic, and amphipathic solvents, and colloids. Depending on the intended use of the solvent and / or product, neutralization and / or washing may be performed, for example, to remove residual solvent and other reagents and prevent undesired contamination.

[0214] As will be appreciated, treatment conditions can be tailored for the particular structure(s) of interest to be extracted. In the examples below, individual structural cells were extracted from apples and pears, and substantially intact vascular bundles were extracted from celery and asparagus. For example, for vascular bundles, treatment conditions may be selected to be relatively less harsh (and / or treatment may be stopped earlier) to prevent destruction of the vascular bundles, while for individual structural cells, relatively more harsh treatment may be desirable.

[0215] Additionally, a detailed study and description of liquid-based extraction / maceration techniques for extracting one or more structures from plant or fungal tissue can be found in PCT International Patent Application No. PCT / CA2020 / 050654, entitled "High Density Microchannels," which is incorporated herein by reference in its entirety.

[0216] In certain embodiments, the extracting step may further comprise mechanically agitating, e.g., stirring, the plant or fungal tissue in a salt solution, a base solution, an acid solution, or an acid and peroxide solution.

[0217] As will be appreciated, in certain embodiments, the one or more structures derived from plant or fungal tissue extracted during the extracting step may include any suitable plant- or fungal-derived three-dimensional structure, such as one or more structures derived from a thixosa or pulp cellulose structure, microchannels such as xylem and / or phloem, or any combination thereof.

[0218] In certain embodiments, a thixosa or pulp structure (or any other plant or fungal structure / component of interest) may comprise an extended 3D structure (which may be composed of any one or more of cellulose, hemicellulose, pectin, lignin, etc.; typically, the extended 3D structure may comprise a lignocellulosic structure / material) (which may, in certain embodiments, comprise a plurality of structural cells), a single structural cell or a group of structural cells derived from the extended 3D structure, or any combination thereof. In certain embodiments, a single structural cell or a group of structural cells derived from the extended 3D structure may comprise an isolated structural cell or a small group of clustered structural cells, where the structural cells have a substantially intact three-dimensional structure that typically resembles a hollow cell or pocket, such as the hollow cell or pocket shown in Figures 27-28. As will be appreciated, such structures may typically comprise lignocellulosic materials, such as cellulose- and / or lignin-based structures. It will be understood that in certain embodiments, such structures may comprise other building blocks, such as chitin and / or pectin.

[0219] In certain embodiments of any one or more of the methods described above, the extracting step may further comprise centrifuging. In certain embodiments, the centrifugation may separate the extended 3D structure and / or microchannels, such as xylem and / or phloem, from single structural cells or groups of structural cells derived from the extended 3D structure. In certain embodiments, the extracting step may further comprise centrifuging to separate the extended 3D structure from single structural cells or groups of structural cells derived from the extended 3D structure. By way of example, in certain embodiments, the centrifugation may separate single structural cells or groups of structural cells derived from the extended 3D structure. In yet another embodiment, the one or more structures derived from the plant or fungal tissue may comprise a single structural cell or group of structural cells derived from the extended 3D structure that are localized in the upper band or pellet.

[0220] In another embodiment of any of the one or more methods above, the extracting step may further comprise washing the one or more structures from the plant or fungal tissue.

[0221] In yet another embodiment of any of the one or more methods above, preparing a scaffold biomaterial from the one or more extracted structures may include mixing, agitating, or physically manipulating the extracted structures to remove any residual undesired plant tissue material; washing the one or more extracted structures derived from plant or fungal tissue; or both.

[0222] In yet another embodiment of any of the one or more methods above, the plant or fungal tissue may comprise decellularized plant or fungal tissue, from which cellular material and nucleic acids have been removed. In another embodiment of any of the one or more methods above, the method may further comprise decellularizing the plant or fungal tissue prior to the extracting step. In yet another embodiment of any of the one or more methods above, the method may further comprise decellularizing one or more structures extracted from the plant or fungal tissue. Decellularization methods have been described in detail herein.

[0223] In certain embodiments, conditions for the extraction step can be selected so that no decellularization, or minimal, partial, or extensive decellularization occurs during extraction. In some applications, no, minimal, or partial decellularization may be sufficient or desirable. In other applications, particularly where complete removal of remaining plant / fungal nucleic acids, proteins, etc. is desired, careful decellularization processes (e.g., decellularization processes described herein, including decellularization processes utilizing SDS) can be performed either before or after the extraction step.

[0224] In yet another embodiment of any of the one or more methods above, the method may further comprise decellularizing the plant or fungal tissue, or decellularizing one or more structures derived from the plant or fungal tissue, or both. In another embodiment of any of the one or more methods above, preparing a scaffold biomaterial from the one or more extracted structures may comprise decellularizing the one or more extracted structures.

[0225] In yet another embodiment of any of the one or more methods above, preparing a scaffold biomaterial from the one or more extracted structures may include grinding the one or more extracted structures. In certain embodiments of any of the one or more methods above, the grinding may be performed on the plant or fungal tissue before extraction and / or on the one or more extracted structures after extraction.

[0226] In yet another embodiment of any of the one or more methods above, preparing a scaffold biomaterial from the one or more extracted structures may include incorporating the one or more extracted structures into a matrix; or gluing or attaching the extracted structures and / or scaffold biomaterial together; drying or freeze-drying the one or more extracted structures or scaffold biomaterial; seeding or culturing cells, such as animal cells, on the extracted structures and / or scaffold biomaterial; joining two or more extracted structures and / or scaffold biomaterials by layering, stacking, or other complementary / interlocking configurations; or any combination thereof.

[0227] In certain embodiments, the matrix may include a hydrogel, forming a composite hydrogel.

[0228] In certain embodiments, the matrix or adhesive may comprise, for example, an alginate matrix, a gelatin matrix, a collagen matrix, or a hyaluronic acid matrix. In certain embodiments, the adhesive or matrix may comprise an alginate matrix.

[0229] In another embodiment, provided herein is a scaffold biomaterial produced by any one or more of the methods described above.

[0230] In yet another embodiment, provided herein is a scaffold biomaterial comprising one or more structures extracted from plant or fungal tissue. Examples of such extracted structures may include those described in detail above.

[0231] In yet another embodiment of the scaffold biomaterial, the one or more structures are extracted from the plant or fungal tissue by liquid-based extraction, as described in detail above. In certain embodiments, the one or more structures can be extracted from the plant or fungal tissue by treatment with a salt solution, a base solution, an acid solution, or an acid and peroxide solution. Examples of such maceration solutions are detailed in the preceding sections above.

[0232] In yet another embodiment, the one or more structures derived from plant or fungal tissue can include three-dimensional structures such as one or more structures derived from a thixotropic or pulp cellulose structure, microchannels such as xylem and / or phloem, or any combination thereof. In certain embodiments, the thixotropic or pulp cellulose structure can include an extended 3D structure, a single structural cell or a group of structural cells derived from an extended 3D structure, or any combination thereof. Such structures are described in detail in the preceding sections above.

[0233] In yet another embodiment of any of the one or more scaffold biomaterials described above, the one or more structures may comprise a single structural cell or a group of structural cells derived from an expanded 3D structure by centrifugation, as previously described.

[0234] In yet another embodiment of any of the one or more scaffold biomaterials described above, the scaffold biomaterial may be a decellularized scaffold biomaterial that is devoid of cellular material and nucleic acids of plant or fungal tissue. Techniques for decellularization have been described in detail herein above.

[0235] In another embodiment of any of the one or more scaffold biomaterials described above, the one or more structures may be milled to reduce particle size, as described above.

[0236] In yet other embodiments of any of the one or more scaffold biomaterials described above, the scaffold biomaterial may comprise a matrix having one or more incorporated structures; a product formed by gluing or attaching extracted structures and / or scaffold biomaterials together; a product formed by drying or freeze-drying one or more extracted structures or scaffold biomaterials; a product formed by seeding or culturing cells, such as animal cells, on or in the extracted structures and / or scaffold biomaterials; a product formed by joining two or more extracted structures and / or scaffold biomaterials by layering, stacking, or other complementary / interlocking configurations; or any combination thereof.

[0237] In yet another embodiment of any one or more of the above scaffold biomaterials, the matrix may comprise a hydrogel. In another embodiment of any one or more of the above scaffold biomaterials, the matrix or adhesive may comprise an alginate or another suitable matrix or adhesive described herein or known to those of skill in the art having regard to the teachings herein.

[0238] In another embodiment, provided herein is a food product comprising a scaffold biomaterial, wherein the scaffold biomaterial comprises one or more three-dimensional structures derived from or extracted from plant or fungal tissue.

[0239] As will be appreciated, a wide variety of scaffold biomaterials (also referred to herein as composite scaffold biomaterials) are described in detail herein, many of which may be suitable for such food products. Those skilled in the art, having regard to the teachings herein, will recognize that scaffold biomaterials may be selected from the scaffold biomaterials described herein to tailor characteristics such as structure and / or texture to suit the particular food application of interest.

[0240] In certain embodiments, the food product may comprise a scaffold biomaterial, wherein the scaffold biomaterial comprises one or more three-dimensional structures derived from or extracted from plant or fungal tissue, and the one or more three-dimensional structures may include any suitable plant- or fungal-derived three-dimensional structures, such as one or more structures derived from thixosa or pulp structures, microchannels such as xylem and / or phloem, or any combination thereof.

[0241] In certain embodiments, a thixosa or pulp structure (or any other suitable plant or fungal structure / component) may comprise an extended 3D structure (which may comprise any one or more of cellulose, hemicellulose, pectin, lignin, etc.; typically, the extended 3D structure may comprise a lignocellulosic structure) (which may, in certain embodiments, comprise an extended network comprising a plurality of structural cells), a single structural cell or a group of structural cells derived from the extended 3D structure, or any combination thereof. In certain embodiments, a single structural cell or a group of structural cells derived from the extended 3D structure may comprise an isolated structural cell or a small group of clustered structural cells, where the structural cells have a substantially intact three-dimensional structure that typically resembles a hollow cell or pocket, such as the hollow cells or pockets shown in Figures 27-28. As will be appreciated, such structures may typically comprise lignocellulosic materials, such as cellulose- and / or lignin-based structures. It will be understood that in certain embodiments, such structures may comprise other building blocks, such as, for example, chitin and / or pectin.

[0242] In yet another embodiment of any of the one or more food products described above, the one or more structures derived from the thixosa or pulp structure may comprise an extended 3D structure, a single structural cell or group of structural cells derived from an extended 3D structure, or any combination thereof, as already described in detail above.

[0243] In another embodiment of any of the one or more food products described above, the scaffold biomaterial (or one or more three-dimensional structures thereof) may be decellularized, such that the one or more three-dimensional structures are devoid of cellular material and nucleic acids of plant or fungal tissue. Techniques for decellularization have already been described in detail above.

[0244] In yet other embodiments of any of the one or more food products described above, the scaffold biomaterial may comprise any of one or more scaffold biomaterials described herein selected to suit a particular or desired application.

[0245] In yet another embodiment of any of the one or more food products, the scaffold biomaterial comprises one or more A matrix having two or more three-dimensional structures incorporated therein; a product formed by gluing or attaching one or more three-dimensional structures and / or scaffold biomaterials together; a product formed by drying or freeze-drying one or more three-dimensional structures or scaffold biomaterials; a product formed by seeding or culturing cells, such as animal cells, on or in a three-dimensional structure and / or scaffold biomaterial; a product formed by joining two or more extracted structures and / or scaffold biomaterials by layering, stacking, or other complementary / interlocking configurations; or any combination thereof.

[0246] In another embodiment of any of the one or more food products described above, the scaffold biomaterial may comprise a matrix in which one or more three-dimensional structures are embedded; a product formed by adhering or attaching one or more three-dimensional structures and / or scaffold biomaterials together; or any combination thereof.

[0247] In yet other embodiments of any of the one or more food products described above, the matrix or adhesive may comprise an alginate or another suitable matrix or adhesive described herein or known to those of skill in the art having regard to the teachings herein.

[0248] In yet another embodiment of any of the one or more food products, the scaffold biomaterial may comprise two or more different three-dimensional structures derived from or extracted from the same or different plant or fungal tissue. In another embodiment, the food product may comprise two or more different scaffold biomaterials having different structural or physical properties. In yet another embodiment, the two or more different three-dimensional structures and / or the two or more different scaffold biomaterials may be selected to provide the food product with a desired stiffness, mouthfeel, and / or texture. Considerations for scaffold biomaterial selection and combinations, and the advantages that may be gained therefrom, particularly with regard to the tunability of structural properties, are described in detail throughout this specification.

[0249] In yet another embodiment of any of the one or more foods described above, one or more cells are seeded or cultured on the scaffold biomaterial and / or three-dimensional structure. In certain embodiments, the one or more cells may comprise animal cells, such as cells of livestock animals, fish, insects, or other animals of interest. In certain embodiments, the animal cells may comprise, for example, bovine, porcine, fish, elk, chicken, turkey, or avian cells. In certain embodiments, the one or more cells may comprise muscle cells, adipocytes, connective tissue cells (i.e., fibroblasts), cartilage, bone, epithelial, or endothelial cells, or any combination thereof.

[0250] In yet another embodiment of any of the one or more food products described above, the food product may comprise at least a first layer of a scaffold biomaterial and a second layer of a scaffold biomaterial, one of the layers optionally seeded with muscle cells and the other layer optionally seeded with adipocytes.

[0251] In yet another embodiment of any of the one or more food products described above, the one or more cells may be aligned along one or more channels or grooves of the scaffold biomaterial and / or three-dimensional structure. In certain embodiments, the one or more cells may comprise muscle cells. Cell alignment is described in further detail in Example 6 below. In certain embodiments, such cell alignment may be used, for example, to more closely mimic meat tissue characteristics.

[0252] In yet another embodiment of any of the one or more food products above, the food product may comprise one or more three-dimensional structures derived from the thixotropic or pulp structure, and one or more microchannel structures. In yet another embodiment of any of the one or more food products above, the three-dimensional structures derived from the thixotropic or pulp structure comprise an extended 3D structure, a single structural cell or a group of structural cells derived from the extended 3D structure, or any combination thereof. In other embodiments of any of the food products, the microchannel structures may comprise xylem and / or phloem. In yet other embodiments of any of one or more of the food products described above, the xylem and / or phloem may be provided in the form of vascular bundles. Such three-dimensional structures have already been described in detail above.

[0253] In yet another embodiment of any of the one or more food products described above, the scaffold biomaterial may comprise any of the composite scaffold biomaterials described herein selected to suit the particular application of interest.

[0254] As will be understood, a food product may refer to any suitable edible product, or a precursor or starting material thereof. Food products may be provided in a variety of different forms. In certain embodiments, a food product may include a scaffold biomaterial and may be in a ready-to-use, ready-to-cook, cooked, smoked, smoked, dried / lyophilized, frozen, vacuum-sealed, or other suitable form or format, with or without one or more additional ingredients, seasonings, dyes, preservatives, or other food-related agents. Food products may be decellularized to remove plant and / or fungal cells, or may be immediately decellularizable. Food products may be pre-seeded with a cell(s) of interest, may be immediately seeded with a cell(s) of interest, or may be cell-free. Those skilled in the art, with the teachings herein in mind, will recognize a variety of different forms and formats for the food products described herein, and will recognize that the food products may be adapted as desired to suit a particular application of interest.

[0255] In certain embodiments, the scaffold biomaterials and / or foods described herein may be for use as a base scaffold that may be used, for example, with one or more other materials, drugs, cells, fillers, etc. to impart one or more additional characteristics, such as color, taste, and / or mouthfeel or texture characteristics.

[0256] In certain embodiments, the foods described herein may be for use as or in the preparation of meat substitutes / meat mimics. While the foods described herein may have a wide variety of applications in the food industry, meat substitutes / meat mimics are particularly interesting given the environmental issues often associated with livestock. The foods described herein may be considered to be close to plant-based meat substitutes / meat mimics; however, the foods described herein may also be fully compatible with the growth / cultivation of animal cells (e.g., muscle cells and / or fat cells from livestock animals) within a scaffold, which in certain examples can be used to improve the mouthfeel, texture, structure, appearance, and / or eating experience of the food. Therefore, in some embodiments, the foods may further comprise animal cells, and thus, in such embodiments, may not be entirely plant-based. In embodiments in which animal cells are used, such foods may be considered to be closer to, for example, artificially cultured foods.

[0257] In another embodiment, there is provided a method for preparing a food product, comprising: providing plant or fungal tissue; deriving or extracting one or more three-dimensional structures from plant or fungal tissue; and Producing a scaffold biomaterial by providing a scaffold biomaterial from one or more derived or extracted three-dimensional structures; and Preparing food from scaffold biomaterials Provided herein is a method comprising:

[0258] In another embodiment of the above method, producing a scaffold biomaterial may comprise performing any one or more of the methods described above. Indeed, methods for producing a scaffold biomaterial have already been described in detail above.

[0259] In yet another embodiment of any of the one or more methods above, the scaffold biomaterial is ingested. The steps of preparing the article may include any one or more of the following: incorporating one or more derived or extracted three-dimensional structures into a matrix; or gluing or attaching the derived or extracted three-dimensional structures and / or scaffold biomaterials together; drying or freeze-drying one or more derived or extracted three-dimensional structures or scaffold biomaterials; seeding or culturing cells on the derived or extracted three-dimensional structures and / or scaffold biomaterials; joining two or more derived or extracted three-dimensional structures and / or scaffold biomaterials by layering, stacking, or other complementary / interlocking configurations; or any combination thereof.

[0260] In yet another embodiment of any of the one or more methods above, preparing a food product from the scaffold biomaterial may include seeding or culturing cells on the derived or extracted three-dimensional structure and / or scaffold biomaterial.

[0261] In another embodiment of any of the above one or more methods, the one or more cells can comprise animal cells, such as cells of a livestock animal, fish, insect, or other animal of interest. In certain embodiments, the cells can comprise, for example, bovine, porcine, fish, elk, chicken, turkey, or avian cells. In certain embodiments, the one or more cells can comprise muscle cells, adipocytes, connective tissue cells (i.e., fibroblasts), cartilage, bone, epithelial, or endothelial cells, or any combination thereof.

[0262] In yet another embodiment of any of the one or more methods above, the method comprises producing at least a first layer of scaffold biomaterial and a second layer of scaffold biomaterial, one of the layers optionally seeded with muscle cells and the other layer optionally seeded with adipocytes.

[0263] In yet another embodiment of any of one or more of the methods above, the method may include aligning one or more cells along one or more channels or grooves of a scaffold biomaterial and / or three-dimensional structure. Techniques for cell alignment are described in detail above and in Example 6 below. In certain embodiments, the one or more cells may include muscle cells.

[0264] In yet another embodiment of any of the one or more methods above, the method may include producing two or more different scaffold biomaterials having different structural or physical properties; producing one or more scaffold biomaterials comprising two or more different three-dimensional structures derived from or extracted from the same or different plant or fungal tissue; or any combination thereof. In yet another embodiment, the two or more different three-dimensional structures and / or the two or more different scaffold biomaterials may be selected to provide a desired stiffness, mouthfeel, and / or texture to the food product, for example. [Example]

[0265] Biological tissues are complex structures composed of a collection of different cell types. Various cell types can interact to perform specialized functions in the body. The organization of cells and extracellular matrices is often directly related to function. Therefore, dysfunction of cells, tissues, and / or organs can result from both biochemical and physical defects, ranging from genetic disorders to physical injuries. The biochemical and physical environments within the body can differ within, at the interfaces between, and between different tissue types and organs. Therefore, it is difficult to recreate or approximate the natural in vivo environment of cells. Considerable research has been directed toward the production of biomimetic constructs. Various approaches have been proposed as potential means to recapture the natural environment, including synthetic biology, regenerative medicine, transplantation, templated, and scaffolding. However, recreating the cellular microenvironment is difficult. Because tissues are themselves complex composites, Simple materials typically cannot achieve the biochemical and physical complexity that mimics the natural environment, and therefore extensive research has been devoted to composite materials developed from one or more of the aforementioned approaches to producing biomaterials.

[0266] An attractive approach to biomaterials design is decellularization, in which damaged tissue is replaced with a scaffold made of ECM proteins that lack cells. This concept is based on the idea that damaged tissue can be replaced with a scaffold of the same tissue or organ, where healthy cells can repopulate and restore proper tissue or organ function. Typically, such organ decellularization approaches have several challenges, including donor tissue dependency, compatibility issues, and practical limitations. As an alternative, synthetic approaches offer several other biomaterial production methods, including 3D printing, casting, and electrospinning, that have been developed to create custom structures that can circumvent certain drawbacks of organ decellularization approaches. Ideally, the high complexity of structures obtained through organ decellularization or transplantation can be combined with the customizability and controllability associated with synthetic biomaterial approaches. Combining both techniques may, for example, enable the production of materials that more closely replicate healthy tissue. [Example]

[0267] Composite biomaterials assembled by gel casting In this example, different scaffold biomaterial subunits are combined by gel casting using a hydrogel to bond the subunits together. While it is contemplated that many different hydrogels may be used, this example primarily relates to gelatin, collagen, and agarose hydrogels. In certain embodiments, the hydrogel may comprise, for example, gelatin, collagen, agarose, hyaluronic acid, alginate, fibrin, fibronectin, agar, PEG, PVA, or any combination thereof. In certain embodiments, two or more scaffold biomaterial subunits may be used as a scaffold for a hydrogel to form around, and the hydrogel may have one or more characteristics tailored to the desired application. In certain embodiments, materials with different properties may be bonded together in this manner. For example, in certain embodiments, different regions of the material may have different topography, density, chemical functionality, mechanical properties, porosity, or any combination thereof.

[0268] In the following studies, to provide accuracy and reproducibility, CNC or biopsy punches were used. The scaffold biomaterial subunits were generated by cutting the scaffold biomaterial into the desired shape using a cutting tool. The scaffold biomaterial was then sliced ​​with a mandolin slicer. The desired shape was extracted from the bulk material by slicing. The thickness of the mandolin slices was used to set the z-thickness of the material. The material was then decellularized and sterilized as described in International Publication No. 2017 / 136950, entitled "Decellularized Cell Wall Structures from Plants and Fungus and Use Thereof as Scaffold Materials," which is incorporated herein by reference in its entirety. The scaffold biomaterial subunits were then ready for cell culture and / or implantation. The scaffold biomaterial subunits can be easily assembled into their final form by coating them with a hydrogel. It is contemplated that either temporary or permanent hydrogels can be used, which may, for example, provide time-dependent biochemical and / or physical anchorages. By way of example, in certain embodiments, it is contemplated that synthetic tubes may be generated by stacking subunits (e.g., ring-shaped subunits) on top of each other and coating the structure with a hydrogel such as agarose. In such instances, agarose is liquid at higher temperatures (i.e., above about 65°C) but remains solid at physiological temperatures, so that after application, the agarose can solidify around the stack of subunits to create a synthetic tube (e.g., a flexible tube-like structure).

[0269] We have developed 3D biocompatible scaffolds composed of decellularized plant tissue. These biomaterials can support cell proliferation, infiltration, and / or growth in vitro and in vivo. As described, we have developed composites of cellulose scaffolds and hydrogels, in which the presence of hydrogels can enable the temporary or permanent introduction of distinct biochemical and physical anchors. Cellulose scaffolds can be attractive substrate materials due to their high biocompatibility, natural abundance, simple production, low cost, and / or structural complexity. Plants have evolved to produce complex, specialized structures. Surprisingly, many of these structures are related to those found in the human body. By creating composite materials containing these plant structures, biomimetic materials for tissue engineering can be created. In certain embodiments, preparation of larger sizes can pose challenges due to the size of the source material. Thus, composite plant-based materials may be a solution to the size limitations imposed by nature. The use of multicomponent systems can expand composability by, for example, designing materials that retain the characteristics of the source material but have different chemical and / or physical environments. For example, composite materials can be engineered to have regions with different porosity and / or mechanics while maintaining the intricate features of the source plant structure. An example of a direct consequence of using materials with different mechanical properties is the phenomenon of stress shielding. Stress shielding can occur in the body and can be a significant issue for bone implants. Inadequate or excessive stress shielding can cause damage or degradation of surrounding healthy tissue. Furthermore, interfaces between different tissues often separate regions with fundamentally different microenvironments. Composite materials can be engineered to mimic the interface of different cell populations and environments.

[0270] In this example, the creation of cellulose-based, plant-derived composite scaffolds is investigated. The scaffolds were assembled by gel casting. Such gel-cast composites can provide an attractive approach to biomaterials design. This example demonstrates that complex biomaterials can be assembled from subunits, which can be held together by biocompatible hydrogels. Cells cultured on and / or within the resulting composite can be exposed to different structural, mechanical, and / or chemical environments. Cells can infiltrate the scaffold and proliferate in the gel connecting the subunits and across their interface. Furthermore, cell migration can be hindered, for example, by the presence of pre-existing cells on adjacent scaffolds or by using a less porous hydrogel. Composite materials in these studies demonstrated stress shielding, supported the growth of different cell types, and created interfaces between distinct tissues.

[0271] Therefore, the results indicate that hydrogels can hold the overall structure together and provide structural integrity. While many different hydrogels can be used, this illustrative example focuses on gelatin, collagen, and / or agarose. First, the starting material was cut into its desired shape. Using a CNC or biopsy punch to perform the cutting allowed for greater accuracy and reproducibility. The desired shape was then removed from the bulk material by slicing with a mandolin slicer. The thickness of the mandolin slice was used to set the z-thickness of the material.

[0272] The material was then decellularized and sterilized according to established protocols. See WO 2017 / 136950 entitled "Materials for Implantation," which is incorporated herein by reference in its entirety. The materials were then ready for cell culture / implantation and easily assembled into their final form by coating with a hydrogel. Transient or permanent hydrogels can be used, for example, to provide time-dependent biochemical and physical footholds. For example, in certain embodiments, synthetic tubes can be created by stacking rings of scaffold biomaterial on top of each other and coating with a hydrogel such as agarose. Agarose can be used at high temperatures ( The agarose can be liquid at temperatures above 65°C (above 65°C) but remain solid at physiologically relevant temperatures. The agarose can solidify around the stack of rings to create a tube or flexible tube-like structure.

[0273] Materials and Methods Scaffold Production: Using a mandolin slicer, McIntosh red apples (Canada Fancy) were sliced ​​into thin sections of 1.2 mm, measured with a vernier caliper. Then, 5 mm diameter disks with a thickness of 1.2 mm were cut from the thipial tissue of the slices. A 2 mm disk was removed from the center of the 5 mm disk using a Carbide 3D Shapeoko 3 CNC machine and Chilipeppr jpadie software. A 0.8 mm diameter drill bit was used to cut the scaffold at a 180° angle at a speed of 1 mm / s. Subunits were designed using Inkscape and converted to G-code using Jscut. Thus, a 2 mm inner diameter, 5 mm outer diameter, and 1 mm thick disk were obtained. Macroscopic rings of 0.2 mm were obtained. The samples were transferred to a 0.1% SDS solution and decellularized for 48 hours with shaking at 180 RPM. After decellularization, the samples were washed three times with dH2O. The rings were then incubated in 100 mM CaCl2 for 24 hours at room temperature to remove any detergent residues. The samples were washed three times with dH2O to remove salt residues and then sterilized by incubation with 70% ethanol. After removing the ethanol, three washes with dH2O were performed to obtain sterile, contaminant-free rings. Alternatively, the samples may be autoclaved for sterilization.

[0274] Construction of artificial tubes: Decellularized apple-derived cellulose scaffold rings were assembled into tubes by stacking them through a needle and coating them with 1.5% agarose. The tubes were allowed to cool for 30 minutes.

[0275] In vivo biocompatibility testing: Tubes containing the two subunits were implanted subcutaneously in rats and later removed to assess biocompatibility and integrity.

[0276] Chemically crosslinked hydrogel adhesive: 5 x 5 x 1.2 mm pieces of decellularized apple were cut and prepared according to established protocols (Hickey, RJ, Modulevsky, DJ, Cuerrier, CM, & Pelling, AE (2018). Customizing the shape and microenvironment biochemistry of biocompatible macroscopic plant-derived cellulose scaffolds. ACS Biomaterials Science & Engineering, doi:10.1021 / acsbiomaterials.8b00178, Customizing the Shape and Microenvironment Biochemistry of Biocompatible Macroscopic Plant-Derived Cellulose Scaffolds Ryan J. Hickey, Daniel J. ACS Biomaterials Science & Engineering 2018 4 (11), 3726-3736, and International Publication No. 2017 / 1369 (See pamphlet No. 50.) The subunits were assembled using gelatin cross-linked with glutaraldehyde adhesive. The glutaraldehyde was reduced with sodium borohydride. Cells were seeded onto the constructs and viability was assessed.

[0277] Results and Discussion: Composite production - We successfully established a process to combine subunits with a hydrogel. The hydrogel used in this example was 1.5% agarose. Apple-sourced rings were assembled by stacking them and coating them with hydrogel (1.5% agarose). The resulting structure was a tube. Figure 8 shows, from left to right, images of the ring subunit, a model of the tube-like structure, the tube-like structure at various stages of assembly, and the final ring-structured artificial tube product.

[0278] Fluid flow test - Congo red solution was passed through the tube to demonstrate the ability of the tube-like structures to flow / transport fluid. Figure 9 shows the results of a fluid transport test, in which Congo red staining solution was successfully passed through the tube depicted in Figure 8.

[0279] Biocompatible—The vessels / units remained intact and biocompatible in vivo, and cells grew on the scaffold. Figure 25 shows an excised composite vessel. The composite vessel was formed by casting 1.5% agarose around two laminated decellularized apple-derived cellulose rings. The specimens were implanted subcutaneously in rats for four weeks. The composite vessel was then excised (removed), and it was observed that the vessel remained intact and showed no signs of infection or encapsulation / rejection.

[0280] Biomaterial Adhesive - In another exemplary construction, subunits were glued together using gelatin cross-linked with glutaraldehyde gel. Intact units supported cell growth in vitro. Figure 10 shows images of adhesive composite biomaterials made by gluing with glutaraldehyde cross-linked gelatin. The biomaterials were glued with glutaraldehyde cross-linked gelatin and reduced with sodium borohydride. (A) (A) shows two subunits of an apple-derived cellulose scaffold glued together, and (B) shows that the adhesive construct supported cell proliferation of GFP 3T3 cells.

[0281] Modifications such as adhesion, gel casting, chemical functionalization, loading with one or more agents (i.e., drugs, signaling molecules, growth factors, metabolites, etc.) can further extend the functionality of the materials and devices described herein.

[0282] Gel-cast composites can exploit the natural complexity of individual subunit scaffold materials while enabling the design of structures and features not found in nature. Furthermore, the approaches described herein can enable more complex physical and / or mechanical properties (i.e., stress shielding and site-specific elastic modulus, channels, pores, etc.). Such composites can be used to combine different cell types in different regions. This approach can provide an alternative or complement to interlocking composites, particularly in applications where physical connections are undesirable, inapplicable, or insufficient. While agarose is an example of a hydrogel that can be used, it will be understood that many others are contemplated, including materials comprising one or more of gelatin, collagen, and hyaluronic acid.

[0283] This example approach may provide the ability for cellulose-, hemicellulose-, chitin-, chitosan-, pectin-, lignin-, and / or lignan-based scaffold biomaterials to have tunable biochemical, biophysical, and mechanical properties. Furthermore, the composite hydrogels may incorporate time-dependent / independent release of drugs, signaling molecules, growth factors, metabolites, and / or ECM proteins and / or components (i.e., ions, sugars, proteoglycans, metabolites, etc.), or any combination thereof. This may also enable, for example, the creation of larger macroscopic objects with varying degrees of flexibility and connectivity.

[0284] Figure 1 shows an example of a contemplated application of the composite materials described herein, which may be generated in the form of a blood vessel. Blood vessels are complex structures with various layers of tissue (top left and center images). In the example provided herein, ring structures are prepared from apple-derived cellulose (bottom right and left images), which may be layered and coated with a hydrogel such as 1.5% agarose to produce a tube (see also top and bottom center images). This tube may be surrounded by different layers or membranes containing different cell types. The sample material for the membrane may be, for example, decellularized orange mesocarp membrane (see top right image). Combinations of multiple elements can be used to more closely replicate complex structures such as blood vessels, where different tissues and cell types are radially organized. The center image in this figure is taken from Blausen.com staff (2014), Medical Gallery of BlausenMedical 2014, Wiki Journal of Medicine 1(2).

[0285] It is contemplated that by assembling subunits in this manner, using hydrogels to provide structural integrity and hold the structure together, a wide variety of larger and / or more diverse structures may be generated. This approach may also allow for significant customization and control of structural properties. Through the selection of scaffold biomaterials (which may be all the same or a mixture of two or more different scaffold biomaterials, as desired), the selection of subunit type, and / or the selection of hydrogel, a high level of control and tunability over the resulting structure may be achieved.

[0286] In certain embodiments, it is contemplated that the present approach may provide structures and / or features not found in nature while leveraging the natural complexity of the scaffold biomaterial source within individual subunits. Similarly, such approaches may enable the control and / or creation of complex physical and mechanical properties (i.e., stress shielding and site-specific elastic modulus, channels, pores, etc., as needed). It is contemplated that such approaches may enable the combination of different cell types in different regions of the resulting structure. Furthermore, by using hydrogels or other gels, it is contemplated that such approaches may enable the combination of subunits even in applications where simple physical connections are undesirable, inapplicable, and / or insufficient. In certain embodiments, it is contemplated that such approaches may be used to provide structures with a tight seal, for example, for fluid flow and / or for creating semipermeable interfaces. In certain embodiments, it is contemplated that the hydrogel may be or include agarose, gelatin, collagen, or hyaluronic acid hydrogel, or any combination thereof.

[0287] In certain embodiments, the scaffold biomaterials described herein may be functionalized and / or loaded with one or more agents depending on the desired application. Such agents may include, for example, any one or more of therapeutic agents, signaling molecules, growth factors, metabolites, ECM proteins or components, or any combination thereof. In certain embodiments, the agents may provide time-dependent or non-time-dependent release of such agents. In certain embodiments, one or more agents may be covalently attached to cellulose-, hemicellulose-, chitin-, chitosan-, pectin-, lignin-, and / or lignan-based scaffold biomaterials, either directly or indirectly via a linker. In certain embodiments, one or more acylation and alkylation-type reactions or other suitable reactions using sulfur, nitrogen, boron, and / or halide compounds (i.e., thiols, imides, amines, amides, borohydrides, borohydrates, and halides) may be used.

[0288] In certain embodiments, it is contemplated that the structures described herein may be designed such that one or more sections of the structure are more or less hydrophobic or hydrophilic relative to one or more other sections of the structure.

[0289] In certain embodiments, it is contemplated that the structures described herein may be designed so that site- and / or cell type-specific attractants and / or repressants are incorporated into appropriate regions of the structure to suit a particular application.

[0290] In certain embodiments, the scaffold biomaterial subunits described herein may be customizable in terms of shape and / or structure, hi certain embodiments, larger and / or more complex structures that may have variable and / or controllable flexibility and / or articulation can be prepared from such subunits.

[0291] Although this example focuses on the use of hydrogels, other methods for holding the subunits together in a structure may also be used. It is also contemplated that any of adhesives, coatings, gels, and / or pastes, or any combination thereof, may be used to achieve this.

[0292] In certain embodiments, the scaffold biomaterials described herein may be cellulose-based, hemicellulose-based, chitin-based, chitosan-based, pectin-based, lignin-based, lignan-based, or any combination thereof. In certain embodiments, the composite structures described herein may be tunable with respect to their biochemical, biophysical, and / or mechanical properties.

[0293] While this example primarily relates to gel casting techniques, in certain embodiments, instead of casting, the scaffold biomaterial is immersed in a hydrogel or other gel, and then a subtractive production method is used to obtain the final product. It is contemplated that this may be possible.

[0294] In this example, gels are used to combine two or more separate scaffold biomaterial subunits to provide composite biomaterial structures with larger and / or more complex architectures with tunable properties.

[0295] In certain embodiments, it is contemplated that structures described herein comprising two or more scaffold biomaterial subunits may overcome and / or ameliorate certain problems associated with larger sizes and correspondingly longer decellularization times, and / or with diffusion in larger constructs. In certain embodiments, structures described herein may allow for the design of certain structures and features not found in nature while leveraging the natural complexity of the source of scaffold material in the individual subunits. In certain embodiments, structures described herein may allow for increased complexity of physical and / or mechanical properties (i.e., stress shielding and site-specific modulus, channels, pores, etc.). In certain embodiments, structures described herein may allow for permeability modification through the use of different adhesives and / or coatings (e.g., tubes, semipermeable membranes, and / or sealants for joints). In certain embodiments, structures described herein may provide tunable mechanical properties and / or joints (i.e., Young's modulus can be varied) based on the adhesive or coating used. In certain embodiments, the structures described herein may be articulated structures (i.e., ring-stacked vessels) that can provide varying degrees of flexibility and mobility as needed. In certain embodiments, appropriate drugs, signaling molecules, growth factors, metabolites, and / or ECM proteins and / or components may be added to the structures described herein to provide a desired response that may be systemic or cell / tissue type specific, and / or positive or inhibitory as needed for a particular application. In certain embodiments, the scaffolds described herein may be loaded or functionalized with drugs and used to administer drugs, thereby providing at least some degree of site-specific drug delivery, which, in certain embodiments, can reduce drug dosage and / or increase efficacy.

[0296] In certain embodiments, the structures described herein can provide time-dependent and / or non-time-dependent release of one or more drugs, which can be achieved by loading the drugs into hydrogels or other gels with various release characteristics used in the scaffold biomaterial subunits and / or by covalently attaching the drugs to the scaffold biomaterial by appropriate chemical functionalization methods. In certain embodiments, the structures described herein can be drug-containing tubes that can be loaded into the tubes covalently linked via linker molecules such as succinic acid (i.e., in certain embodiments, a transient or permanent anchorage can be added to induce a desired outcome, e.g., promoting endothelial growth). Agents that promote the growth of the blood may be added, for example, to reduce clotting and / or inflammation, etc. (Drugs may be added). Such conduits may be targeted to site-specific locations and may have time-specific release properties. In certain embodiments, such chemical modifications may allow for the reduction or resolution of steric hindrance issues and / or may allow for increased complexity of site-specific modifications. In certain embodiments, linker molecules may be used to mediate connections between drugs, signaling molecules, growth factors, metabolites, ECM proteins and components, etc., and conduits containing such compounds, or any combination thereof.

[0297] Steric hindrance can present a problem in certain situations. For example, in the absence of any other biochemical or biophysical modifications or signaling, different cell attachment densities can lead to different cellular responses. In certain embodiments of the structures described herein, the degree of substitution can now be tailored to suit, for example, a particular function.

[0298] In certain embodiments, the structures described herein may be for use in complex tissue engineering, as biomaterial implants for tissue repair / regeneration, for drug delivery, for incorporation of growth factors, or any combination thereof.

[0299] In certain embodiments, the structures described herein may be for use in non-therapeutic applications, such as articulated components for synthetic biorobotics and / or electrical circuit integration.

[0300] In certain embodiments, the materials described herein may be suitable for 3D printing of biomaterials in gel and / or paste form. For example, if carboxymethyl and hydroxylethyl groups are present, the gel or paste can be crosslinked with chemical crosslinkers (i.e., citric acid, glutaraldehyde, etc.) and temperature. Dissolution of the biomaterial with a polar solvent (i.e., dimethylacetamide / LiCl) can be followed by reconstitution (by casting, printing, or molding) by solvent evaporation, dilution, or leeching. Multiple applications are contemplated, such as 3D printing, injectable hydrogels, molds, and adhesives. [Example]

[0301] Composite occlusal biomaterials In this example, different scaffold biomaterial subunits were combined using shape-based interlocking. The shape of the subunits was used to hold the assembly structure together. While a wide variety of interlocking shapes are contemplated, this example primarily used peg-and-hole type interlocking, similar to Lego blocks. Interlocking can achieve customization of shape and / or structure, providing larger macrostructures with varying degrees of flexibility and articulation, as desired for a particular application.

[0302] We have developed 3D biocompatible scaffolds composed of decellularized plant tissue. These biomaterials can support cell proliferation, infiltration, and / or growth in vitro and in vivo. As described, we have developed composites of cellulose scaffolds and hydrogels, in which the presence of hydrogels can allow for the transient or permanent introduction of distinct biochemical and physical anchors. Cellulose scaffolding can be an attractive substrate material due to its high biocompatibility, natural abundance, simple production, low cost, and / or structural complexity. Plants have evolved to produce complex, specialized structures. Surprisingly, many of these structures are related to those found in the human body. By creating composite materials containing these plant structures, biomimetic materials for tissue engineering can be created. In certain embodiments, preparation of larger sizes can pose challenges due to the size of the source material. Thus, composite plant-based materials offer a solution to the size limitations imposed by nature. This may be a solution. The use of multi-component systems allows for the expansion of constructibility by designing materials that retain the characteristics of the source material but have, for example, different chemical and / or physical environments. For example, composite materials can be designed to have regions with different porosity and / or mechanics while maintaining the intricate characteristics of the source plant structure. An example of a direct consequence of using materials with different mechanical properties is the phenomenon of stress shielding. Stress shielding can occur in the body and can be a significant issue for bone implants. Inadequate or excessive stress shielding can cause damage or degradation of surrounding healthy tissue. Furthermore, interfaces between different tissues often separate regions with fundamentally different microenvironments. Composite materials can be engineered to mimic the interface of different cell populations and environments.

[0303] In these studies, to provide accuracy and reproducibility, scaffold biomaterials were first cut into subunits of the desired shape using a CNC. The desired shapes were then extracted from the bulk material by slicing with a mandolin slicer. The thickness of the mandolin slices was used to set the z-thickness of the material. The scaffold biomaterial subunits were then decellularized and sterilized. See International Publication No. 2017 / 136950, entitled "Decellularized Cell Wall Structures from Plants and Fungus and Use Thereof as Scaffold Materials," which is incorporated herein by reference in its entirety. The material was then ready for cell culture and / or implantation and was easily assembled into the final structure by pressing the subunits together using tweezers.

[0304] In this example, the creation of composite cellulose-based plant-derived scaffolds is explored. The scaffolds were assembled solely by geometry, without the use of external agents such as adhesives, crosslinkers, and hydrogels (although any or all of these external agents could also be used in combination with this geometric construct). Such interlocking composites offer an attractive approach to biomaterials design. The results of this example demonstrate that complex biomaterials can be assembled from subunits, similar to assembling Lego blocks. Cells can infiltrate the scaffold and cross subunit interfaces. Furthermore, cells can integrate separate subunits through matrix deposition, and cell migration can be hindered, for example, by the presence of pre-existing cells on adjacent scaffolds. The composites demonstrated stress shielding, supported the growth of various cell types, and created interfaces between distinct tissues. This approach may be attractive, for example, for creating larger and / or more complex composites.

[0305] Materials and Methods: Scaffold Production: A McIntosh red apple (Canada Fancy) was cut to create two flat parallel surfaces. Using a Carbide 3D Shapeoko 3 CNC machine and Chilipeppr jpadie software, the apple was cut into Lego-style pieces with pegs (5 mm x 5 mm x 2 mm, with a 2 mm peg extending from the center) and holes (5 mm x 5 mm x 2 mm, with a 2 mm diameter hole in the center). A 0.8 mm diameter drill bit was used to cut the scaffold at a 180° angle at a speed of 1 mm / s. Subunits were designed using Inkscape and JScut. The code was converted to G-code using a 3D printer. Samples were removed from the bulk apple tissue by slicing upside down against a mandolin slicer set to the appropriate thickness (4 mm for pegs and 2 mm for holes). Samples were transferred to a 0.1% SDS solution and decellularized for 72 hours with shaking at 180 RPM. After decellularization, the samples were washed three times with dH2O. The subunits were then incubated in 100 mM CaCl2 for 24 hours at room temperature to remove any detergent residues. The samples were then washed three times with dH2O to remove salt residues and then sterilized by incubation with 70% ethanol. After removing the ethanol, three washes with dH2O were performed to obtain a sterile, contaminant-free scaffold. For stress-shielding experiments, carrots were cut into the hole subunit shapes described above.

[0306] Cell culture: GFP NIH 3T3 mouse fibroblasts, NIH 3T3 mouse fibroblasts MC 3T3 E1 cells and MC 3T3 E1 subclone 4 preosteoblasts were maintained at 37°C and 5% CO2. GFP 3T3 and 3T3 cells were cultured in Dulbecco's Modified Eagle Medium - High Glucose (DMEM) (Hyclone Laboratories Inc.) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin (100 U / mL and 100 μg / mL, respectively). MC 3T3 E1 cells The medium was cultured in Minimum Essential Medium α (MEMα) without ascorbic acid. The cells were cultured in PBS (α). Serum supplementation and antibiotics were the same as those previously mentioned. The cells cultured on the cell culture plates were rinsed with phosphate-buffered saline, trypsinized, and resuspended in medium. The cells were counted and centrifuged to separate the cells from the trypsin and medium. The supernatant was aspirated and 5 × 10 4 The cell-containing pellet was resuspended in 25 μL of fresh culture medium. The cells were seeded onto the scaffolds and incubated for 1 hour. After 1 hour, the culture plate was filled with the appropriate medium to submerge the samples. Seeding was repeated 1 week later. Cells were grown for 2 weeks to allow for infiltration into the scaffolds before subunit assembly and migration analysis. The culture medium was changed daily, and after 1 week of growth, the samples were transferred to new culture plates.

[0307] For preosteoblast differentiation, cells were grown on each subunit for an additional 3 weeks before assembly during the differentiation process. GFP 3T3 cells were maintained in DMEM, while MC 3T3 E1 cells were cultured in differentiation medium (MEMα with 4 mM disodium hydrogen phosphate and 50 μg / mL ascorbic acid). The medium was changed daily. After combining the subunits and culturing for 1 week, mechanical investigation, fixation, and imaging were performed.

[0308] Subunit assembly: Complementary peg-and-hole Lego-style pieces / subunits were combined to create composite units. Two subunits were assembled by manually pressing them together with tweezers until they clicked into place. For experiments involving different cell types, cells were grown on each subunit for two weeks before assembly.

[0309] Staining: Decellularized scaffolds were imaged using a biocompatible live cell scaffold stain. Two different stains were used: calcofluor white (0.1 mg / mL in medium) and Congo red (0.1 mg / mL in medium) to obtain blue and red scaffolds, respectively. Staining was performed without fixation for migration experiments. Before staining, cells were washed three times with PBS and then fixed with 3.5% paraformaldehyde for 10 minutes for higher resolution imaging. Cells were stained with 10 μg / mL Hoechst 33342 (Invitrogen), 5 μM cell tracker red (Thermo Fisher), and 1 M SiR-actin (Spirochrome Cytoskeleton) for 1 hour. , samples were washed with PBS and mounted in VectaShield mounting medium during imaging.

[0310] Microscopy: Cells and biomaterials were imaged with epifluorescence and laser scanning confocal microscopes. Samples were imaged with a Nikon TiE A1-R high-speed resonant scanning confocal microscope equipped with 4x and 60x objectives. Confocal imaging was performed with a confocal microscope. Images were processed using ImageJ (Fiji). The confocal images presented here are maximum intensity projections of the confocal volume as well as 3D reconstructions. Brightness / contrast settings were adjusted to maximize fluorophore signal. No other image manipulations were performed.

[0311] Cell migration quantification: Phase-contrast images allowed the (x, y) coordinates of the subunits to be recorded in Fiji (ImageJ). Scaffold coverage area was monitored by the projected area of ​​GFP 3T3 cells. Confocal images were thresholded using the Fiji (ImageJ) compatible threshold plugin, and the (x, y) coordinates of the cell coverage area on the desired bound subunit were measured using the particle analysis plugin. The cell:scaffold area ratio was calculated weekly for 3 weeks after assembly. Values ​​presented are the mean ± standard error of the mean (sem).

[0312] Mechanics: Custom-manufactured Dynamic Mechanical Analysis (DMA) devices and Using NIRS and NIRS software, the materials were strained at a strain rate of 50 μm / s after 2 weeks of culture. Young's modulus was measured by compressing to 10% strain. The force-indentation curves were converted into stress-strain curves, which were then fitted in Origin 8.5 to calculate Young's modulus. In addition, stress shielding was studied by compressing the Lego blocks parallel and perpendicular to the subunit interface. This analysis yielded a mixed Young's modulus. The integration strength mediated by cell migration through the interface was investigated by tensile measurements. For tensile measurements, the biomaterials were fixed to the DMA device using Ultra Gel (LePage). Prior to mechanical testing, the biomaterials were The device was left attached for 10 minutes, the strain rate was the same as for the compression device, and the force at which the subunits were pulled apart was recorded.

[0313] Histological examination: For histological analysis, serial 5 μm thick sections were cut, starting from the inner 1 mm of the cellulose scaffold. The sections were stained with Masson's trichrome stain. A Zeiss MIRAX MIDI slide scanner (Zeiss, Toronto, Canada) with a 40x objective was used. Slices were imaged for cell invasion, extracellular matrix deposition, and angiogenesis (vasculogenesis). Photomicrographs were visualized using a Pannoramic Viewer (3DHISTECH Ltd., Budapest, Hungary). The data were analyzed using the Lee software.

[0314] Statistical analysis: One-way ANOVA test was used to evaluate statistical differences between cells cultured on biomaterials under different conditions. Tukey post-hoc analysis was performed to determine statistical differences between individual samples. To compare more than two samples, one-way ANOVA was used instead of multiple Student's t-tests to reduce the risk of type 1 statistical error. When only two samples were compared, Student's t-test was used. All values ​​presented are mean ± standard error of the mean (sem). Statistically significant differences (indicated by an asterisk) indicate P<0.05.

[0315] Results and Discussion: Composite Lego Fabrication: Proof-of-concept Lego-style subunit blocks were designed to create composite biomaterials assembled solely by shape. Fabrication of such shapes was a relatively time-consuming process using manual cutting techniques. Therefore, Computer Numerical Control router cutting (CNC) was preferred to provide reproducible materials in minutes. Complementary "peg" and "hole" subunits were fabricated. A small piece of trego-style tissue was cut from apple thip tissue (Figure 11). The resulting material was decellularized, processed, and then repopulated with mammalian cells to form a composite biomaterial for tissue engineering and 3D cell culture. The subunits were easily assembled by hand and clicked into place (Figure 11). The tight-fitting geometry of the Lego design kept the units intact throughout weeks of culture and manipulation.

[0316] Cell migration through the composite: To evaluate cell migration through the interface between the two subunits of the composite, we conducted single-cell-type and dual-cell-type migration studies. In the single-cell-type migration assay, GFP-positive 3T3 cells were loaded into one subunit and allowed to grow and infiltrate the scaffold for two weeks, while the second subunit was left bare. Once the materials were mated, cell coverage of the bare scaffold was monitored weekly for three weeks by confocal microscopy (Figure 12). In contrast, in the dual-cell-type assay, 3T3 fibroblasts (lacking GFP) were seeded into the complementary subunit, and the experiment was repeated to investigate the effect of the presence of pre-existing cells on the migration profile (Figure 13).

[0317] Single cell type migration resulted in a rapid increase in cell coverage on bare scaffolds (Figures 12 and 14). After 3 weeks, the coverage area was comparable to that of preloaded subunits. The results showed that cells were able to cross the interface and invade and grow into the adjacent biomaterial.

[0318] In dual-cell-type migration experiments, the area of ​​cell coverage increased, but coverage was much less than that observed in single-cell-type migration. The presence of cells on the second subunit hindered cell migration (Figure 13). The composite biomaterial allowed the two distinct populations of cells to interact and integrate. The cell types chosen for this study were contact-inhibited. Thus, the two populations not only intermingled to a small extent, but also largely remained two distinct groups (Figure 14).

[0319] Integration of click biomaterials: The migration studies described above clearly demonstrated that cells can migrate from one subunit to another when the appropriate structure is used. As confirmed by histological analysis, cells accumulate ECM proteins as they infiltrate and proliferate into the scaffold. This migration and ECM deposition led to the integration of the two subunits. The Lego-like peg-and-hole design held the units together through friction of the tightly interlocking geometry. To assess whether the presence of cells caused stronger integration of the subunits, the force required for disassembly of the composite was measured. Measurements were performed using a custom-fabricated dynamic mechanical analysis (DMA) device. The top and bottom of the sample were glued to parallel plates of the DMA device. The presence of cells resulted in a higher tensile force required for subunit separation compared to a bare scaffold lacking cells. Consequently, in these studies, cell infiltration and migration through the interface reinforced the composite unit (see Figure 15).

[0320] Stress Shielding and Mechanics: The phenomenon of stress shielding is the differential application of force to each object within a composite system. The different elastic moduli of the components can induce different strains and stresses in each part of the system. The effective Young's modulus of a composite can be derived from its geometry, the direction of the applied force, and / or the elastic moduli of the constituents. A direct consequence of this phenomenon is its ability to have an effective Young's modulus that can depend on the direction of the applied force. The click / interlocking biomaterials described herein can allow stress shielding to occur by choosing source materials with different elastic properties. The design used in this example demonstrated that applying stress parallel or perpendicular to the plane of the interface resulted in different effective elastic moduli (see Figure 16). To explore this concept, two subunits were selected to have different elastic moduli. The two source materials were apple and carrot. When combined, the effective Young's modulus depended on the direction of the applied force, resulting in a stress-shielding biomaterial (see Figure 16). In this model, to simplify this study, the viscous effects of the medium within the constructs were neglected and they were treated as two elastic bodies.

[0321] Tissue Interface: As supported by migration, integration, and stress-shielding results, composites containing different tissue types can be fabricated. Here, MC 3T3 E1 subclone 4 cells were grown in one subunit for two weeks. Preosteoblasts were differentiated in MEMα containing ascorbic acid and inorganic phosphate for three weeks. Calcium and collagen matrix deposition on the scaffold was observed. The biomaterial was then combined with a complementary Lego-style subunit loaded with GFP 3T3 fibroblasts. A bone-fibroblast tissue interface was effectively created. As previously observed in single-tissue type studies, cells migrated across the interface. In addition, preosteoblast differentiation was linked to an increase in the material's elastic modulus. As a result, the two subunits had different elastic moduli, and stress-shielding was added to the bone-fibroblast composite. Thus, stress-shielding was achieved both by using different subunit source materials and by using different cell types to create distinct tissues. Here, osteocytes and fibroblasts were selected. This work supports the feasibility of fabricating interlocking composites containing different cell types and tissues with defined interfaces (see Figure 17).

[0322] The use of composite materials is attractive for designing biomaterials that mimic the natural environment of cells or tissues. Various strategies have been used to integrate different components of the natural cellular environment. Various components can be combined, for example, with crosslinkers, hydrogels, adhesives, and / or chemical modifications. Because it is often desirable to create composite biomaterials without the use of such secondary components, in this example, we developed a method for creating composite materials that bond together without the need for secondary elements. In this example, interlocking "click" biomaterials were designed based solely on shape. The shape used was a peg-and-hole Lego-style block design. Using a CNC milling machine, rapid, highly reproducible cuts were made for relatively high-throughput production of subunits. The tightly interlocking shapes were sufficient to keep the units intact for several weeks from manual manipulation and handling.

[0323] The subunits were derived from decellularized plant tissue. The complexity and diversity of plant structures make them well suited to biomaterials. Although these organisms belong to separate kingdoms, plant structures can be engineered to fit specific structures in animal and human bodies. This type of biomaterial is highly biocompatible, tunable, and customizable. In vivo, angiogenesis has been observed without template or pre-loading with external agents such as growth factors. Similarly, cells can infiltrate and grow into the scaffold in vitro and in vivo. Matrix deposition and the usual minimal immune response can be observed. The basic structure of the material is typically cellulose. Chemical linkage of glucose subunits results in a permanent scaffold that does not degrade to a significant extent in the body. These characteristics, combined with the ability to tailor chemical and physical properties, relatively cost-effective production, and abundant source materials, make these biomaterials attractive for tissue engineering. Interlocking biomaterial methods can enable the creation of complex structures and products not typically found in nature. Of particular note is that different structures derived from different plants or plant structures can be combined to produce materials with different sizes, mechanical properties, surface chemistries, porosities, and / or degradation rates.

[0324] In this study, we investigated cell migration across the interface between two subunits of a composite to examine how cells respond to the interface between the two components. Both single-cell-type and dual-cell-type migration studies were performed. In the single-cell-type migration assay, one subunit was loaded with GFP 3T3 cells and combined with a bare scaffold. Cells crossed the interface, invaded, and proliferated on the bare scaffold. A rapid increase in cell coverage area was observed on the bare scaffold. After 3 weeks, the second subunit had coverage equivalent to that of the preloaded scaffold. The primary implications of these findings lie in wound healing. In this study, collective cell migration was three-dimensional. 3D collective migration has been shown to differ from 2D surface migration. These results support the use of composite cellulose-based biomaterials in wound healing and further support the possibility that cells may generally be able to move from one unit to another and fill secondary structures without the use of additional agents to mediate migration across the interface. In contrast, the experiment was repeated using two subunits preloaded with two distinct cell populations. Results indicate that cells migrated from one subunit to the other, but with much less coverage than observed with single-cell-type migration. The cell types used here were contact-inhibited. Thus, preloading the scaffold effectively prevented extensive migration into adjacent scaffolds. The composite biomaterial allowed two distinct cell populations to interact and integrate at the interface, and the selection of contact-inhibited cell types demonstrates that composites can be used to create tissue interfaces that separate two distinct cell groups. Other applications also contemplate using non-contact-inhibited cells to create regions of overlapping cells and varying degrees of cell type intermixing for specific tissues of interest.

[0325] The tight fitting geometry of the interlocking design used in this example ensures that the pieces fit neatly into one unit. The force required to separate the materials was then investigated. The measurements were then repeated with a scaffold containing cells. As discussed, the cells crossed the interface and infiltrated into the adjacent scaffold. Thus, the two subunits were integrated to some degree by the cells and the ECM they deposited. The integration of the subunits by the cells resulted in a higher tensile force required to separate the units. This supports various tissue engineering applications. These results indicate that the composite interface was reinforced as the cells infiltrated the material. In regenerative medicine and medical applications involving biomaterials, tissue integration is a key concern. For example, in bone implants, a lack of tissue integration can result in loose implants that may ultimately fail as a result of damage to surrounding tissues and residue formation.

[0326] Similarly, stress shielding is a key concern in bone implants. Typical materials used have stiffnesses that significantly exceed that of natural bone tissue. Previous approaches are often far from ideal. However, alternative materials are used because they typically lack the mechanical properties to withstand the loads placed on bone. Stress shielding can cause the degradation of surrounding healthy bone tissue through osteoclast resorption. In the absence of stress, osteoclasts are signaled to resorb bone tissue. This lack of stress can be achieved by shielding the implant with a higher elastic modulus. Stress shielding has been identified as a key challenge in bone implants and bone tissue engineering. However, stress shielding occurs throughout other tissues as well. Stress shielding exists at tissue interfaces. Therefore, properly mimicking stress shielding is desirable to recreate certain microenvironments. The interlocking biomaterial approach described herein can enable targeted stress shielding, allowing materials to be engineered to have a specific effective elastic modulus. In this example, we neglected the viscous effects of the medium within the constructs and treated them as two elastic bodies for simplicity in this study.

[0327] An attractive application of composite biomaterial products with different cell types and / or mechanical properties may be a bone-fibroblast tissue interface. This example demonstrates that pre-osteoblasts can be differentiated in an apple-derived scaffold and combined with a secondary scaffold loaded with fibroblasts. In this example, the bone tissue exhibited calcium and collagen matrix deposition. At the interface, cells migrated through the interface, integrating the two tissues. In addition, pre-osteoblast differentiation was associated with an increase in the material's elastic modulus. Thus, the produced bone-fibroblast composite exhibited stress shielding. This study demonstrates that complex entities can be produced. Biomaterials can have different cell types and / or mechanical profiles. This concept can be extended or combined with further modification, for example, with chemical modification and / or hydrogels and / or crosslinkers, but this approach does not rely on such additional factors to remain structurally intact. Interestingly, this approach may enable the design of custom interfaces.

[0328] In this example, a composite cellulose-based plant-derived scaffold was successfully assembled by geometry alone, without the use of external agents such as adhesives, crosslinkers, and hydrogels. Interlocking subunits offers an attractive approach to biomaterial design because it can reduce the need for additional components. The click biomaterial presented here is a Lego-style design. The material in this example supported cell proliferation and migration through the interface, as well as the integration of separate subunits through matrix deposition. Cell migration was hindered by the presence of pre-existing cells in the adjacent scaffold. The composite material demonstrated the ability to demonstrate stress shielding when the subunits had different elastic moduli. It was also demonstrated that bone-fibroblast composites and interfaces could be created. The biomaterial interlocking composite approach demonstrated here may provide the ability to create complex composites that more closely mimic the natural in vivo environment.

[0329] Figure 2 shows an image of the assembly structure containing two subunits interlocked together. Figure 2 shows an interlocking cellulose-based biomaterial in which blocks can be assembled using a Lego-style peg-and-hole geometry as shown. Tightly interlocking subunits can be assembled / combined to provide a biocompatible intact structure.

[0330] It is contemplated that, using the interlocking techniques described herein, structures described herein comprising two or more scaffold biomaterial subunits may overcome and / or ameliorate certain problems associated with larger sizes and correspondingly longer decellularization times, and / or with diffusion in larger constructs. In certain embodiments, the structures described herein may allow for the design of certain structures and features not found in nature while leveraging the natural complexity of the scaffold material source in the individual subunits. In certain embodiments, the structures described herein may allow for increased complexity in physical and / or mechanical properties (i.e., stress shielding and site-specific modulus, channels, pores, etc.). In certain embodiments, the structures described herein may be articulated structures that can impart varying degrees of flexibility and mobility as needed. In certain embodiments, the structures described herein may allow for the combination of different cell types in different regions.

[0331] In certain embodiments, adhesives, hydrogel (or other gel) casting, coating, cross-linking, and / or smearing can be used to further secure and / or hold the subunits in the structure.

[0332] In certain embodiments, appropriate drugs, signaling molecules, growth factors, metabolites, and / or ECM proteins and / or components may be attached to (i.e., functionalized or loaded onto and / or within) the structures described herein to provide a desired response, which may be systemic or cell / tissue type specific, and / or positive or inhibitory as needed for a particular application. In certain embodiments, the scaffolds described herein may be loaded or functionalized with a drug and used to administer the drug, thereby providing at least some degree of site-specific drug delivery, which, in certain embodiments, can reduce drug dosage and / or increase efficacy. In certain embodiments, the structures described herein can provide time-dependent and / or non-time-dependent release of one or more agents, such as appropriate drugs, signaling molecules, growth factors, metabolites, and / or ECM proteins and / or components.

[0333] In certain embodiments, the scaffold biomaterials described herein may be cellulose-based, hemicellulose-based, chitin-based, chitosan-based, pectin-based, lignin-based, lignan-based, or any combination thereof. In certain embodiments, the composite structures described herein may be tunable with respect to their biochemical, biophysical, and / or mechanical properties.

[0334] In certain embodiments, the structures described herein are used in custom in vitro 3D cell culture devices; in vivo applications and / or basic research studies; complex tissue engineering; biomaterial implants for tissue repair and / or regeneration; medical devices such as devices or implants for bone, connective tissue, skin, muscle, nerve, and / or interfaces; complex tissue repair and / or replacement; membranes and / or filters (e.g., artificial kidneys and / or simple biochemical separation columns); vectors for site-specific and time-specific drug delivery; increasing the biocompatibility of existing medical devices by coating or fabricating composites with the materials described herein; vectors for primary cell culture; cosmetic procedures (e.g., implants and / or subcutaneous topographies); or stents and / or shunts; synthetic biorobotics. non-medical applications, such as electrical circuit integration or interconnect components for medical devices; or any combination thereof. [Example]

[0335] Composite biomaterials of plant and bacterial cellulose In this example, a composite biomaterial containing both plant and bacterial cellulose is provided. In certain embodiments, guided assembly-based biolithography (GAB) techniques can be used to grow bacterial cellulose on a decellularized or cast / printed plant cellulose source, resulting in a composite biomaterial in which tunable ratios of cellulose with different crystalline structures are arranged in distinct configurable regions; the guided assembly-based biolithography (GAB) technique can enable different microtopographies and densities. In certain embodiments, a bacterial source can deposit cellulose on a plant-derived cellulose-based scaffold biomaterial, e.g., two different celluloses with different ratios and / or crystalline structures. Thus, the resulting product can contain different celluloses that may have different functional group chemistries, densities, porosities, and / or mechanical properties. By combining celluloses, the resulting product can offer increased complexity, configurability, and / or additional features over either cellulose alone. In certain embodiments, the manner in which bacterial cellulose grows on a plant cellulose scaffold can be dictated or influenced by the shape of the plant cellulose scaffold, the culture conditions, or both. In certain embodiments, the resulting scaffold biomaterial may be customizable in terms of shape and / or structure and / or can be used to provide macrostructures with varying degrees of flexibility and / or connectivity.

[0336] 3D biocompatible scaffolds containing decellularized plant tissues have been developed. See International Publication No. 2017 / 136950, entitled "Decellularized Cell Wall Structures from Plants and Fungus and Use Thereof as Scaffold Materials," which is incorporated herein by reference in its entirety. These biomaterials can support cell growth, infiltration, and proliferation in vitro and in vivo. Cellulose scaffolding can be an attractive substrate material due to its high biocompatibility, natural abundance, ease of production, low cost, and structural complexity. Plants have evolved to produce complex, specialized structures. Remarkably, many of these structures are related to those found in the human body. By creating composite materials from these plant structures, biomimetic materials for tissue engineering can be created.

[0337] Such an approach poses challenges when larger substrates and / or implants are desired. Therefore, composite plant-based materials can provide a solution to these size challenges imposed by nature. Multicomponent systems can retain the advantages of plant-derived materials, but potential applications can be expanded by engineering materials with different chemical and / or physical environments. For example, composite materials can be engineered to have regions with different porosity and / or mechanics while maintaining the intricate characteristics of plant-derived structures. The use of materials with different mechanical properties can provide stress-shielding. Stress-shielding occurs in the body and is a common issue with bone implants. Inadequate or excessive stress-shielding can cause damage or degradation of surrounding healthy tissue. Furthermore, interfaces between different tissues often separate regions with radically different microenvironments. Composite materials can be engineered to mimic the interface of different cell populations and environments.

[0338] In certain embodiments, the present approach can utilize the guided assembly-based biolithography (GAB) technique, which allows bacterial cellulose to be grown on decellularized or cast / printed plant-derived cellulose sources to allow for the formation of different cell types. Composite materials can be obtained in which different ratios of cellulose with different crystalline structures are located in different regions. This technique can also enable the design of different microtopographies and / or densities. Bacterial sources can deposit cellulose onto plant-based cellulose scaffolds (or other such scaffolds). The two different sources may have different ratios of crystalline structures (e.g., cellulose Iα / β). As a result, the cellulose can have different functional group chemistries, densities, porosities, and / or mechanical properties. Such composites can be used to expand the functionality of each material. The manner in which bacterial cellulose grows on a plant cellulose source or scaffold can be determined, at least in part, by, for example, the geometry of the plant cellulose source or scaffold and the culture conditions.

[0339] method: Scaffold Production: Decellularized materials were prepared according to established protocols. Decellularized Cell Wall Structures from Plants and Fungus and Their Use as Scaffolds See WO 2017 / 136950 entitled "Materials for Implantable and Non-Platable Cells," which is incorporated herein by reference in its entirety.

[0340] Bacterial cellulose production and (GAB): Several organisms can be used for this task. Bottan, S., Robotti, F., Jayathissa, P., Hegglin, A., Bahamonde, N., Heredia-Guerrero, JA, et al. (2015). Surface-structured bacterial cellulose with guided assembly-based biolithography (GAB). ACS Nano 9, 206-219, which is incorporated herein by reference in its entirety. The scaffold biomaterial may be decellularized and can serve as a template structure, allowing for the combination of two different types of cellulose from two different sources in the resulting composite.

[0341] In certain embodiments, provided herein are scaffold biomaterials comprising both plant-derived (or fungal-derived) and bacterial-derived cellulose. Such constructs can exploit the natural complexity of the individual subunit scaffold materials used in composites while enabling the creation of structures and features not found in nature. In certain embodiments, it is contemplated that such approaches can enable more complex and / or tunable physical and / or mechanical properties (i.e., stress shielding and site-specific modulus, channels, pores, etc.). In certain embodiments, such approaches can be used to provide scaffold biomaterials that allow the combination of different cell types in different regions. In certain embodiments, such approaches can provide subunit combinations in applications where simple physical connections are undesirable, inapplicable, or insufficient. In certain embodiments, it is contemplated that such approaches can be used to create seals for fluid flow and / or to create semipermeable interfaces. In certain embodiments, the subunits can be glued, coated, or cast together using adhesives, gels, or pastes, such as, for example, agarose-based, gelatin-based, collagen-based, and / or hyaluronic acid-based agents, to increase structural integrity.

[0342] In certain embodiments, adhesives, hydrogel (or other gel) casting, coating, cross-linking, and / or smearing can be used to further secure and / or hold the subunits into a structure.

[0343] In certain embodiments, appropriate drugs, signaling molecules, growth factors, metabolites, and / or ECM proteins and / or components may be infused into the structures described herein to provide a desired response, which may be systemic or cell / tissue type specific, and / or may be positive or inhibitory as needed for the particular application.

[0013] In certain embodiments, the scaffolds described herein may be loaded or functionalized with drugs and used to administer the drugs, thereby providing at least some degree of site-specific drug delivery, thereby, in certain embodiments, reducing drug dosage and / or increasing efficacy. In certain embodiments, the structures described herein may provide time-dependent and / or non-time-dependent release of one or more agents, such as appropriate drugs, signaling molecules, growth factors, metabolites, and / or ECM proteins and / or components.

[0344] In certain embodiments, it is contemplated that time-dependent and / or time-independent release may be provided by loading drugs into gels with various release characteristics and / or by covalently attaching drugs to the scaffold via chemical functionalization methods. For example, in certain embodiments, drug-containing tubes may be loaded into tubes that are covalently linked via linker molecules such as succinic acid. Such tubes may be directed to site-specific locations and may have time-specific release properties. In certain embodiments, chemical modifications may provide steric hindrance suitable for increasing the complexity of site-specific modifications. In certain embodiments, linker molecules may be used to mediate the connection of drugs, signaling molecules, growth factors, metabolites, ECM proteins and / or components, or any combination thereof, with tubes containing such compounds. In the absence of other biochemical or biophysical modifications or signaling, different cell attachment densities may lead to different cellular responses, and this approach may allow, for example, the degree of substitution to be tailored for specific functions.

[0345] In certain embodiments, the scaffold biomaterials described herein may be cellulose-based, hemicellulose-based, chitin-based, chitosan-based, pectin-based, lignin-based, lignan-based, or any combination thereof. In certain embodiments, the composite structures described herein may be tunable with respect to their biochemical, biophysical, and / or mechanical properties.

[0346] In certain embodiments, the scaffold biomaterials described herein can be prepared by using a plant-derived scaffold biomaterial structure as a scaffold and / or guide onto which bacterial cellulose can be grown or deposited. In other embodiments, bacterial cellulose can be used as a template, or a synthetic source such as plastic can be used as a template. In certain embodiments, guided assembly-based biolithography (GAB) can be used, in which a template can be used to transfer topography to the biomaterial. In certain embodiments of GAB, the template can act as a guide for bacterial cellulose to grow on the biomaterial, providing a composite structure of plant and bacterial-derived cellulose with a designed topography and / or density.

[0347] In certain embodiments, the scaffold biomaterials described herein may be functionalized and / or loaded with one or more agents to suit the desired application. Such agents may include, for example, any one or more of a therapeutic agent, a signaling molecule, a growth factor, a metabolite, an ECM protein or component, or any combination thereof. In certain embodiments, the agent may provide for time-dependent or non-time-dependent release of such agent. In certain embodiments, one or more agents may be covalently attached to the cellulose-based, hemicellulose-based, chitin-based, chitosan-based, pectin-based, lignin-based, and / or lignan-based scaffold biomaterial, either directly or indirectly via a linker. In certain embodiments, the ... via one or more acylation and alkylation type reactions, or via the addition of sulfur, nitrogen, boron, and / or halide compounds (i.e., thiol, imide, imine, amine, amide, borohydride, borohydrate, and halogenated Other suitable reactions using the compound (a) can be used.

[0348] In certain embodiments, it is contemplated that the structures described herein may be designed such that one or more compartments of the structure are more or less hydrophobic or hydrophilic relative to one or more other compartments of the structure. In certain embodiments, it is contemplated that the scaffold biomaterials described herein may be configured to have site and / or cell type specificity using functionalization with appropriate attractants and / or repressants.

[0349] It is contemplated that, using the approaches described herein, the structures described herein comprising plant and bacterial cellulose may overcome and / or ameliorate certain problems associated with larger sizes and correspondingly long decellularization times, and / or with diffusion in larger constructs. In certain embodiments, the structures described herein may allow for the design of certain structures and features not found in nature while leveraging the natural complexity of the scaffolding material source in the individual subunits. In certain embodiments, the structures described herein may allow for increased complexity in physical and / or mechanical properties (i.e., stress shielding and site-specific modulus, channels, pores, etc.). In certain embodiments, the constructs described herein may allow for modification or tuning of permeability through the use of different adhesives and / or coatings (i.e., tubes, semipermeable membranes, and sealants for junctions). In certain embodiments, the constructs described herein may be tunable with respect to mechanical properties and / or junctions (i.e., Young's modulus can be changed) based on the use of adhesives or coatings. In certain embodiments, the structures described herein may be articulated structures that can impart varying degrees of flexibility and mobility as needed. In certain embodiments, the structures described herein can allow for the combination of different cell types in different regions.

[0350] In certain embodiments, the scaffold biomaterials described herein may be for use in complex tissue engineering and / or biomaterial implants for tissue repair and / or regeneration, for drug delivery, and / or for incorporation of growth factors. In certain embodiments, the scaffold biomaterials described herein may be for use in non-medical applications, such as articulating components for synthetic biorobotics, or electrical circuit integration, or any combination thereof. [Example]

[0351] Crosslinking and chemical functionalization of plant-derived cellulose In this example, the crosslinkable cellulose is derived from and produced from a plant source. In embodiments where carboxymethyl and hydroxyethyl cellulose are present, the cellulose gel or paste may be crosslinked with citric acid and heat. Carboxymethyl and hydroxyethyl cellulose functional groups are not generally found in plant-derived cellulose. Therefore, functional group addition may be performed. Collagen may then be covalently attached to the cellulose. In this example, such a synthetic strategy allows for the use of chemical linker molecules with carboxyl terminal groups to provide the desired functional groups on the cellulose. The introduction of citric acid in the presence of heat can create crosslinks between these functional groups, an advantage of this approach being that a non-toxic crosslinking agent is used (i.e., citric acid is a natural metabolite). Such an approach may have applications, for example, in 3D printing, injectable hydrogels, molds, and adhesives.

[0352] We have developed 3D biocompatible scaffolds containing decellularized plant tissue. These biomaterials can support cell growth, infiltration, and proliferation in vitro and in vivo. Composites of cellulose scaffolds and hydrogels have also been investigated. The presence of hydrogels allows for distinct biochemical and biological functions. Cellulose scaffolds can enable the temporary or permanent introduction of structural anchorages. Cellulose scaffolds are attractive substrate materials because they are highly biocompatible, abundant in nature, simple to produce, low-cost, and / or can provide complex structures. Plants have evolved to produce complex, specialized structures. Surprisingly, many of these structures are related to those found in the human body. Creating composites of these plant structures can produce biomimetic materials for tissue engineering, for example. However, creating large-sized implants and / or scaffolds can be difficult, for example, due to the size of the source material. Thus, composite plant-based materials can provide a solution to the size limitations imposed by nature. Multicomponent systems can retain the characteristics of the material, but by engineering materials with different chemical and / or physical environments, customization and / or applicability can be expanded. For example, composite materials can be engineered to have regions with different porosity and / or mechanics while maintaining the intricate characteristics of the plant structures from which they are derived. An example of a direct consequence of using materials with different mechanical properties is the stress-shielding phenomenon. Stress shielding occurs in the body and is a key issue for bone implants, for example. Inadequate or excessive stress shielding can cause damage or degradation of surrounding healthy tissue. Furthermore, interfaces between different tissues often separate regions with radically different microenvironments. Composite materials can be engineered to more closely mimic the interface of different cell populations and environments.

[0353] Additionally, modifications such as adhesion, gel casting, chemical functionalization, and / or loading with one or more agents (i.e., drugs, signaling molecules, growth factors, metabolites, etc.) can further expand the functionality of the scaffolds and biomaterials described herein. For example, chemically functionalized and crosslinked materials can exploit the natural complexity of the source material in individual subunits while enabling the design of certain structures and features not found in nature. Furthermore, they can provide more complex physical and / or mechanical properties (i.e., stress shielding and site-specific elastic modulus, channels, pores, etc.). Such composites can be used, for example, to combine different cell types in different regions. This approach can provide an alternative to composite interlocking, adhesion, or gel casting, for example, when a more permanent and stronger covalent bond is desired. Of course, the chemical crosslinking approach described in this example represents an illustrative example, and a wide variety of suitable alternative chemical crosslinking approaches can also be used. Many other reactions can be used, incorporating different functional groups and / or crosslinkers as needed.

[0354] Materials and Methods Scaffold Production: Decellularized materials were prepared according to established protocols. Decellularized Cell Wall Structures from Plants and Fungus and Their Use as Scaffolds See WO 2017 / 136950, entitled "Decellularized Materials," which is incorporated herein by reference in its entirety. In certain embodiments, the decellularized material can be converted into a paste or gel by bleach treatment, basic solvent, and / or physical shear steps.

[0355] Chemical functionalization: Chemical functionalization of cellulose can be carried out according to the method described in Ribeiro-Viana, RM, Faria-Tischer, PCS, and Tischer, CA (2016). Preparation of succinylated cellulose membranes for functionalization purposes. Carbohydr. Polym. 148, 21-28, which is incorporated herein by reference in its entirety.

[0356] Briefly, in certain embodiments, cellulose may be succinylated with succinic anhydride. Prior to the reaction, the cellulose-based scaffold biomaterial may be subjected to solvent exchange to remove excess water. The cellulose-based scaffold biomaterial may then be immersed in methanol. The cellulose-based scaffold biomaterial may be immersed in dichloromethane and gently soaked for approximately 5 minutes. The solvent may then be discarded, and this procedure may be repeated two more times. The cellulose-based scaffold biomaterial may then be immersed in dichloromethane and gently soaked for approximately 5 minutes. The solvent may then be discarded, and the same procedure may be repeated two more times. Succinylated cellulose-based scaffold biomaterial may be prepared by soaking the cellulose in 10 mL of dichloromethane, 174 μL of pyridine, and 216 mg of succinic anhydride, which may be performed in a static system. The reaction time may vary (approximately 2 to 24 hours), and two different temperatures: 25°C and reflux may be used in different experiments. The reaction may then be stopped by adding 2 mL of methanol. The material may then be washed three times with water, then ethanol, and finally dried at room temperature.

[0357] If coupling to one or more functional moieties is desired, it may be carried out in a flat-bottom flask to which succinylated cellulose, 8 mL of anhydrous DMF, 40 mg of 3-(ethyliminomethyleneamino)-N,N-dimethylpropan-1-amine (EDC), and 8 mg of 4-dimethylaminopyridine (DMAP) are added and mixed. The reaction may be kept at room temperature for 1 hour. An appropriately functionalized functional moiety may then be added and stirred on a tumbling table for 18 hours at room temperature. The material may then be washed three times with water and finally dried at room temperature.

[0358] Citric Acid Crosslinking: Chemical functionalization of cellulose can allow different functional groups to be covalently linked to the material. Linking functional groups such as carboxymethyl cellulose and hydroxyethyl cellulose can allow for crosslinking using citric acid and heat. Other crosslinking agents are available and may also be used. This example is for illustrative purposes; citric acid may be preferred for certain applications because it is generally non-toxic and a natural by-product of cellular metabolism.

[0359] Citrate cross-linking is Raucci, M. Alvarez-Perez, Demitri, C. et al.(2015). Effect of Citric acid crosslinking cellulose-based hydrogels on osteogenic differentiation. This may be performed according to the method described in Journal of Biomedical Materials Research - Part A. 103 (6), 2045-2056, which is incorporated herein by reference in its entirety.

[0360] Briefly, two different cellulose derivatives may be prepared and used in this preparation: sodium salt of carboxymethyl cellulose (CMCNa) and hydroxyl ethyl cellulose (HEC). CMCNa is a cellulose derivative in which a carboxymethyl group (-CH2-COOH) is attached to some of the hydroxyl groups of the glucopyranose monomers that make up the cellulose backbone. This functional group may be responsible for the high sorption capacity due to the Donnan effect. HEC is a nonionic polymer that can form a stable network with a lower sorption capacity. Samples may be prepared by double esterification crosslinking using citric acid (CA). Briefly, hydrogel samples can be obtained by reacting CMCNa and HEC, respectively, with CA (20% [wt / wt] based on the polymer weight) as a crosslinker in distilled water. For each of the two cellulose derivatives, a total concentration of 2 wt% of the polymer in water can be prepared by gently stirring at room temperature until a clear solution is obtained. This final solution can be used to mold 10 mm thick samples. The samples can be pre-dried at 30 °C for 24 hours to remove absorbed water, and then kept at 80 °C for 12 hours for the crosslinking reaction.

[0361] In this example, hydrolyzed collagen was covalently attached to modified cellulose, which involves modification of cellulose followed by a reaction involving crosslinking or coupling to the modified cellulose. For example:

[0362] Results and Discussion: As shown in Figure 6, a succinic acid linker molecule was covalently attached to cellulose. Hydrolyzed collagen was then covalently attached to the linker group of cellulose, as shown in Figure 7. Figure 6 shows cellulose with a monoester of succinic acid, where the succinic acid is covalently attached to the cellulose. The FTIR spectrum in Figure 6 shows carbonyl and carboxylic acid peaks, indicating a successful reaction. The gray spectrum is the control, and the black spectrum is the functionalized scaffold. Figure 7 shows cellulose to which hydrolyzed collagen has been covalently linked. The hydrolyzed collagen was covalently linked to a succinic acid linker molecule. The FTIR spectrum shows amide peaks, indicating a successful reaction. The gray spectrum is the succinated control, and the black spectrum is the functionalized scaffold.

[0363] In the absence of carboxymethyl cellulose (CMCNa) and hydroxyethyl cellulose (HEC), citric acid treatment did not crosslink manually crushed plant-derived cellulose gels. However, the cellulose was successfully pressed into a gel / paste, as shown in Figure 3. Figure 3 illustrates injectable cellulose gels and pastes, demonstrating that cellulose can be dehydrated, ground into a powder form, and then rehydrated to produce a gel or paste of the desired consistency. Cellulose does not crosslink in the presence of citric acid and heat in the absence of carboxymethyl and hydroxyethyl cellulose.

[0364] This example can provide crosslinkable cellulose derived from plant sources. When carboxymethyl and hydroxyethyl cellulose are present, the cellulose gel or paste can be crosslinked with citric acid and heat. These functional groups (carboxymethyl and hydroxyethyl cellulose) are not typically found in plant-derived cellulose. Therefore, the addition of functional groups may be implemented. This example successfully covalently binds collagen to cellulose, supporting the approach proposed herein. Desired functional groups can be obtained by utilizing chemical linker molecules with carboxyl terminal groups. Crosslinks between these functional groups can be created by introducing citric acid in the presence of heat. An advantage of this approach is the use of a substantially nontoxic crosslinking agent (citric acid is a natural metabolite). Multiple applications are contemplated, including, but not limited to, 3D printing, injectable hydrogels, molds, and adhesives. Modifications such as adhesion, gel casting, chemical functionalization, and loading with one or more agents (i.e., drugs, signaling molecules, growth factors, metabolites, etc.) can further expand the functionality of these materials.

[0365] Chemically functionalized and crosslinked materials can exploit the natural complexity of the scaffold source material in individual subunits while allowing for the design of certain structures and features not found in nature. Furthermore, they can enable more complex physical and mechanical properties (i.e., stress shielding and site-specific elastic modulus, channels, pores, etc.). Such composites can be used to combine different cell types in different regions. This crosslinking approach can provide an alternative or complement to interlocking, adhesive, or gel-cast composites when more permanent and / or strong and / or covalent bonds are required.

[0366] The described approaches can provide tunable biochemical, biophysical, and / or mechanical properties of cellulose, hemicellulose, chitin, chitosan, pectin, lignin-based, and / or lignan-based scaffolds. Furthermore, in certain embodiments, the time course of drugs, signaling molecules, growth factors, metabolites, and / or ECM proteins and components can be enhanced. It is contemplated that intercellular or intercellular release may be achieved with or without the use of additional hydrogels. The described approach may also enable the creation of larger macroscopic objects, for example, with varying degrees of flexibility and / or connectivity.

[0367] This approach can be used to create custom shapes with customizable features while still preserving the complex natural structure of plant-derived scaffold biomaterials. By way of example, it is contemplated that customizable shapes and / or structures can be prepared, which in certain embodiments may include the use of composites, adhesives, coatings, gels, and / or pastes to provide the desired structure. Similarly, in certain embodiments, it is contemplated that complex physical and / or mechanical properties (such as crystalline structure, porosity, ductility, toughness, strength, elasticity, plasticity, or any combination thereof) can be adjusted or controlled by varying the concentration and crosslinker concentration. It is contemplated that mechanical properties can be controlled / altered by varying the amount of crosslinking. Typically, fewer crosslinks may result in a more flexible material. Furthermore, crosslinking can also alter porosity. Typically, a higher degree of crosslinking can decrease porosity. In certain embodiments, such an approach, when used in vivo, can provide an adhesive approach that may be minimally invasive for freeform injection and allow for control of diverse biochemical and / or biophysical properties.

[0368] In certain embodiments, the crosslinking method can include providing shredded cellulose, adding functional groups to the shredded cellulose, and then crosslinking the shredded cellulose via the added functional groups. Also, in certain embodiments, the method can include 3D printing to provide the desired structure. The material can be molded or printed into the desired shape, and then crosslinked to maintain the desired shape and provide structural integrity. It is contemplated that the density and / or directionality of the resulting structure can be controlled by adjusting the degree and conditions of crosslinking.

[0369] In certain embodiments, the scaffold biomaterials described herein may be cellulose-based, hemicellulose-based, chitin-based, chitosan-based, pectin-based, lignin-based, lignan-based, or any combination thereof. In certain embodiments, the composite structures described herein may be tunable with respect to their biochemical, biophysical, and / or mechanical properties.

[0370] In certain embodiments, appropriate drugs, signaling molecules, growth factors, metabolites, and / or ECM proteins and / or components may be attached to (i.e., functionalized or loaded onto and / or within) the structures described herein to provide a desired response, which may be systemic or cell / tissue type specific, and / or positive or inhibitory as needed for a particular application. In certain embodiments, the scaffolds described herein may be loaded or functionalized with a drug and used to administer the drug, thereby providing at least some degree of site-specific drug delivery, which, in certain embodiments, can reduce drug dosage and / or increase efficacy. In certain embodiments, the structures described herein can provide time-dependent and / or non-time-dependent release of one or more agents, such as appropriate drugs, signaling molecules, growth factors, metabolites, and / or ECM proteins and / or components.

[0371] In certain embodiments, the biomaterial scaffolds described herein may have customizable shapes and / or structures and / or may allow for the creation of larger macrostructures with varying degrees of flexibility and / or connectivity as needed.

[0372] Examples of succinylated cellulose membranes and crosslinking of cellulose-based hydrogels with citric acid are given in Ribeiro-Viana, Renato & Faria, Tischer, Paula & Tischer, Cesar. (2016). Title: Preparation of succinylated cellulose membranes for functionalization purposes. Carbohydrate Polymers. 148.10.1016 / j.carbpol.2016.04.033; and Raucci, MG & Alvarez-Perez, MA & Demitri, Christian & Giugliano, D & DeBenedictis, Vincenzo & Sannino, A & Ambrosio, Luigi. (2014). Effect of citric acid crosslinking cellulose-based hydrogels on osteogenic differentiation: Effect of Cellulose-Based Hydrogels on Osteogenic Differentiation. Journal of Biomedical Materials Research Part A. 103.10.1002 / jbm.a.35343, each of which is incorporated herein by reference in its entirety.

[0373] It is contemplated that, using the approaches described herein, the structures described herein may overcome and / or ameliorate certain problems associated with larger sizes and correspondingly longer decellularization times, and / or with diffusion in larger constructs. In certain embodiments, the structures described herein may allow for the design of certain structures and features not found in nature while leveraging the natural complexity of the source of scaffold material in the individual subunits. In certain embodiments, the structures described herein may allow for increased complexity in physical and / or mechanical properties (i.e., stress shielding and site-specific modulus, channels, pores, etc.). In certain embodiments, the constructs described herein may allow for modification or tuning of permeability through the use of different adhesives and / or coatings (i.e., tubes, semipermeable membranes, and sealants for junctions). In certain embodiments, the constructs described herein may be tunable with respect to mechanical properties and / or junctions (i.e., Young's modulus can be changed) based on the use of adhesives or coatings. In certain embodiments, the structures described herein may be articulated structures that can impart varying degrees of flexibility and mobility as needed. In certain embodiments, the structures described herein can allow for the combination of different cell types in different regions.

[0374] In certain embodiments, suitable drugs, signaling molecules, growth factors, metabolites, and / or ECM proteins and / or components may be attached to (i.e., functionalized or loaded onto and / or within) the structures described herein to provide a desired response, which may be systemic or cell / tissue type specific, and / or positive or inhibitory as needed for a particular application. In certain embodiments, the scaffolds described herein may be loaded or functionalized with a drug and used to administer the drug, thereby providing at least some degree of site-specific drug delivery, which, in certain embodiments, can reduce drug dosage and / or increase efficacy. In certain embodiments, the structures described herein can provide time-dependent and / or non-time-dependent release of one or more agents, such as suitable drugs, signaling molecules, growth factors, metabolites, and / or ECM proteins and / or components, which may be provided by loading the agent into a suitable gel with various release characteristics or by covalently attaching the agent to the structure by chemical functionalization methods. In certain embodiments, drug-containing tubes may be loaded into the tubes, which may be covalently linked via a linker molecule, such as succinic acid. In certain embodiments, the tubes may be used to store one or more drugs or compounds that are released at a specific time and / or location. In certain embodiments, the tubes may be tethered to cellulose in specific regions using linker molecules that may be covalently attached to the cellulose. Such tubes may be, for example, targeted to site-specific locations and may have time-specific release properties.

[0375] In certain embodiments, such chemical modifications reduce or eliminate steric hindrance problems. In certain embodiments, linker molecules may be used to mediate connections with drugs, signaling molecules, growth factors, metabolites, ECM proteins and components, and the like, as well as vessels containing such compounds, or any combination thereof.

[0376] Steric hindrance may present a problem in certain circumstances. For example, in the absence of any other biochemical or biophysical modifications or signaling, different cell attachment densities may lead to different cellular responses. In certain embodiments of the structures described herein, the degree of substitution can now be tailored to suit, for example, a particular function.

[0377] In certain embodiments, the crosslinked plant-derived scaffold biomaterials can be used for 3D printing and / or injectable hydrogel applications. In certain embodiments, the crosslinked plant-derived scaffold biomaterials described herein may be for use in non-medical applications, such as synthetic biorobotics, or electrical circuit integration, or any combination thereof.

[0378] Cellulose for crosslinking and / or chemical functionalization can be prepared by a number of different methods. See, for example, Ribeiro-Viana, RM, Faria-Tischer, PCS, and Tischer, CA (2016). Preparation of succinylated cellulose membranes for functionalization purposes. Carbohydr. Polym. 148, 21-28, which is incorporated herein by reference in its entirety. By way of example, in certain embodiments, cellulose can be reacted to form a cellulose monoester of succinic acid, which can facilitate crosslinking and / or functionalization with one or more agents, such as proteins, enzymes, or therapeutic agents. In certain embodiments, succinylation may be performed by: (a) storing the cellulose material in dimethylacetamide containing 5-20% by weight cellulose and 5-10% by weight LiCl at a temperature of 30°C to 85°C; (b) storing the cellulose material in this solvent for 10 minutes to 1 hour, followed by reaction with succinic anhydride at a molar ratio of 1:2 to a maximum of 1:12 at 30°C to 90°C; (c) thoroughly washing the resulting cellulose material with distilled water for 1-3 days, and then subjecting the resulting solid cellulose monoester material to a step of crosslinking and / or linking proteins, enzymes, and / or drugs. In certain embodiments, dimethylacetamide and LiCl may be used as a solvent in an amount appropriate to avoid dissolving the cellulose, allowing a significant amount of succinic anhydride to react with the cellulose. The succinic acid molecules, combined with the hydroxyl groups, primarily at C6, on the cellulose surface, can provide an activated cellulose surface for preparing commercial products from cellulose, which have applications in, for example, tissue regeneration and pharmaceutical fields. In certain embodiments, the reaction to produce succinate esters of cellulose may be carried out in such a way that the esterification can occur in one step without any dissolution of the cellulose, preserving its crystalline structure and its original form.A succinylation reaction using dimethylacetamide and LiCl as solvents can provide a process for producing succinic acid esters of cellulose, in which esterification occurs in one step without dissolving the cellulose. This reaction can provide an alternative method for producing cellulose ester materials, and can introduce carboxylic acid functional groups that can enable functionalization of cellulose by creating specific biochemical modifications on the cellulose surface while substantially maintaining the original cellulose structure. As an example, in one embodiment, succinylated cellulose can be functionalized with collagen. This was performed in the Examples, and spectra demonstrating collagen linkage are shown in Figure 7.

[0379] Figure 7 shows the results of chemical functionalization of cellulose with denatured collagen. IR spectra of a control scaffold (gray) and a functionalized scaffold (black). The control scaffold was functionalized with a succinic acid linker molecule but lacked collagen. The absorption peaks corresponding to amide (I and II) bonds indicate the covalent attachment of collagen to the cellulose chain via the linker molecule succinic acid. Indicates that the append was successful.

[0380] Figure 24 shows a composite cellulose-based material. Two subunits were glued together with gelatin cross-linked with glutaraldehyde and reduced with sodium borohydride to obtain a composite structure. These structures were biocompatible after reduction. These structures support the growth of GFP 3T3 fibroblast cells. [Example]

[0381] 3D cellulose-based hydrogels with channels In this example, cellulose hydrogels containing built-in channels derived from plant or fungal sources are provided. The cellulose of the hydrogel can be crosslinked according to methods previously described herein, allowing for the preparation of scaffold biomaterials with custom channels configured in the crosslinked hydrogel. Of course, this approach can also be adapted to other hydrogels, such as hemicellulose-based, chitin-based, chitosan-based, pectin-based, lignin-based, lignan-based, or cellulose-based hydrogels, or any combination thereof. In certain embodiments, the components may be sourced from plants and / or fungi. In certain embodiments, the plants and / or fungi may be grown and sterilized. In certain embodiments, the plant components may be enzymatically isolated and concentrated.

[0382] This example provides hydrogels with built-in channels, including materials derived from plant or fungal sources. Cross-linking of the cellulose (or other such structures, such as hemicellulose, chitin, chitosan, pectin, lignin, lignan, or any combination thereof) of the hydrogel can be achieved by the cross-linking techniques described herein. The resulting biomaterial (i.e., 3D cross-linked hydrogel) can have custom channels built into the material. The gel can be printed or cast with a fugitive gel or filler (such as an alginate hydrogel). The fugitive gel can act as a space-filler. Once the cellulose (or other such structures, such as hemicellulose, chitin, chitosan, pectin, lignin, lignan, or any combination thereof) gel has been cross-linked around the fugitive gel, the secondary fugitive gel can be removed. The secondary template hydrogel can be removed using several techniques, such as, for example, a solution change and / or temperature change sufficient to cause a change in the fugitive gel that allows for its removal. Such an approach can provide a non-invasive method for creating channels of desired configuration within cross-linked gels, thus creating more complex 3D networks of channels and / or pores.

[0383] An example of this approach may be the use of alginate as a transient hydrogel that acts as a channel template. Cellulose (or other such structures, such as hemicellulose, chitin, chitosan, pectin, lignin, lignan, or any combination thereof) can be printed around such a channel template and then cross-linked. A calcium-free medium, such as PBS, may then be introduced. Alginate dissolves because it relies on a calcium-rich environment to maintain cross-linking. The resulting structure may be a material with defined channels and pores.

[0384] The ability to template and control channel density and / or placement within a scaffold and / or crosslinked matrix may be desirable for several different applications. For example, in cell culture and / or in vivo implantation applications, channels may be desirable because, in certain circumstances, diffusion alone may be insufficient to provide adequate solution and nutrient exchange, especially for larger structures and / or implants that do not have channels. Insufficient diffusion in such cases can risk causing a necrotic core in which cells cannot survive, even when placed in the center. By introducing appropriately configured channels into a material, it is possible to Such challenges can be reduced or overcome by providing flow through them. For surgical applications where an immediate blood supply is desired, there may not be time to wait for angiogenesis to provide the channels, and therefore in such cases it may be desirable to provide the predetermined vasculature through templated channels.

[0385] Materials and Methods: Scaffold Gel Production: Decellularized materials can be prepared according to the methods and protocols detailed herein. See also International Publication No. WO 2017 / 136950, entitled "Decellularized Cell Wall Structures from Plants and Fungus and Use Thereof as Scaffold Materials," which is incorporated herein by reference in its entirety. The material can then be converted into a gel or paste and extruded or 3D printed. The gel state can be achieved, for example, mechanically or by treatment with bleach or basic solvents. Additionally, the gel can be crosslinked by chemical functionalization techniques described herein.

[0386] Channel templating: Some fugitive gels can act as a template for channels. Fugitive gels can be used to block scaffold gels. This blockage can be achieved by The transient gel can be patterned to leave channels perforating the material upon removal. The method for removing the transient gel can be selected based on the nature and properties of the transient gel (also taking into account the nature and properties of the scaffold gel). Some methods can include, for example, temperature-change-based methods (e.g., using heat to change the state of the transient gel and allow for its removal), ionic solution washing (e.g., washing with a solution or agent that disintegrates or removes the transient gel), and salt buffer exchange (e.g., salt exchange that disintegrates or removes the transient gel). In certain embodiments, the transient gel can include, for example, an alginate gel that can remain relatively solid in the presence of calcium, but can be dissolved and washed away upon solvent exchange with a sodium-based solvent.

[0387] 3D Printing: In certain embodiments, the manufacturing process may involve, for example, a dual extruder system (one for the primary scaffold gel and the other for the temporary gel mold). Materials can be assembled layer by layer, as in traditional 3D printing or resin printing, and channels can be molded throughout the scaffold gel as needed and / or appropriate for the particular intended application.

[0388] In certain embodiments, cellulose hydrogels may be printed or cast with a fugitive hydrogel (e.g., alginate, etc.). The fugitive hydrogel can act as a space filler. After crosslinking the cellulose around the fugitive hydrogel (space filler), the fugitive hydrogel can be removed. Several techniques can be used to remove the fugitive hydrogel, such as by subjecting the material to solution and / or temperature changes. Such approaches can be relatively non-invasive while providing the desired channels for in vivo applications. In certain embodiments, complex 3D networks of channels and / or pores can be engineered.

[0389] In certain embodiments, an alginate-based fugitive hydrogel can be used, which can act as a channel template. Cellulose can be printed or introduced around such an alginate-based fugitive hydrogel channel template, and the cellulose can then be crosslinked. A calcium-free medium, such as PBS, can then be introduced. Since alginate hydrogels typically prefer a calcium-rich environment to maintain crosslinking, the alginate-based fugitive hydrogel may dissolve in a calcium-free medium. The resulting structure then resembles a 3D crosslinked cellulose hydrogel with predetermined channels and / or pores organized according to the geometry of the original fugitive hydrogel. We can provide you with a guide.

[0390] In certain embodiments, such an approach can allow for the creation of custom shapes while substantially preserving the structure of the plant-derived scaffold biomaterial.

[0391] This approach can be used to create custom shapes with customizable features while still preserving the complex architecture of plant-derived scaffold biomaterials. In certain embodiments, it is contemplated that by varying the concentration and crosslinker concentration, complex physical and / or mechanical properties (such as crystalline structure, porosity, ductility, toughness, strength, elasticity, plasticity, or any combination thereof) can be tuned or controlled. In certain embodiments, such an approach can be used, for example, to provide a template for vascularization. In certain embodiments, providing configurable channels can reduce or eliminate problems associated with diffusion dependence in certain applications.

[0392] In certain embodiments, the techniques described herein may include the steps of providing a fractured cellulose gel; functionalizing the cellulose of the fractured cellulose gel with functional moieties for cross-linking; positioning the cellulose gel and fugitive gel such that the fugitive gel provides a template for a desired channel on and / or within the cellulose gel; cross-linking the cellulose gel; and removing the fugitive gel to provide a cross-linked cellulose gel containing a channel templated by the fugitive gel.

[0393] In certain embodiments, the approaches described herein may allow materials to be molded or printed into a desired shape or structure, followed by crosslinking to provide structural integrity to the desired shape or structure, and providing channels and / or pores in the structure at predetermined locations and orientations through the placement of a fugitive gel. In certain embodiments, it is contemplated that the density, directionality, or both of the microstructures, channels, and / or pores may be controllable as desired.

[0394] In certain embodiments, the cellulose gel or hydrogel may be or comprise cellulose, or may be replaced or combined with another similar material, and the cellulose gel or hydrogel described above may be cellulose-based, hemicellulose-based, chitin-based, chitosan-based, pectin-based, lignin, lignan-based, or any combination thereof. In certain embodiments, the 3D hydrogels described herein may be tunable with respect to their biochemical, biophysical, and / or mechanical properties.

[0395] In certain embodiments, suitable drugs, signaling molecules, growth factors, metabolites, and / or ECM proteins and / or components may be attached to (i.e., functionalized or loaded onto and / or within) the 3D hydrogels described herein to provide a desired response, which may be systemic or cell / tissue type specific, and / or positive or inhibitory depending on the particular application. In certain embodiments, the 3D hydrogels described herein may be loaded or functionalized with drugs and used to administer the drug, thereby providing at least some degree of site-specific drug delivery, which, in certain embodiments, can reduce drug dosage and / or increase efficacy. In certain embodiments, the 3D hydrogels described herein can provide time-dependent and / or non-time-dependent release of one or more drugs, such as suitable drugs, signaling molecules, growth factors, metabolites, and / or ECM proteins and / or components, which can be provided by loading the drug into suitable gels with various release characteristics or by covalently attaching the drug to the structure via chemical functionalization methods. In certain embodiments, drug-containing tubes may be loaded into tubes that may be covalently linked via linker molecules such as succinic acid. Such tubes may be, for example, targeted to site-specific locations and may have time-specific release properties.

[0396] In certain embodiments, chemical modifications may provide suitable steric hindrance to allow for increased site-specific modification complexity. In certain embodiments, linker molecules may be used to mediate connections with drugs, signaling molecules, growth factors, metabolites, ECM proteins and / or components, or any combination thereof, as well as tubes containing such compounds. In the absence of other biochemical or biophysical modifications or signaling, different cell attachment densities may lead to different cellular responses, and this approach may allow for tuning the degree of substitution for specific functions, for example.

[0397] In certain embodiments, the 3D hydrogels described herein can be used to provide customizable shapes and / or structures, which may include composites, adhesives, coatings, gels, pastes, or any combination thereof. In certain embodiments, larger macrostructures can be prepared with varying degrees of flexibility and / or connectivity.

[0398] It is contemplated that, using the approaches described herein, the 3D hydrogels described herein may overcome and / or ameliorate certain problems associated with larger sizes and correspondingly long decellularization times, and / or with diffusion in large constructs. In certain embodiments, the structures described herein may allow for the design of certain structures and features not found in nature, while leveraging the natural complexity of the source of scaffolding material (i.e., cellulose, etc.) in the individual subunits. In certain embodiments, the structures described herein may allow for increased complexity in physical and / or mechanical properties (i.e., stress shielding and site-specific modulus, channels, pores, etc.). In certain embodiments, the constructs described herein may allow for modification or tuning of permeability through the use of different adhesives and / or coatings (i.e., tubes, semipermeable membranes, and sealants for junctions). In certain embodiments, the constructs described herein may be tunable with respect to mechanical properties and / or junctions (i.e., Young's modulus can be varied) based on the use of adhesives or coatings. In certain embodiments, the structures described herein may be articulated structures that can provide varying degrees of flexibility and mobility as needed, hi certain embodiments, the structures described herein may allow for the combination of different cell types in different regions.

[0399] In certain embodiments, the 3D hydrogels described herein can be used for 3D printing and / or injectable hydrogel applications. In certain embodiments, the crosslinked plant-derived 3D hydrogels described herein can be for use in non-medical applications, such as synthetic biorobotics, or electrical circuit integration, or any combination thereof. [Example]

[0400] Decellularized celery scaffolds for guided cell alignment of C2C12 mouse myoblasts Cell culture on 2D flat Petri dishes cannot recapitulate cell orientation in vitro, which is a key attribute of functional tissue. A wide variety of methods have been used to induce alignment, including topographical footing, cyclic strain, and electrical stimulation. However, these methods typically involve time-consuming and expensive procedures. In this example, the alignment of C2C12 mouse myoblasts onto decellularized vascular bundles of celery (Apium graveolens) is demonstrated.

[0401] The alignment and orientation of cells in vivo is called anisotropy and plays an important role in tissue functionality. Multinucleated structures in muscle tissue, known as myofibers, rely on uniaxial alignment to generate force along their axis. Airways, arteries, and veins rely on circumferential alignment of smooth muscle to facilitate fluid and gas transport. White matter in the brain relies on anisotropic axonal fibers for proper function. However, in the laboratory, 2D Petri dishes cannot replicate anisotropy, which in turn can lead to differences in gene expression and inaccurate results. To overcome this discrepancy, alignment has been induced using a wide variety of methods, including topographical footholds, cyclic strain, and electrical stimulation. However, these methods are laborious and often rely on the use of specialized equipment. This example demonstrates the alignment of C2C12 mouse myoblasts into decellularized vascular bundles in celery. Xylem channels (38.50 μm ± 6.86 μm) and phloem channels (21.52 μm ± 5.0 μm) fall within the 10–100 μm diameter range appropriate for optimal myoblast alignment. After 10 days in growth medium, the apical ends of C2C12 actin fibers and nuclei were observed to be oriented parallel to the vascular furrow. Subsequently, after 5 days in differentiation medium, myotubes with an average length of 308.08 ± 169.44 μm (N = 103) were angled -2.44° ± 3.83° (N = 14) from the average orientation of the vascular bundles. These results indicate that the microtopography of the vascular bundles guided muscle cell alignment. The results presented here highlight the potential of this plant-derived scaffold for in vitro applications of muscle myogenesis and / or when structural anisotropy is desired to more closely resemble in vivo organization and / or conditions.

[0402] The multinucleated structures of muscle tissue, known as myofibers, allow for the generation of axial force (Chal, J., & Pourquie, O. (2017). Making muscle: Skeletal myogenesis in vivo and in vitro. Development (Cambridge, England), 144(12),2104. doi: 10.1242 / dev.151035; Narayanan et al 2002). Airways, arteries, and veins depend on the circumferential alignment of smooth muscle to facilitate fluid and gas transport (Clark, JF, & Pyne-Geithman, G. (2005). Vascular smooth muscle function: The physiology and pathology of vasoconstriction. Pathophysiology, 12(1), 35-45. doi:10.1016 / j.pathophys.2005.02.007; Komuro, T., Desaki, J., & Uehara, Y. (1982). Three-dimensional organization of smooth muscle Muscle cells in blood vessels of laboratory rodents. Cell and Tissue Research, 227(2), 429-437. doi:10.1007 / BF00210897). The white matter in the brain needs different functions to function properly. Feng, Y., Okamoto, RJ, Namani, R., Genin, GM, & Bayly, PV (2013). Measurements of mechanical anisotropy in brain tissue and implications for transversely isotropic material models of white matter. Journal of (The Mechanical Behavior of Biomedical Materials, 23, 117-132. doi:10.1016 / j.jmbbm.2013.04.007). However, in the laboratory, in vitro studies are typically performed on flat 2D Petri dishes that lack biologically active adhesion sites, dimensionality, microtopography, and proper mechanical stimuli. This, in turn, causes cells to appear randomly scattered, which manifests as dissimilarity in proliferation, differentiation, and global gene expression.To further assimilate 2D cell cultures into the in vivo environment, pillars (Goedecke, N., Bollhalder, M., Bernet, R., Silvan, U., & Snedeker, J. (2015). Easy and accurate mechano-profiling on micropost arrays MyJoveCorporation. doi:10.5167 / uzh-120364), microchannels (Hume, SL, Hoyt, SM, Walker, JS, Sridhar, BV, Ashley, JF, Bowman, CN, & Bryant, SJ (2012). Alignment of multi-layered muscle cells within three-dimensional hydrogel macrochannels. Acta Biomaterialia, 8(6), 2193-2202. doi:10.1016 / j.actbio.2012.02.001), and nanofibers (Fee, T., Surianarayanan, S., Downs, C., Zhou, Y., & Berry, J. (2016). Nanofiber alignment regulates NIH3T3 cell orientation and cytoskeletal gene expression on electrospun PCL+Gelatin nanofibers. PloS One, 11(5), e0154806. doi:10.1371 / journal.pone.0154806) and other substrates with a wide variety of topographical structures have been developed (Goedecke, N., Bollhalder, M., Bernet, R., Silvan, U., & Snedeker, J. (2015). Easy and accurate mechano-profiling on micropost arrays MyJoveCorporation. doi:10.516). 7 / uzh-120364). To further understand the role of directionality, or in other words, to induce cellular anisotropy, grooves, microchannels, annular strain, and electrical stimulation have been shown to induce such phenomena (Tanaka, T., Hattori-Aramaki, N., Sunohara, A., Okabe, K., Sakamoto, Y., Ochiai, H., . . . Kishi, K. (2014). Alignment of skeletal muscle cells cultured in collagen gel by mechanical and electrical stimulation. International Journal of Tissue Engineering, 2014, 1-5.doi:10.1155 / 2014 / 621529, Liu, B., Qu, M., Qin, K., Li,Z., Li, H., Shen, B., & Jiang, Z. (2008;2007;). Role of cyclic strainfrequency in regulating the alignment of vascular smooth muscle cells in vitro.Biophysical Journal, 94(4), 1497-1507. doi:10.1529 / biophysj.106.098574, Hume, SL, Hoyt, SM, Walker, JS, Sridhar, BV, Ashley, JF, Bowman, CN, & Bryant, SJ (2012). Alignment of multi-layered muscle cells within three-dimensional hydrogel macrochannels.Acta Biomaterialia, 8(6), 2193-2202. doi:10.1016 / j.actbio.2012.02.001, Altomare, L., Gadegaard, N., Visai, L., Tanzi, M. C., & Fare, S. (2010). Biodegradable microgrooved polymeric surfaces obtained by photolithography for skeletal muscle cell orientation and myotube development. Acta Biomaterialia, 6(6), 1948 - 1957. doi:10.1016 / j.actbio.2009.12.040, Charest, J.L., Garcia, A. J., & King, W. P. (2007). Myoblast alignment and differentiation on cell culture substrates with microscale topography and model chemistries. Biomaterials, 28(13), 2202 - 2210. doi:10.1016 / j.biomaterials.2007.01.020). Smooth muscle cells (Kuppan, P., Sethuraman, S., & Krishnan, U. M. (2016). Interaction of human smooth muscle cells on random and aligned nanofibrous scaffolds of PHBV and PHBV - gelatin. International Journal of Polymeric Materials and Polymeric Biomaterials, 65(16), 816. doi:10.1080 / 00914037.2016.1163562), bone Muscle cells (Cooper, A., Jana, S., Bhattarai, N., & Zhang, M. (2010). Aligned chitosan-based nanofibers for enhanced myogenesis. Journalof Materials Chemistry, 20(40), 8904. doi:10.1039 / c0jm01841d), neurons (Basso, JMV, Simon, M., & Staii, C. (2018). Neuronal dynamics on patterned substrates measured by fluorescence microscopy. MRS Communications, 8(2), 487-492. doi:10.1557 / mrc.2018.52), and tendon-derived cells (Foolen, J., Wunderli, SL, Loerakker, S., & Snedeker, JG (2018). Tissue alignment enhances remodeling potential of tendon-derived cells - lessons from a novel microtissue model often don scarring. Matrix Biology, 65, 14-29. doi:10.1016 / j.matbio.2017.06.002) compared to the smooth surface of a tissue culture dish. Note that uniaxial grooves exhibited differences in gene expression. Skeletal muscle cells exhibited upregulation of troponin T, myosin heavy chain, and myogenin on uniaxial grooves (Cooper, A., Jana, S., Bhattarai, N., & Zhang, M. (2010). Aligned chitosan-based nanofibers for enhanced myogenesis. Journal of Materials Chemistry, 20(40), 8904. doi:10.1039 / c0jm01841d). Substrate topography has also been shown to influence the lineage differentiation of mesenchymal stem cells. Mesenchymal stem cells cultured on grooves and ridges committed to myogenic and adipogenic lineages, whereas smooth surfaces induced osteogenic differentiation (Wang, P., Li, W., Yu, J., & Tsai, W. (2012). Modulation of osteogenic, adipogenic, and myogenic differentiation of mesenchymal stem cells by submicron grooved topography. Journal of MaterialsScience: Materials in Medicine, 23(12), 3015-3028. doi:10.1007 / s10856-012-4748-6).

[0403] Microchannel development was achieved using photolithography (Camelliti, P., Kohl, P., McCulloch, AD, & Gallagher, JO (2006). Micropatterned cell cultures on elastic membranes as an in vitro model of myocardium. Nature Protocols, 1(3), 1379-1391. doi:10.1038 / nprot.2006.203, Leclerc, A., Tremblay, D., Hadjiantoniou,S., Bukoreshtliev, NV, Rogowski, JL, Godin, M., & Pelling, AE (2013). Three dimensional spatial separation of cells in response to microtopography. Biomaterials, 34(33), 8097-8104.doi:10.1016 / j.biomaterials.2013.07.047), femtosecond pulsed laser Users (Yeong, W. Y., Yu, H., Lim, K. P., Ka Lai Gary Ng, Yin Chiang Freddy Boey, Subbu, V. S., & Tan, L. P. (2010). Multiscale topological guidance for cell alignment via direct laser writing on biodegradable polymer. Tissue Engineering Part C: Methods, 16(5), 1011. doi:10.1089 / ten.tec.2009.0604), 3D printing (Tan, Z., Liu, T., Zhong, J., Yang, Y., & Tan, W. (2017). Control of cell growth on 3D‐printed cell culture platforms for tissue engineering. Journal of Biomedical Materials Research Part A, 105(12), 3281-3292. doi:10.1002 / jbm.a.36188, Tijore, A., Irvine, S. A., Sarig, U., Mhaisalkar, P., Baisane, V., & Venkatraman, S. (2018). Contact guidance for cardiac tissue engineering using 3D bioprinted gelatin patterned hydrogel. Biofabrication, 10(2), 025003. doi:10.1088 / 1758-5090 / aaa15d), and electron beam lithography (Wang, P., Yu, H., & Tsai, W. (2010). Modulation of alignment and differentiation of skeletal myoblasts by submicron ridges / grooves surface structure. Biotechnology and Bioengineering, 106(2), 285-n / a.doi:10.1002 / bit.22697; Goto et al. 2007) to provide tunability and comparison Due to its ease of use, it has proven to be a popular method. Microchannel widths ranging from 5 to 200 μm have been shown to be able to induce myoblast alignment, with channels between 20 and 100 μm wide allowing optimal myotube maturation (Hume, SL, Hoyt, SM, Walker, JS, Sridhar, BV, Ashley, JF, Bowman, CN, & Bryant, SJ (2012). Alignment of multi-layered muscle cells within three-dimensional hydrogels). macrochannels. Acta Biomaterialia,8(6), 2193-2202. doi:10.1016 / j.actbio.2012.02.001, Sun, Y., Duffy, R., Lee, A., & Feinberg, AW (2013). Optimizing the structure and contractility of engineered skeletal muscle thin films. Acta Biomaterialia, 9(8), 7885-7894.doi:10.1016 / j.actbio.2013.04.036, Altomare, L., Gadegaard, N., Visai, L., Tanzi, MC, & Fare, S. (2010). Biodegradable microgrooved polymeric surfaces obtained by photolithography for skeletal muscle cell orientation and myotube development. Acta Biomaterialia, 6(6),1948-1957. doi:10.1016 / j.actbio.2009.12.040, Charest, JL, Garcia, AJ, & King, WP (2007). Myoblast Alignment and differentiation on cell culture substrates with microscale topography and model chemistries. Biomaterials, 28(13), 2202-2210. doi:10.1016 / j.biomaterials.2007.01.020). Channel depth has also been shown to play a role in cell alignment. Microgrooves 2 μm deep provide a transient foothold for cell alignment, while channels greater than 5 μm deep have been shown to induce permanent alignment (Zhao, Y., Zeng, H., Nam, J., & Agarwal, S. (2009). Fabrication of skeletal muscle constructs by topographic activation of cell alignment. Biotechnology and Bioengineering, 102(2), 624-631. doi:10.1002 / bit.22080, Hume, SL, Hoyt, SM, Walker, JS, Sridhar, BV, Ashley, JF, Bowman, CN, & Bryant, SJ (2012). Alignment of multi-layered muscle cells within three-dimensional hydrogel macrochannels. Acta Biomaterialia, 8(6), 2193-2202.doi:10.1016 / j.actbio.2012.02.001). This observation This appears to be cell line specific: C2C12 responded to grooves smaller than 0.5 μm, unlike primary myoblasts (Altomare, L., Gadegaard, N., Visai, L., Tanzi, M.C., & Fare, S. (2010). Biodegradable microgrooved polymeric surfaces obtained by photolithography for skeletal muscle cell orientation and myotube development. Acta Biomaterialia, 6(6), 1948-1957. doi:10.1016 / j.actbio.2009.12.040). On the considered substrates Cell alignment in the smear may be due to localization and contact guidance. Cells were considered aligned when the average angle of the cells relative to the substrate pattern direction was less than 10° (Altomare, L., Gadegaard, N., Visai, L., Tanzi, MC, & Fare, S. (2010). Biodegradable Microgrooved polymeric surfaces obtained byphotolithography for skeletal muscle cell orientation and myotube development.Acta Biomaterialia, 6(6), 1948-1957. doi:10.1016 / j.actbio.2009.12.040, Charest, JL, Garcia, AJ, & King, WP (2007). Myoblast alignment and differentiationon cell culture substrates with m icroscale topography and model chemistries.Biomaterials, 28(13), 2202-2210. doi:10.1016 / j.biomaterials.2007.01.020).

[0404] The biocompatibility of decellularized plant tissue in vitro and in vivo makes such a substrate attractive. The use of detergents such as SDS can damage cell membranes, leading to cell lysis (Fontana, G., Gershlak, J., Adamski, M., Lee, J., Matsumoto, S., Le, H.D., . . . Murphy, W. L. (2017). Biofunctionalized plants as diverse biomaterialsfor human cell culture. Advanced Healthcare Materials, 6(8), n / a.doi:10.1002 / adhm.201601225、Modulevsky, D. J., Lefebvre, C., Haase, K., Al-Rekabi,Z., & Pelling, A. E. (2014). Apple derived cellulose scaffolds for 3Dmammalian cell culture. PLoS One, 9(5), e97835.doi:10.1371 / journal.pone.0097835, Brown, R. B., & Audet, J. (2008). Current techniques for single-cell lysis. Journal of the Royal Society Interface, 5(Suppl 2), S131. doi:10.1098 / rsif.2008.0009.focus, Modulevsky, D. J., Cuerrier, C. M., & Pelling, A. E. (2016). Biocompatibility of subcutaneously implanted plant-derived cellulose biomaterials. PLoS One, 11(6), e0157894.doi:10.1371 / journal.pone.0157894, Hickey, R. J., Modulevsky, D. J., Cuerrier, C. M., & Pelling, A. E. (2018). Customizing the shape and microenvironment biochemistry of biocompatible macroscopic plant-derived cellulose scaffolds. ACS Biomaterials Science & Engineering, doi:10.1021 / acsbiomaterials.8b00178). Immortalized cell line has been shown to grow throughout relatively porous decellularized apple tissue without the need for biofunctionalization (Modulevsky, DJ, Lefebvre, C., Haase, K., Al-Rekabi, Z., & Pelling, AE (2014). Apple derived cellulose scaffolds for 3D mamm...

Claims

1. A hydrogel, such as a cellulose-based hydrogel, comprising: a crosslinked matrix, such as a crosslinked cellulose matrix, and One or more channels formed in the crosslinked matrix by removing the fugitive space-filling material from the crosslinked matrix. The hydrogel comprising:

2. The hydrogel of claim 1, wherein the crosslinked matrix is ​​prepared from decellularized plant or fungal tissue, from which cellular material and nucleic acids of the tissue have been removed, and wherein the decellularized plant or fungal tissue comprises a three-dimensional porous structure.

3. A hydrogel as described in claim 1 or 2, wherein the temporary space filling material comprises a temporary gel such as an alginate hydrogel.

4. A hydrogel described in any of claims 1 to 3, wherein a matrix is ​​crosslinked around a transient space filler, and then the transient space filler is removed from the crosslinked matrix, thereby forming one or more channels.

5. The hydrogel of claim 4, wherein the transient space-filling material has been removed by dissolution, heating, a change in salt concentration, decomposition, or any combination thereof.

6. A hydrogel described in any of claims 1 to 5, wherein a transient space-filling material is disposed within the crosslinked matrix so as to form a template for a network of channels within the matrix.

7. The hydrogel described in claim 6, wherein the transient space-filling material is disposed within the matrix by deposition via 3D printing.

8. A hydrogel described in any of claims 1 to 7, further comprising one or more agents such as a therapeutic agent, a signaling molecule, a growth factor, a metabolite, an ECM protein or component, or any combination thereof.

9. A hydrogel described in any of claims 1 to 8, wherein the matrix is ​​prepared from decellularized plant or fungal tissue that is cellulose-based, hemicellulose-based, chitin-based, chitosan-based, pectin-based, lignin-based, lignan-based, or any combination thereof.

10. A hydrogel according to any one of claims 1 to 9, wherein the crosslinked matrix comprises at least two structurally different celluloses.

11. The hydrogel of claim 10, wherein the at least two structurally different celluloses are derived from different plant or fungal sources, from different parts of the same plant or fungal source, from two different species of plant or fungal source, exhibit different Young's modulus properties, comprise different cell types, comprise different hydrogels, or any combination thereof.

12. Use of a hydrogel according to any of claims 1 to 11 for tissue repair or regeneration; in an implant; for culturing one or more cell types in vitro or in vivo; for mimicking in vivo tissue or tissue interfaces; for bone tissue engineering; for bone repair or regeneration; for transporting fluids or liquids; for mimicking tissue interfaces; for wound healing; for delivering a drug such as a therapeutic agent, a signalling molecule, a growth factor, a metabolite, an ECM protein or component, or any combination thereof; or for any combination thereof.

13. A method for repairing or regenerating tissue in a subject in need thereof; for providing an implant; for culturing one or more cell types; for mimicking an in vivo tissue or tissue interface; for bone tissue engineering; for repairing or regenerating bone; for transporting fluids or liquids; for mimicking a tissue interface; for wound healing; for delivering a drug such as a therapeutic agent, a signaling molecule, a growth factor, a metabolite, an ECM protein or component, or any combination thereof; or any combination thereof, comprising: Providing a hydrogel according to any one of claims 1 to 11, and introducing the hydrogel into the subject at a site in need thereof; The method comprising:

14. A method for preparing a hydrogel having one or more channels, such as a cellulose-based hydrogel having one or more channels, comprising: providing a first crosslinkable material or gel, such as a natural or modified cellulose, chitin, lignin, lignan, hemicellulose, or pectin-based material or gel; providing a transient space-filling material; generating a three-dimensional structure comprising the first crosslinkable material or gel having the fugitive space-filling material dispersed therein such that the fugitive space-filling material forms a template for one or more channels in the first crosslinkable material or gel, and crosslinking the first crosslinkable material or gel during or after generating the three-dimensional structure; and removing the temporary space-filling material from the three-dimensional structure to provide a hydrogel having the one or more channels. The method comprising:

Images