Filament hydrogels and uses thereof

Abstract

The present invention relates to a fibril hydrogel comprising an aqueous solution and substantially aligned fibrils comprising collagen and at least one proteoglycan, said fibril hydrogel having viscoelastic characteristics such that it can be used in a wide range of industries, such as in the field of use, as well as in the field of use. Including but not limited to the food industry, the biomaterial industry, the pharmaceutical industry and the cosmetic industry, and for research purposes.

Description

Field of the Invention

[0001] The present invention relates to a fibrillar hydrogel comprising an aqueous solution and substantially aligned fibrils comprising collagen and at least one proteoglycan, the fibrillar hydrogel having viscoelastic properties enabling its use in a wide range of industries, including but not limited to the food industry, biomaterials industry, pharmaceutical industry and cosmetics, as well as for research purposes. Background of the Invention

[0003] The extracellular matrix (ECM) is an aggregate of macromolecules arranged in a three-dimensional network designed to give structural and functional support to the cells embedded therein. The ECM contains various types of macromolecules, including but not limited to collagen, elastin, fibronectin, laminin, glycoproteins, proteoglycans, and glycosaminoglycans (GAGs). The exact composition of the ECM varies from tissue to tissue, and adequately mimicking this highly critical supportive environment is essential for fields such as tissue engineering and ex vivo cell culture. There has been increasing success in producing artificial ECM-like hydrogels in a variety of ways, although the characteristics of the produced hydrogels are inconsistent. Challenges in creating hydrogels that closely resemble the authentic ECM present in living tissues include batch-to-batch compositional variations, the inability to accurately recreate the structural integrity of the natural ECM, and reliance on chemical regimens that render the hydrogels biologically incompatible.

[0004] Fibrillar hydrogels are a class of artificial hydrogels that successfully recreate the filamentous architecture of the in vivo ECM, as well as the compositional precision imparted by the controlled nature of the synthetic process. However, structural integrity alone is not sufficient to create functional ECM-mimicking hydrogels. The correct macromolecular composition, which in turn affects biophysical properties, is very important for the functionality and biological relevance of fibrillar hydrogels.

[0005] Collagen is the major ECM component of certain animal species, including mammals. The primary role of collagen is to provide a scaffold to support tissue, although many other functions of collagen have been elucidated, including roles in cell attachment, cell migration, filtration, and morphogenesis. Skin or animal hide contains large amounts of collagen.

[0006] "Collagen" refers to the name of a superfamily of proteins characterized by a triple helical domain that constitutes more than 95% of the molecule. This domain is composed of three alpha chains, each containing approximately 1,000 amino acids, wound in a rope-like manner to form a tight triple helical structure. Each alpha chain contains a repeating triplet of amino acids -(Gly-XY)n-, with glycine accounting for approximately one-third of the amino acid residues in collagen. Although there may be up to 400 possible Gly-XY triplets, X is usually proline and Y is usually hydroxyproline. Hydroxylation of proline residues is essential for helical integrity, and defects in this process lead to widespread defects throughout connective tissue. Different animals can produce different collagen amino acid compositions, which can result in different collagen properties. The collagen structure consists of three interwoven peptide chains of varying lengths that form the collagen triple helix.

[0007] During the production of extracellular matrix by skin fibroblasts, triple helical monomers are synthesized, and these monomers can self-assemble into fiber forms. These triple helices are held together by electrostatic interactions, which include salt bridging, hydrogen bonding, van der Waals interactions, dipole-dipole forces, polarization forces, hydrophobic interactions and / or covalent bonds. The triple helices can be bound together into bundles, called protofibrils, and the protofibrils can be further assembled to produce fibers and fiber bundles. Due to the alternating overlap of collagen monomers, protofibrils have a characteristic ribbon-like appearance. Protofibrils and fibers usually branch and interact throughout the skin layer or hide layer. Changes in the organization or cross-linking of protofibrils and fibers can provide strength to the material.

[0008] At present, there are 28 known different collagen types. The collagen types are numbered by Roman numerals, and the chains found in each collagen type are identified with Arabic numerals. Detailed descriptions of the structure and biological functions of various types of naturally occurring collagen are available in the art (e.g., Ayad et al. 1998. The Extracellular Matrix Facts Book, Academic Press, San Diego, CA).

[0009] Type I collagen is the most common form of collagen in mammalian species and is widely distributed in the skin, bones, muscles, tendons and lungs throughout the body. It is the main structural macromolecule present in the extracellular matrix of multicellular organisms and accounts for approximately 20% of the total protein mass. Type I collagen is a heterotrimeric molecule comprising two α1 (I) chains and one α2 (I) chain. Other collagen types are not as abundant as type I collagen and show different distribution patterns. For example, type II collagen is the main collagen in cartilage and vitreous humor. Type III collagen is found as the main structural component of hollow organs such as large blood vessels, uterus and intestine. It is also found in many other tissues together with type I collagen.

[0010] In addition to animal skin or hides, collagen has been successfully isolated from various areas of the mammalian body. In recent years, collagen has been harvested from bacteria and yeast using recombinant technology.

[0011] The international (PCT) application publication No. WO 2022 / 043994 of the applicant of the present invention provides a system and method for producing acellular animal collagen and / or a culture medium containing the collagen. The system and method of this invention include a cell culture that continuously produces a soluble collagen secreted into the culture medium, so that the soluble collagen can be easily collected. Thereafter, the soluble collagen can be processed to form collagen fibrils and fibers, and the collagen product can be used in medicine or cosmetics, in the cell culture food industry, or can be further biomanufactured to form leather-like textiles.

[0012] International (PCT) application publication No. WO 2023 / 017509 of the applicant of the present invention provides compositions and methods for generating lineage-committed progenitor cells (especially including ECM-producing cells, including collagen-producing cells) from pluripotent stem cells and cells differentiated therefrom.

[0013] The beneficial characteristics of collagen have led to its use in tissue engineering and a variety of biomedical applications, and great efforts have been made to produce high-quality collagen. Medical and cosmetic applications of collagen include, for example, dermal fillers, wound dressings, and guided tissue regeneration.

[0014] For example, International (PCT) Application Publication No. WO 2018 / 046920 discloses a method for producing a jellyfish collagen hydrogel and a kit for producing the hydrogel. The jellyfish collagen hydrogel can be used in the preparation of 3D cell culture scaffolds and in the preparation of medical devices.

[0015] Collagen scaffolds have been widely used in tissue engineering due to their low immunogenicity, porous structure, good permeability, biocompatibility, and biodegradability. These scaffold structures serve as templates with specific mechanical and biological properties similar to the natural extracellular matrix (ECM).

[0016] Collagen is also a major component in many cosmetic preparations, as it is a natural humectant and moisturizer. Hydrolyzed collagen is primarily used in hair preparations and skin care products, but can also be found in cosmetics, shampoos, and bath products. Hydrolyzed collagen can also be used in hair dyes.

[0017] Leather is used in a wide variety of applications, including furniture upholstery, clothing, shoes, luggage, handbags and accessories, and automotive applications. The global trade value of leather is estimated to be $100 billion per year, and there is a continuous and increasing demand for leather products. However, due to increased awareness of animal welfare, the use of environmentally harmful chemicals, and health issues, the use of natural leather from slaughtered animals has been rejected by the public. Therefore, new methods are needed to meet the demand for leather materials that are suitable for large-scale production and exhibit at least equal or better properties than natural leather (e.g., International (PCT) Application Publication Nos. WO 2017 / 003999 and WO 2017 / 142887; U.S. Application Publication No. 2019 / 0203000).

[0018] U.S. Patent No. 9,644,177 discloses membranes formed from type I collagen for use as a basal layer model, potentially for forming complex three-dimensional structures. The membranes described therein are formed from type I collagen and one or more additional ECM proteins, such as additional types of collagen, laminin, vitronectin, fibronectin, and chondroitin sulfate proteoglycans, and comprise a bilayer surface.

[0019] There is an unmet need for adequate mimetic hydrogels for use as synthetic ECMs that possess the qualities of the in vivo ECM, including structural integrity, compositional accuracy, correct biophysical properties, and biocompatibility. Fibrillar hydrogels of this nature are essential for countless industries, including but not limited to tissue engineering and cell culture, the cosmetic and food industries, the imitation leather industry, and the like. SUMMARY OF THE INVENTION

[0021] The present invention provides a fibrillar hydrogel comprising: an aqueous solution, typically a buffer or water; and more than one substantially aligned fibrils comprising animal collagen and at least one type of proteoglycan. In an exemplary embodiment, the fibrils and / or the hydrogel further comprise additional extracellular matrix (ECM) proteins, and the pH of the hydrogel aqueous solution is non-acidic. In certain embodiments, the animal is a mammal, including humans and non-human mammals. According to certain exemplary embodiments, the animal is a non-human animal. Advantageously, according to certain embodiments, the fibrillar hydrogel of the present invention is substantially free of pyrogens. According to certain exemplary embodiments, the fibrillar hydrogel of the present invention is substantially free of endotoxins.

[0022] The present invention is based in part on the unexpected discovery that collagen and proteoglycans secreted from bovine embryonic fibroblasts and / or collagen-producing cells differentiated from bovine-derived pluripotent stem cells can form fibrils that advantageously maintain their fibril structure in non-acidic aqueous solutions having a pH of at least about 6.00 to form stable fibrillar hydrogels.

[0023] Another advantage of the fibril hydrogel of the present invention is that the fibrils are substantially aligned parallel to each other, forming an ordered multi-axis alignment in the hydrogel, which contributes to the viscoelastic characteristics of the hydrogel and the viscoelasticity of the products produced therefrom. As another advantage, the fibril hydrogel of the present invention has a filamentous structure similar to the filamentous structure of the natural ECM. This advantageously makes the fibril hydrogel of the present invention suitable for use as a raw material in a variety of industries, including the pharmaceutical industry, consumer products (e.g., non-animal leather), the food industry, cosmetics, etc., without the need for additional priming procedures. In a variety of applications, the fibril hydrogel can be used directly without significant priming.

[0024] According to certain aspects, the present invention provides a fibrillar hydrogel comprising an aqueous solution and more than one fibril comprising collagen and at least one type of proteoglycan, wherein the pH of the hydrogel aqueous solution is at least about 6.00, and wherein at least a portion of the fibrils are orderly aligned within the hydrogel.

[0025] According to some embodiments, the ordered alignment is a dual-axis alignment. According to certain embodiments, the ordered alignment is a multi-axis alignment.

[0026] According to some embodiments, the fibrils are aligned in parallel.

[0027] According to some embodiments, at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the fibrils are orderly aligned within the hydrogel. Each possibility represents a separate embodiment of the present invention.

[0028] According to some embodiments, the fibrils are orderly aligned in segments with a fibril length of about 100 nm to about 10 mm.

[0029] According to certain embodiments, the collagen molecule comprises hydroxylated proline and hydroxylated lysine residues of at least 40% of the total number of proline and lysine residues of said collagen molecule.

[0030] According to certain embodiments, the aqueous solution is selected from the group consisting of: water, a buffer solution and a cell culture medium. Each possibility represents a separate embodiment of the present invention.

[0031] According to certain exemplary embodiments, the aqueous solution is water.

[0032] According to further exemplary embodiments, the aqueous solution is a buffer solution capable of maintaining a pH of 6.0 and above. According to some embodiments, the buffer solution is capable of maintaining a pH in the range of about 6.0 to about 8.0.

[0033] According to certain embodiments, the aqueous solution content of the fibril hydrogel is about 30% to 99%, 35% to 99%, 40% to 99%, 45% to 99%, 45% to 99%, 50% to 99%, 55% to 99%, 60% to 99%, 65% to 99%, 70% to 99%, 75% to 99%, 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% w / w of the total wet weight of the hydrogel. Each possibility represents a separate embodiment of the present invention.

[0034] According to certain embodiments, the aqueous solution content of the fibril hydrogel is about 90% to about 99%. According to these embodiments, the fibril hydrogel comprises a dry weight content of about 1% to about 10% of the total weight of the hydrogel. Each possibility represents a separate embodiment of the present invention.

[0035] According to certain embodiments, the aqueous solution content of the fibril hydrogel is about 95% to about 99%. According to these embodiments, the fibril hydrogel comprises a dry weight content of about 1% to about 5% of the total wet weight of the hydrogel. Each possibility represents a separate embodiment of the present invention.

[0036] According to some embodiments, type I collagen is the main type of collagen contained in the fibrils. According to certain embodiments, type I collagen forms about at least 60% w / w of the dry weight of the total collagen based on the hydrogel. According to certain embodiments, the fibrils comprise at least one additional collagen selected from the group consisting of: collagen III, collagen VI, collagen XII, collagen IV, collagen V, collagen XI, collagen XIV, collagen XVIII and any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0037] According to certain embodiments, the at least one additional collagen is selected from the group consisting of: collagen III, collagen VI, collagen XII, and any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0038] According to certain embodiments, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, or at least 50% or more of the total number of proline and lysine residues in collagen I are hydroxylated. Each possibility represents a separate embodiment of the present invention.

[0039] According to certain embodiments, the collagen I molecule comprises hydroxylated lysine residues of at least 25% of the total number of lysine residues on the molecule. According to certain embodiments, the percentage of hydroxylated lysine residues is from about 25% to about 35% of the total lysine residues in collagen I. Each possibility represents a separate embodiment of the present invention.

[0040] According to certain embodiments, the collagen I molecule comprises hydroxylated proline residues of at least 40% of the total number of proline residues on the molecule. According to certain embodiments, the percentage of hydroxylated proline residues is from about 40% to about 60% of the total proline residues in the collagen I molecule. Each possibility represents a separate embodiment of the present invention.

[0041] According to certain embodiments, the at least one proteoglycan is selected from the group consisting of decorin, biglycan, lumican, prolargin, asporin, fibromodulin, versican, and any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0042] According to some embodiments, the proteoglycan core protein is associated with at least one glycosaminoglycan (GAG) selected from the group consisting of chondroitin sulfate, dermatan sulfate, heparin, heparan sulfate and keratan sulfate. Each possibility represents a separate embodiment of the present invention.

[0043] According to some embodiments, at a temperature in the range of about 22° C. to about 26° C., the yield stress of the fibril hydrogel comprising about 90% to about 99% aqueous solution content is about 1 Pa to about 50 Pa, or about 1 Pa to about 20 Pa, or about 5 Pa to about 20 Pa. Each possibility represents a separate embodiment of the present invention.

[0044] According to certain exemplary embodiments, at a temperature of 25°C, a fibril hydrogel comprising an aqueous solution content of about 90% to about 99% has a yield stress of about 2 Pa to about 20 Pa.

[0045] According to some embodiments, the phase angle (°) of the fibril hydrogel comprising about 90% to about 99% aqueous solution content is in the range of about 1° to about 50° or about 5° to about 20° at a temperature of about 22° to about 26° C. Each possibility represents a separate embodiment of the present invention.

[0046] According to some embodiments, the phase angle of the fibril hydrogel comprising about 90% to about 99% aqueous solution content is about 12° to about 20° at a temperature of about 22° to about 26° C. Each possibility represents a separate embodiment of the present invention.

[0047] According to some embodiments, the total amount of collagen is about 5% to about 95% w / w (on a dry weight basis) of the total dry weight of the fibril hydrogel. According to certain embodiments, the total amount of collagen is about 20% to about 90% w / w (on a dry weight basis) of the total dry weight of the fibril hydrogel. According to certain exemplary embodiments, the total amount of collagen is about 70% to about 80% (on a dry weight basis) of the total dry weight of the fibril hydrogel.

[0048] According to some embodiments, the fibrillar hydrogel further comprises hyaluronic acid.

[0049] According to certain embodiments, the fibrillar hydrogel further comprises at least one glycoprotein selected from the group consisting of fibronectin, serpin H1, galectin-1, periostin, laminin, peroxidasin, Elastin Microfibril Interface Localized Protein (EMILIN), fibrillin, tenascin C, Secreted protein acidic and rich in cysteine ​​(SPARC), and any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0050] According to certain embodiments, the fibrillar hydrogel further comprises at least one protein of cytoskeletal filaments. According to certain exemplary embodiments, the protein of cytoskeletal filaments is selected from the group consisting of: actin, vimentin, myosin, filament protein, tropomyosin, transgelin, tubulin and any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0051] According to certain embodiments, the fibrillar hydrogel further comprises at least one cell membrane protein. According to certain exemplary embodiments, the cell membrane protein is an annexin.

[0052] According to certain embodiments, the fibrillar hydrogel is produced by animal cells cultured in culture under conditions capable of synthesizing collagen.

[0053] According to some embodiments, the conditions capable of synthesizing collagen include the following steps: (i) growing the animal cells under conditions capable of at least one of cell proliferation, cell differentiation, cell maturation, and any combination thereof; and (ii) adding at least one agent that induces collagen synthesis to the culture medium.

[0054] According to certain embodiments, the agent inducing collagen synthesis is selected from the group consisting of: iron, insulin, ascorbic acid or salts thereof, and combinations thereof. Each possibility represents a separate embodiment of the present invention.

[0055] According to certain exemplary embodiments, the agent that induces collagen synthesis is ascorbic acid or a salt thereof.

[0056] Collagen-producing cells can be obtained from humans as well as from any non-human animal that has collagen-producing cells.

[0057] According to certain embodiments, the collagen producing animal is selected from the group consisting of: mammals, poultry, aquatic animals, invertebrates and reptiles. Each possibility represents a separate embodiment of the present invention.

[0058] According to certain embodiments, the collagen producing cells are human cells. According to other embodiments, the collagen producing cells are of a non-human animal.

[0059] According to some embodiments, the non-human animal is an animal known to be used for its skin or hide, including but not limited to bovine, sheep, horse, crocodile, and the like.

[0060] According to some embodiments, the collagen producing non-human mammal is an ungulate.

[0061] According to some embodiments, the ungulate is selected from the group consisting of bovine, buffalo, ovine, equine, pig, giraffe, camel, deer, hippopotamus and rhino. Each possibility represents a separate embodiment of the present invention.

[0062] According to certain exemplary embodiments, the ungulate is a bovine.

[0063] According to certain other exemplary embodiments, the reptile is of the family Crocodylidae.

[0064] According to certain embodiments, the cells are stromal cells. According to certain exemplary embodiments, the stromal cells are fibroblasts.

[0065] According to certain additional or alternative embodiments, the cells are collagen-producing cells differentiated from pluripotent stem cells.

[0066] The fibril hydrogels of the present invention can be used in a variety of applications. According to certain embodiments, the fibril hydrogels are used to form biomaterials (for the textile industry and for medical purposes), foods, nutritional products (including dietary supplements and food additives), cosmetics or medical products. Each possibility represents a separate embodiment of the present invention.

[0067] According to certain exemplary embodiments, the fibril hydrogels of the present invention are used in the production of textiles, including leather-like textiles. The natural acellular fibril hydrogels of the present invention are suitable for use in a variety of textile production methods currently known and to be known in the art.

[0068] According to further exemplary embodiments, the fibrillar hydrogels of the present invention are used in the production of food products, in particular cultured meat products.

[0069] According to further exemplary embodiments, the fibril hydrogels of the present invention are used in the production of medical materials and devices.

[0070] According to a further exemplary embodiment, the fibril hydrogel of the present invention is used in the production of a cosmetic product.

[0071] According to certain additional aspects, the present invention provides a textile comprising at least 10% of the fibril hydrogel of the present invention. According to certain embodiments, the textile has skin / hide / leather-like characteristics. According to certain embodiments, the textile comprises about 20% to about 60% of the fibril hydrogel. Each possibility represents a separate embodiment of the present invention.

[0072] According to yet other certain aspects, the present invention provides a food product comprising at least 0.5% of the fibril hydrogel of the present invention. According to certain embodiments, the food product comprises about 1% to about 5% of the fibril hydrogel. Each possibility represents a separate embodiment of the present invention. According to certain embodiments, the food product is a cell cultured meat product.

[0073] According to certain further aspects, the present invention provides a medical material comprising at least 0.1% of the fibrillar hydrogel of the present invention. According to certain embodiments, the medical material comprises from about 0.1% to about 1% of the fibrillar hydrogel. Each possibility represents a separate embodiment of the present invention.

[0074] According to yet certain other aspects, the present invention provides a cosmetic product comprising at least 0.01% of the fibrillar hydrogel of the present invention. According to certain embodiments, the cosmetic comprises from about 0.1% to about 1% of the fibrillar hydrogel. Each possibility represents a separate embodiment of the present invention.

[0075] According to certain further aspects, the present invention provides a nutritional product comprising at least 1% of the fibrillar hydrogel of the present invention. According to certain embodiments, the nutritional product comprises from about 1% to about 20% of the fibrillar hydrogel. Each possibility represents a separate embodiment of the present invention.

[0076] It should be understood that any combination of each of the aspects and embodiments disclosed herein is expressly encompassed within the present disclosure.

[0077] Additional embodiments and the full scope of the applicability of the present invention will become apparent from the detailed description given below. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only, because various changes and modifications within the spirit and scope of the present invention will be apparent to those skilled in the art from the detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 shows a magnification of x4 ( Figure 1A )、x10( Figure 1B ) and x20( Figure 1C ) (taken by an Olympus CKX53 cell culture microscope).

[0080] Figure 2 Depicted is a Coomassie-stained SDS-PAGE gel of proteins obtained from fibrillar hydrogels. The left lane shows a kDa scale standard.

[0081] Figure 3 Depicted is the analysis of type I collagen within the fibrillar hydrogel proteins. Proteins separated by SDS-PAGE were transferred to a PVDF membrane and membrane proteins were analyzed using anti-COL1 antibodies. A major band of the expected size was observed (130 / 139 kDa).

[0082] Figure 4 Depicted is the analysis of type III collagen within fibrillar hydrogel proteins. Proteins separated by SDS-PAGE were transferred to PVDF membranes and membrane proteins were analyzed using anti-COL3A1 antibodies. A band of the expected size was observed (140 kDa).

[0083] Figure 5 Depicted is the analysis of type VI collagen within the fibrillar hydrogel protein. The protein purified by SDS-PAGE was transferred to a PVDF membrane and the membrane protein was analyzed using an anti-COL16A2 antibody. A band of the expected size (120-160 kDa) was observed.

[0084] Figure 6 Depicted is the analysis of type XII collagen within fibrillar hydrogel proteins. Proteins purified by SDS-PAGE were transferred to PVDF membranes and membrane proteins were analyzed using anti-COL12A1 antibodies. Several bands >185 kDa were observed (expected size: 333 kDa).

[0085] Figure 7 Schematic representation of the major protein types and protein classes revealed by LC-MS / MS is depicted.

[0086] Figure 8The MS-MS proteomic analysis of the fibrillar hydrogel-collagen 1 alpha chain 1 (COL1α1) is shown. Grey letters-amino acid residues identified by MS-MS; Black letters-amino acid residues not identified by MS-MS; Shaded (P)-hydroxylation observed on proline residues; Shaded (K)-hydroxylation observed on lysine residues; (▼)-hydroxylation on lysine residues in the literature; (●)-hydroxylation on proline residues in the literature.

[0087] Fig. 9 The MS-MS proteomic analysis of the fibrillar hydrogel-collagen 1 α chain 2 (COL1α2) is shown. Grey letters-amino acid residues identified by MS-MS; Black letters-amino acid residues not identified by MS-MS; Shaded (P)-hydroxylation observed on proline residues; Shaded (K)-hydroxylation observed on lysine residues; (▼)-hydroxylation on lysine residues in the literature; (●)-hydroxylation on proline residues in the literature.

[0088] Fig.10 A graph depicting the relationship between viscosity (Pa·s) and shear stress (Pa) of a fibrillar hydrogel produced from bovine fibroblasts is shown. The graph demonstrates a complex nonlinear relationship between viscosity and shear stress, with viscosity increasing with increasing shear stress until a yield stress value of 16.2 (Pa), after which viscosity decreases with further increases in shear stress.

[0089] Fig.11 A rheological diagram representing the viscoelastic behavior of a fibrillar hydrogel produced from bovine fibroblasts is shown. The diagram shows the elastic modulus (G'), the viscous modulus (G") Pa in Pa, the phase angle (δ) in degrees °, and the Y* parameter in %.

[0090] Fig.12 A graph depicting the relationship between viscosity (Pa·s) and shear stress (Pa) of three batches of fibrillar hydrogels produced from bovine pluripotent stem cells is shown. Batch 1 (○), Batch 2 (◇), Batch 3 (□). The graph shows a complex nonlinear relationship between viscosity and shear stress, with viscosity increasing with increasing shear stress until the yield stress value is 4 (Pa), after which the viscosity decreases with further increase in shear stress.

[0091] Fig.13 A rheological graph representing the viscoelastic behavior of a fibrillar hydrogel produced from bovine pluripotent stem cells is shown. The graph displays the elastic modulus (G') in Pa, the viscous modulus (G") in Pa, the phase angle (δ) in degrees °, and the Y* parameter in %. DETAILED DESCRIPTION OF THE INVENTION

[0093] The present invention discloses a fibrillar hydrogel, which comprises an aqueous solution and more than one fibril, wherein the fibril comprises collagen and at least one type of proteoglycan. In an exemplary embodiment, the collagen is non-human animal collagen, particularly type I collagen, the hydrogel further comprises additional ECM proteins, and the hydrogel fibril structure is stable at non-acidic pH.

[0094] The fibril hydrogels of the present invention are superior to the collagen-containing hydrogels known to date, at least in that the hydrogels themselves can be used in a variety of applications and the preparation process does not require further initiation. The fibril hydrogels of the present invention are the best raw materials for the sustainable preparation of products based on animal-derived materials. According to certain embodiments of the present invention, the fibril hydrogels can be used in a variety of applications in the textile, pharmaceutical, food, nutrition and cosmetic industries. In particular, the fibril hydrogels of the present invention can have a large number of medical uses, such as for the preparation of wound dressings and / or scaffolds for tissue regeneration.

[0095] The collagen-containing hydrogel of the present invention can also be used in the preparation of textiles, mainly in the preparation of skin-like or animal-like or leather-like textiles, and in the food industry, for the production of cultured meat.

[0096] With reference to the accompanying description, the principles and operation of the present invention can be better understood. Before explaining in detail at least one embodiment of the present invention, it should be understood that the present invention is not necessarily limited to the details shown in the following description or illustrated by the examples in its application. The present invention can carry out other embodiments, or can be practiced or carried out in a variety of ways. In addition, it will be understood that the wording and terminology used herein are for the purpose of description and should not be considered as limiting.

[0097] Collagen biosynthesis is a complex process involving intracellular and intercellular steps. The intracellular steps include gene translation and polypeptide synthesis, especially the synthesis of collagen α chain; post-translational modifications, including proline and lysine hydroxylation and glycosylation; and procollagen triple helix formation, including proline cis-trans isomerization and stabilization by heat shock protein 47 (HSP47). Soluble procollagen is then secreted into the intercellular space, where it is processed by N-terminal and C-terminal propeptide cleavage to form tropocollagen. Covalent cross-linking then occurs within and between triple helical collagen molecules to form fibrils, which are assembled to form collagen fibers as part of the ECM.

[0098] According to the teaching of the present invention, most of the collagen present in the hydrogel is in the form of fibrils. Therefore, as used herein, the term "collagen" refers to any known collagen type, including type I to type XX collagen. The term also encompasses procollagen and any collagenous protein comprising the motif (Gly-XY) n, where n is an integer. It encompasses collagen molecules, trimers of collagen molecules and collagen fibrils. Collagen types are interchangeably labeled with Arabic numerals (1, 2, 3, etc.) and Roman numerals (I, II, III, etc.) herein.

[0099] As used herein, the term "fibril" or "fibrils" refers to a filamentous structure of a fibrous structural protein, primarily type 1 collagen and at least one type of proteoglycan, including but not limited to decorin, biglycan, lumican, prolargin, asporin, fibromodulin and versican. Each possibility represents a separate embodiment of the present invention. According to certain embodiments, the fibrous structural protein further comprises at least one of type 3 collagen, type 4 collagen, type 5 collagen, type 6 collagen, type 11 collagen, type 12 collagen, type 14 collagen, type 18 collagen, fibronectin, fibrillin, vitronectin, elastin, and any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0100] The term "proteoglycan" is used herein in its broadest sense as known in the art. The basic proteoglycan unit consists of a "core protein" and one or more covalently attached glycosaminoglycan (GAG) chains. The attachment point is a serine residue to which the glycosaminoglycan is connected by a tetrasaccharide bridge. According to some embodiments, the proteoglycan core protein associates with at least one glycosaminoglycan (GAG) selected from the group consisting of chondroitin sulfate, dermatan sulfate, heparin, heparan sulfate and keratan sulfate. Each possibility represents a separate embodiment of the present invention. According to further embodiments, the fibrillar hydrogel and / or the fibrils contained therein comprise GAGs that do not form part of the proteoglycan. According to certain embodiments, at least one proteoglycan is selected from the group consisting of decorin, biglycan, photocan, prolargin, asporin, fibromodulin, versican and any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0101] The fibrillar hydrogels of the present invention may further comprise one or more cytoskeletal filament proteins, including but not limited to actin, vimentin, filamin, and myosin, which contribute to the formation of filamentous structures.

[0102] According to certain embodiments, the fibrils of the fibrillar hydrogels of the present invention have diameters ranging from nanometers to micrometers, typically from about 100 nm to about 10 μm, and lengths up to millimeters, typically ranging from about 100 nm to 10 mm.

[0103] As used herein, the term "hydrogel" refers to any dispersion of molecules, fibrils or particles in a liquid in which a solid is a discontinuous phase and a liquid is a continuous phase. According to certain exemplary embodiments, the term "hydrogel" refers to a dispersion of more than one fibril in an aqueous solution as described above, wherein the hydrogel comprises at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% aqueous solution and at most about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70% dry weight. Each possibility represents a separate embodiment of the present invention. Therefore, the hydrogel of the present invention is referred to as a "fibrillar hydrogel". It should be clearly understood that the advantage of the fibrillar hydrogels of the present invention is that the fibrils are generally or nearly orderly aligned within the hydrogel, typically parallel to each other in a multi-axial order, contributing to the stability and viscoelastic characteristics of the hydrogel, and therefore to the elasticity of the products produced therefrom.

[0104] According to some embodiments, at least a portion of the fibrils are orderly aligned within the fibril hydrogel. According to some embodiments, the aligned fibrils exhibit parallel alignment within the hydrogel. According to some embodiments, the fibrils exhibit tilted alignment within the hydrogel.

[0105] According to some embodiments, at least a portion of the fibrils exhibit a networked alignment. According to some embodiments, at least a portion of the network-aligned fibrils are arranged to form a complex interconnected lattice or mesh structure within the hydrogel. According to some embodiments, the network-aligned fibrils exhibit a non-linear interconnected alignment within the hydrogel.

[0106] According to some embodiments, the aligned fibrils are biaxially aligned within the hydrogel. According to some embodiments, the aligned fibrils are multiaxially aligned within the hydrogel.

[0107] According to some embodiments, between about 30% to 95%, 35% to 90%, 30% to 85%, 30% to 80%, 30% to 75%, 30% to 75%, 30% to 70%, 30% to 65%, 30% to 60%, 30% to 55%, 30% to 50%, 30% to 45%, or 30% to 40% of the fibrils are aligned within the hydrogel. Each possibility represents a separate embodiment of the invention.

[0108] According to some embodiments, between about 30% to 35%, 35% to 40%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, or 90% to 95% of the fibrils are aligned within the hydrogel. Each possibility represents a separate embodiment of the invention.

[0109] According to some embodiments, the fibrils are aligned with a fibril length of about 100 nm to about 10 mm. According to some embodiments, the fibrils are aligned with a fibril length of about 100 nm to 500 nm, 500 nm to 1 μm, 1 μm to 5 μm, 5 μm to 10 μm, 10 μm to 20 μm, 20 μm to 50 μm, 50 μm to 100 μm, 100 μm to 200 μm, 200 μm to 300 μm, 300 μm to 400 μm, 400 μm to 500 μm, 500 μm to 600 μm, 600 μm to 700 μm, 700 μm to 800 μm, 800 μm to 900 μm, 900 μm to 1 mm, 1 mm to 2 mm, 2 mm to 3 mm, 3 mm to 4 mm, 4 mm to 5 mm, 5 mm to 6 mm, 6 mm to 7 mm, 7 mm to 8 mm, 8 mm to 9 mm, or 9 mm to 10 mm. Each possibility represents a separate embodiment of the present invention.

[0110] According to some embodiments, the fibrils are aligned with a fibril length of at least about 100 nm, 500 nm, 1 μm, 5 μm, 10 μm, 20 μm, 50 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, or 9 mm. Each possibility represents a separate embodiment of the invention.

[0111] According to some embodiments, the fibrils are aligned with a fibril length of no more than about 100 nm, 500 nm, 1 μm, 5 μm, 10 μm, 20 μm, 50 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, or 9 mm. Each possibility represents a separate embodiment of the invention.

[0112] As used herein, the term "more than one" means two or more.

[0113] As used herein, the term "substantially or approximately aligned" refers to a hydrogel in which the fibrils show more directional uniformity in the matrix compared to a "random" fibril hydrogel.

[0114] As used herein, the term "about" refers to ± 10%.

[0115] The terms "comprise," "comprising," "includes," "including," "having" and their cognates mean "including but not limited to."

[0116] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "fibril" or "protein" may include more than one fibril and protein, including mixtures thereof.

[0117] Throughout this application, various embodiments of the present invention can be presented in the form of ranges.It should be understood that the description in the form of ranges is only for convenience and brevity and should not be interpreted as an unchangeable limitation to the scope of the present invention.Therefore, the description of the scope should be considered to have specifically disclosed all possible sub-ranges and the individual numerical values ​​within the scope.No matter how wide the scope is, this is applicable.No matter when indicating a numerical range, it is intended to include any quoted numerical value (fraction or integer) within the scope of the indication.The wording "ranging / ranges between" the first indicating number and the second indicating number "between" and "ranging / ranges from" the first indicating number "to" the second indicating number are used interchangeably herein and are intended to include the first indicating number and the second indicating number and all fractions and integers therebetween.

[0118] According to certain aspects, the present invention provides a fibrillar hydrogel comprising an aqueous solution and more than one fibril, the fibrils comprising collagen and at least one type of proteoglycan, wherein the pH of the hydrogel aqueous solution is at least about 6.00, and wherein at least a portion of the fibrils are orderly aligned within the hydrogel.

[0119] According to certain aspects, the present invention provides a fibrillar hydrogel comprising an aqueous solution and more than one fibril, the fibrils comprising collagen and at least one type of proteoglycan, wherein at least 40% of the collagen proline and lysine residues of the collagen molecules are hydroxylated, and wherein the pH of the hydrogel aqueous solution is at least about 6.00.

[0120] According to certain aspects, the present invention provides a fibrillar hydrogel comprising an aqueous solution and more than one fibril, wherein the fibrils comprise collagen and at least one type of proteoglycan, wherein the collagen molecules comprise hydroxylated proline and hydroxylated lysine residues of at least 40% of the total number of proline and lysine residues of the collagen molecules, wherein the pH of the hydrogel aqueous solution is at least about 6.00, and wherein at least a portion of the fibrils are orderly aligned within the hydrogel.

[0121] According to certain embodiments, the ordered alignment is a multi-axis alignment.

[0122] As used herein, the term "multi-axial alignment" with respect to fibrils within the hydrogels of the present invention refers to the arrangement or orientation of the fibrils along multiple axes or directions, wherein more than one type of fibril is oriented along a single axis or direction, forming an ordered alignment of the fibrils within the hydrogel.

[0123] According to certain embodiments, the water content of the fibril hydrogel is at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, %, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. Each possibility represents a separate embodiment of the present invention.

[0124] According to certain exemplary embodiments, the content of the aqueous solution of fibril hydrogel is in the range of about 90% to about 99%.Each possibility represents a separate embodiment of the present invention.

[0125] According to certain exemplary embodiments, the content of the aqueous solution of fibril hydrogel is in the range of about 95% to about 99%.Each possibility represents a separate embodiment of the present invention.

[0126] According to some embodiments, the aqueous solution is selected from the group consisting of: water, a buffer solution and a cell culture medium. According to some embodiments, the buffer is selected from the group consisting of: phosphate buffered saline (PBS), tris buffered saline (TBS) or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer. Each possibility represents a separate embodiment of the present invention.

[0127] The cell culture medium can be any culture medium known in the art that is suitable for growing animal cells, particularly mammalian cells. Common cell culture media used in the art include Minimal Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), and Roswell Park Memorial Institute-1640 (RPMI-1640). According to certain exemplary embodiments, the aqueous solution is a buffered solution.

[0128] According to certain additional exemplary embodiments, the aqueous solution is water.

[0129] According to certain embodiments, the pH of the aqueous solution is at least 6.1, at least 6.2, at least 6.3, at least 6.4, at least 6.5, at least 6.6, at least 6.7, at least 6.8, at least 6.9, at least 7.0, at least 7.1, at least 7.2, at least 7.3, at least 7.4, at least 7.5, at least 7.6, at least 7.7, at least 7.8, at least 7.9 or at least 8.0 or more. Each possibility represents a separate embodiment of the present invention.

[0130] According to certain embodiments, the pH of the aqueous solution is in the range of about 6.0 to about 8.0, or about 6.0 to about 7.5, or about 6.0 to about 7.0.

[0131] According to some embodiments, type I collagen is the major type of collagen contained in the fibrils.

[0132] According to certain embodiments, type I collagen forms about at least 60% of the total collagen of the hydrogel. According to some embodiments, type I collagen forms about at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80% or more of the total collagen of the hydrogel. Each possibility represents a separate embodiment of the present invention.

[0133] According to certain exemplary embodiments, type I collagen forms about 60% to about 80% of the total collagen in the hydrogel.

[0134] According to certain embodiments, the fibrils comprise type I collagen and at least one additional collagen selected from the group consisting of: collagen III, collagen IV, collagen VI, collagen XII, collagen V, collagen XI, collagen XIV, collagen II, collagen XI, collagen XVIII, and any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0135] According to certain embodiments, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, or at least 50% or more of the total number of proline and lysine residues in collagen I are hydroxylated. Each possibility represents a separate embodiment of the present invention.

[0136] According to certain embodiments, hydroxylated proline and hydroxylated lysine residues account for about 40% to about 60%, or about 40% to about 55%, or about 40% to about 50% of the total number of proline and lysine residues on the collagen I molecule. Each possibility represents a separate embodiment of the present invention.

[0137] According to certain embodiments, the hydroxylated lysine residues account for at least 25% of the total number of lysine residues on the collagen I molecule. According to certain embodiments, the range of hydroxylated lysine residues is from about 25% to about 45%, or from about 25% to about 40%, or from 25% to about 35% of the total lysine residues in the collagen I molecule. Each possibility represents a separate embodiment of the present invention.

[0138] According to certain embodiments, the hydroxylated proline residues account for at least 40% of the total number of proline residues on the collagen I molecule. According to certain embodiments, the range of hydroxylated proline residues is from about 40% to about 70%, or from about 40% to about 65%, or from about 40% to about 60%, or from about 40% to about 50% of the total proline residues in the collagen I molecule. Each possibility represents a separate embodiment of the present invention.

[0139] Without wishing to be bound by any theory or mechanism of action, the degree of hydroxylation of collagen proline and lysine residues to the above values ​​contributes to fibril formation and alignment and, therefore, to the viscoelastic properties of the fibrillar hydrogels.

[0140] According to certain embodiments, the fibril hydrogel dry weight is from about 1% to about 10% of the total wet weight of the fibril hydrogel.

[0141] According to certain embodiments, the fibril hydrogel dry weight is about 30%, about 35%, about 40%, about 45%, about 50% or about 55% to about 60%, about 65%, about 70% of the total wet weight of the fibril hydrogel. Each possibility represents a separate embodiment of the present invention.

[0142] According to certain embodiments, the fibril hydrogel dry weight is about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, or about 4.0% to about 6%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, or about 8.5% of the total wet weight of the fibril hydrogel. Each possibility represents a separate embodiment of the present invention.

[0143] According to certain exemplary embodiments, the fibril hydrogel dry weight is from about 2% to about 6% of the total wet weight of the fibril hydrogel.

[0144] According to some embodiments, the total amount of collagen is about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% to about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% w / w of the total dry weight of the fibrillar hydrogel. Each possibility represents a separate embodiment of the present invention.

[0145] According to certain embodiments, the total amount of collagen is about 20% to about 90%, about 30% to about 90% w / w, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90% of the dry weight of the fibrillar hydrogel.

[0146] According to certain exemplary embodiments, the total amount of collagen is about 70% to 80% w / w of the dry weight of the fibrillar hydrogel.

[0147] According to some embodiments, the fibrillar hydrogel further comprises hyaluronic acid.

[0148] The fibrillar hydrogels of the present invention are produced by animal cells cultured in a culture medium (generally in suspension). Therefore, the fibrillar hydrogels generally also contain additional proteins secreted into the culture medium. Without wishing to be bound by any theory or mechanism of action, additional proteins may contribute to the fibrillar formation and / or fibrillar properties of the hydrogel.

[0149] According to certain embodiments, the fibrillar hydrogel further comprises at least one protein selected from the group consisting of: glycoproteins, proteins of cytoskeletal filaments, cell membrane proteins, and any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0150] According to certain embodiments, the glycoprotein is selected from the group consisting of: fibronectin, serpin H1, neuroblast differentiation-associated protein AHNAK (desmoyokin), galectin-1, periostin, laminin, peroxiredoxin, EMILIN, fibrillin, tenascin C, secreted protein acidic and rich in cysteine ​​(SPARC), and any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0151] According to certain embodiments, the fibrillar hydrogel further comprises at least one protein of a cytoskeletal filament. According to certain exemplary embodiments, the protein of a cytoskeletal filament is selected from the group consisting of actin, vimentin, myosin, filamin, tropomyosin, lamin, transcollin, tubulin, and any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0152] According to certain exemplary embodiments, the amount of proteins of cytoskeletal filaments is about 33% w / w of the total dry weight of the hydrogel. In other exemplary embodiments, the proteins of cytoskeletal filaments are present in the following percentages of the total dry weight of the hydrogel: actin (10%), vimentin (6%), myosin (2.5%), filamin A (2%), tropomyosin (1.3%), and lamins, elongation factor 1-α1, transglutamin and tubulin account for the remaining 11.2%.

[0153] According to certain embodiments, the amount of cytoskeleton filaments forming proteins in the total dry weight of the hydrogel can be reduced by decellularization. According to certain embodiments, the amount of cytoskeleton filaments forming proteins in the fibrillar hydrogel undergoing decellularization can be changed in the range of about 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, 9% to 10%, 10% to 11%, 11% to 12%, 12% to 13%, 13% to 14%, 14% to 15%, 15% to 16%, 16% to 17%, 17% to 18%, 18% to 19% or 19% to 20%. Each possibility represents a separate embodiment of the present invention.

[0154] According to certain embodiments, the fibrillar hydrogel further comprises at least one cell membrane protein. According to certain exemplary embodiments, the cell membrane protein is an annexin.

[0155] According to certain embodiments, the fibrillar hydrogel is produced by animal cells cultured in a culture medium under conditions capable of synthesizing collagen. According to certain exemplary embodiments, the conditions capable of synthesizing collagen include adding at least one collagen synthesis inducer to the cell culture medium.

[0156] According to certain embodiments, the agent inducing collagen synthesis is selected from the group consisting of: iron, insulin, ascorbic acid or salts thereof, and combinations thereof. Each possibility represents a separate embodiment of the present invention.

[0157] According to certain exemplary embodiments, the agent that induces collagen synthesis is ascorbic acid or a salt thereof.

[0158] Collagen-producing cells can be obtained from any non-human animal that has collagen-producing cells as well as from human cells.

[0159] According to certain exemplary embodiments, the collagen-producing cells are human cells.

[0160] According to certain additional or alternative embodiments, the collagen-producing cells are of a non-human animal.

[0161] According to certain embodiments, the non-human animal is selected from the group consisting of: non-human mammals, poultry, aquatic animals, invertebrates and reptiles. Each possibility represents a separate embodiment of the present invention.

[0162] According to some embodiments, the non-human animal is an animal known to be used for its skin or hide, including but not limited to bovines, sheep, horses, crocodiles, and the like.

[0163] According to some embodiments, the collagen producing cells are of a non-human ungulate.

[0164] According to some embodiments, the ungulate is selected from the group consisting of: Bovinae, buffalo, Ovis, Equine, pig, giraffe, camel, deer, hippopotamus and rhino. Each possibility represents a separate embodiment of the present invention.

[0165] According to certain exemplary embodiments, the ungulate is a bovine.

[0166] According to certain other exemplary embodiments, the reptile is of the family Crocodile.

[0167] According to certain embodiments, the cells are stromal cells. According to certain exemplary embodiments, the stromal cells are fibroblasts.

[0168] According to certain additional or alternative embodiments, the cells are collagen-producing cells differentiated from pluripotent stem cells.

[0169] According to some embodiments, the fibril hydrogels of the present invention have shape retention at physiological temperature.

[0170] The yield stress value may be a value obtained from a yield stress analysis measurement recorded at a temperature of about 22°C to about 25°C and at a strain value of 0.04 Pa to 40 Pa.

[0171] According to some embodiments, the fibrillar hydrogels of the invention having a liquid content of about 90% to about 99% exhibit yield stress values ​​in the range of 1 Pa to 50 Pa, 5 Pa to 50 Pa, 10 Pa to 50 Pa, 10 Pa to 40 Pa, 10 Pa to 35 Pa, 10 Pa to 30 Pa, 10 Pa to 25 Pa, or 10 Pa to 20 Pa, as determined by yield stress analysis at a temperature of 25° C. Each possibility represents a separate embodiment of the present invention.

[0172] According to certain exemplary embodiments, a fibril hydrogel comprising about 90% to about 99% aqueous solution has a yield stress in the range of about 10 Pa to about 20 Pa at 25°C.

[0173] The elastic modulus value (G') of the hydrogel may be the value recorded from an oscillation amplitude sweep analysis at a temperature of about 22° C. to about 25° C. Each possibility represents a separate embodiment of the present invention.

[0174] According to some embodiments, at a temperature of about 22° C. to about 25° C., the fibril hydrogel of the present invention having a liquid content of about 90% to about 99% has an elastic modulus (G') (Pa) of at least about 200 Pa, 210 Pa, 230 Pa, 250 Pa, 270 Pa, 290 Pa, 310 Pa, 330 Pa or 350 Pa. Each possibility represents a separate embodiment of the present invention.

[0175] According to some embodiments, at a temperature of about 22° C. to about 25° C., a fibril hydrogel of the invention having a liquid content of about 90% to about 99% has an elastic modulus (G') (Pa) of no more than about 200 Pa, 210 Pa, 230 Pa, 250 Pa, 270 Pa, 290 Pa, 310 Pa, 330 Pa or 350 Pa. Each possibility represents a separate embodiment of the present invention.

[0176] According to some embodiments, at a temperature of about 22°C to about 25°C, the fibril hydrogel of the present invention having a liquid content of about 90% to about 99% has an elastic modulus (G') (Pa) in the range of about 200 Pa to about 350 Pa. Each possibility represents a separate embodiment of the present invention. According to certain exemplary embodiments, at a temperature of about 22°C to about 25°C, the fibril hydrogel of the present invention having a liquid content of about 90% to about 99% has an elastic modulus (G') (Pa) in the range of about 250 Pa to about 300 Pa. Each possibility represents a separate embodiment of the present invention.

[0177] The elastic modulus (G') values ​​mentioned above are derived from oscillation amplitude sweep analysis.

[0178] According to some embodiments, the fibrillar hydrogels of the present invention have a range of elastic moduli (from oscillation amplitude sweep analysis), wherein the minimum and maximum amounts are selected from the above-described embodiments.

[0179] The phase angle (°) of the hydrogel may be the value recorded from the oscillation amplitude sweep analysis at temperatures of about 22° C. to about 25° C. Each possibility represents a separate embodiment of the present invention.

[0180] According to some embodiments, at a temperature of about 22° C. to about 25° C., a fibril hydrogel of the invention having a liquid content of about 90% to about 99% has a phase angle (°) of at least about 1°, 5°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, or 50°. Each possibility represents a separate embodiment of the present invention.

[0181] According to some embodiments, at a temperature of about 22° C. to about 25° C., a fibril hydrogel of the invention having a liquid content of about 90% to about 99% has a phase angle (°) of no more than about 1°, 5°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, or 50°. Each possibility represents a separate embodiment of the present invention.

[0182] According to certain exemplary embodiments, at a temperature of about 22°C to about 25°C, a fibril hydrogel of the present invention having a liquid content of about 90% to about 99% has a phase angle (°) ranging from about 5° to about 20°.

[0183] The above phase angle (°) values ​​are derived from oscillation amplitude sweep analysis.

[0184] According to some embodiments, at a temperature in the range of about 22° C. to about 26° C., the fibril hydrogel of the present invention having a liquid content of about 90% to about 99% is characterized by at least one of the following: a yield stress of about 1 Pa to about 50 Pa, an elastic modulus (G') of about 200 Pa to about 350 Pa, a phase angle (°) of about 1° to about 50°, and any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0185] According to certain embodiments, the fibril hydrogel of the present invention having a liquid content of about 90% to about 99% at a temperature in the range of about 22° C. to about 26° C. is characterized by at least one of: a yield stress of about 16 Pa, an elastic modulus (G') in the range of about 250 Pa to about 300 Pa, a phase angle (°) of about 15°, and any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0186] According to certain exemplary embodiments, at a temperature in the range of about 22° C. to about 26° C., the fibril hydrogel of the present invention having a liquid content of about 90% to about 99% has a yield stress of about 16 Pa, an elastic modulus (G') in the range of about 250 Pa to about 300 Pa, and a phase angle (°) of about 15°.

[0187] According to some embodiments, the rheological properties of the fibrillar hydrogel of the present invention remain substantially the same after at least one deformation and reorganization cycle, such as two deformation and reorganization cycles. The rheological properties may be one or more properties selected from the group consisting of: yield stress, elastic modulus, and phase angle.

[0188] According to some embodiments, the fibrillar hydrogels of the present invention exhibit a high elastic modulus (G') compared to their viscous modulus (G"), indicating a more solid-like behavior while still maintaining some liquid-like characteristics.

[0189] According to some embodiments, the fibril hydrogel is substantially free of pyrogens. According to some embodiments, the fibril hydrogel is substantially free of endotoxins. According to some embodiments, the fibril hydrogel of the present invention is substantially non-cytotoxic. According to some embodiments, the fibril hydrogel of the present invention is substantially free of residual monomers.

[0190] The fibril hydrogels of the present invention are suitable for a variety of uses. According to certain embodiments, the fibril hydrogels of the present invention are used as biomaterials. According to certain embodiments, the biomaterials are used in the production of textiles (particularly skin-like or animal hide-like or leather-like textiles). The fibril hydrogels are suitable for use in a variety of textile production methods currently known and to be known in the art. According to certain embodiments, the biomaterials are used for medical purposes, such as for the construction of artificial organs or prostheses or for the replacement of bones or tissues. The fibril hydrogels of the present invention are also suitable for use in the food industry. In certain exemplary embodiments, the fibril hydrogels of the present invention are used in the production of cultured meat. The fibril hydrogels can be used in their hydrogel form, or can be biomanufactured as a scaffold for cell growth.

[0191] According to some embodiments, the fibril hydrogels of the invention are suitable for use in formulating nutraceuticals, dietary supplements and food additives. Each possibility represents a separate embodiment of the invention.

[0192] According to some embodiments, the fibrillar hydrogels of the present invention are suitable for use as fat replacers in low-calorie or low-fat food products, providing a similar mouthfeel and texture without adding calories or fat content.

[0193] According to some embodiments, the fibrillar hydrogels of the present invention are suitable for use as thickeners or gelling agents in various food products such as sauces, soups, and dressings.

[0194] According to some embodiments, the fibrillar hydrogels of the present invention are suitable for use as binders in meat, plant-based protein products, or cultured meat products to improve texture and cohesion.

[0195] According to some embodiments, the fibril hydrogels of the present invention are suitable for use as humectants in baked goods, enhancing their texture and shelf life. According to some embodiments, the fibril hydrogels of the present invention are suitable for use as texture modifiers in gluten-free food products, improving overall mouthfeel and consistency.

[0196] According to some embodiments, the fibrillar hydrogels of the present invention are suitable for use as moisture absorbers in the production of crispy food products, such as dried cultured meat products, maintaining their desired texture.

[0197] According to some embodiments, the fibril hydrogels of the present invention are suitable for medical use.

[0198] According to some embodiments, the fibril hydrogel is suitable for use as a wound dressing to promote healing and tissue regeneration. According to these embodiments, the fibril hydrogel may further comprise at least one active agent selected from the group consisting of an antimicrobial agent, a debridement agent, a coagulation promoter, a cell growth and / or tissue regeneration factor, an anti-inflammatory agent, an analgesic, and any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0199] According to some embodiments, the fibrillar hydrogel is suitable for use as a joint filler to provide support, cushioning and promote cartilage regeneration in damaged joints. According to some embodiments, the joint is a knee joint.

[0200] According to some embodiments, the fibrillar hydrogel is suitable for use as a surgical mesh for tissue reinforcement.

[0201] According to some embodiments, the fibrillar hydrogels are suitable for use as a controlled-release drug delivery system, releasing therapeutic agents to a targeted area over time.

[0202] According to some embodiments, the fibrillar hydrogel is suitable for use as a scaffold for angiogenesis.

[0203] According to some embodiments, the fibrillar hydrogel is suitable for use as a supportive scaffold for a skin graft.

[0204] According to some embodiments, the fibrillar hydrogel is suitable for use as an injectable filler for soft tissue augmentation, such as in facial rejuvenation procedures or breast augmentation.

[0205] According to some embodiments, the fibrillar hydrogels are suitable for use as cell culture scaffolds, providing a supportive environment for the growth and differentiation of various cell types.

[0206] According to some embodiments, the fibrillar hydrogels are suitable for use in tissue engineering applications to create customized tissue constructs for implantation, such as in reconstructive surgery.

[0207] According to some embodiments, fibrillar hydrogels are used to guide tissue regeneration. Collagen-based membranes can be used in periodontal and implant therapy to promote the growth of specific cell types.

[0208] According to some embodiments, the fibril hydrogel of the present invention is suitable for cosmetic use. According to some embodiments, the fibril hydrogel is used as a skin filler. Fillers containing collagen can be used in cosmetics to remove fine lines and wrinkles from the face, and can also improve scars.

[0209] According to some embodiments, the fibrillar hydrogels of the present invention are suitable for use in non-surgical nasal remodeling.

[0210] According to some embodiments, the fibril hydrogels of the present invention are suitable for use in brow enhancement, creating a natural-looking semi-permanent solution for thicker brows.

[0211] According to some embodiments, the fibrillar hydrogels of the present invention are suitable for use in skin tightening treatments.

[0212] According to some embodiments, the fibrillar hydrogels of the present invention are suitable for use in cosmetic augmentation including the breasts and buttocks.

[0213] According to some embodiments, the fibril hydrogel of the present invention is suitable for use in treating stretch marks. According to some embodiments, the fibril hydrogel of the present invention is suitable for use in treating dark circles under the eyes.

[0214] A further advantage of the fibrillar hydrogel of the present invention is that it can be used in the above-mentioned applications without requiring any further processing.

[0215] According to some embodiments, the fibril hydrogel is used to produce synthetic leather. According to some embodiments, the fibril hydrogel is used to produce leather-like textiles.

[0216] According to some embodiments, the fibril hydrogel is used to produce a woven textile.

[0217] According to some embodiments, the fibril hydrogels are used to produce nonwoven textiles.

[0218] According to some embodiments, the fibril hydrogels are used in the production of textiles for medical applications, such as wound dressings or sutures.

[0219] According to some embodiments, the fibril hydrogels are used in producing environmentally friendly textiles.

[0220] According to certain other aspects, the present invention provides textiles comprising at least 10% of the fibril hydrogel of the present invention. According to certain embodiments, the textile has skin / hide / leather characteristics. According to certain embodiments, the textile comprises about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% to about 30%, 35%, 40%, 45%, 50% or 60% of the fibril hydrogel. Each possibility represents a separate embodiment of the present invention.

[0221] According to some other aspects, the invention provides a food product comprising at least 0.5% of the fibril hydrogel of the present invention. According to some embodiments, the food product comprises about 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.3%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2% to about 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% of the fibril hydrogel. According to some embodiments, the food product is a meat product of cell culture. Each possibility represents a separate embodiment of the present invention.

[0222] According to certain other aspects, the present invention provides medical materials comprising at least 0.1% of the fibril hydrogel of the present invention. According to certain embodiments, the medical material comprises about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8% or 0.9% to about 1%, 1%, 1.1%, 1.3%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2% of the fibril hydrogel. Each possibility represents a separate embodiment of the present invention.

[0223] According to yet other certain aspects, the present invention provides a cosmetic product comprising at least 0.01% of the fibril hydrogel of the present invention. According to certain embodiments, the cosmetic comprises from about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1% to about 1%, 1%, 1.1%, 1.3%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2% of the fibril hydrogel. Each possibility represents a separate embodiment of the present invention.

[0224] According to some other aspects, the invention provides a nutritional product comprising at least 1% of the fibril hydrogel of the present invention. According to some embodiments, the nutritional product comprises about 1%, 2%, 3%, 3%, 4% or 5% to about 15%, 16%, 17%, 18%, 19% or 20% of the fibril hydrogel. Each possibility represents a separate embodiment of the present invention.

[0225] It should be understood that certain features of the invention described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention described in the context of a single embodiment for brevity may also be provided individually or in any suitable subcombination, or suitably in any other described embodiment of the invention. Certain features described in the context of various embodiments are not considered essential features of those embodiments, unless the embodiment would not function without those elements.

[0226] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. Example

[0227] Example 1: Cell culture and harvesting of fibrillar hydrogels

[0228] 1.1 Production of fibrillar hydrogels using bovine fibroblasts

[0229] Bovine embryonic fibroblasts were expanded in standard culture medium (DMEM + 10% fetal bovine serum). 24 hours after inoculation, the culture medium was changed to basal medium supplemented with ascorbic acid (150 mg / L) to increase the induction of collagen. After 6 days of induction, the culture medium was aspirated and the cells were washed with DPBS without calcium and magnesium. Preheated recombinant trypsin EDTA was added and the cells were incubated at 38.5°C for 5 minutes until a thin fibrillar hydrogel appeared. To dilute the trypsin, an equal volume of DMEM was added to the trypsin and the fibrillar hydrogel was collected into a sterile tube stored at 4°C.

[0230] An exemplary optical microscopy picture (Olympus CKX53 cell culture microscope) showing the organized structure of fibrils within the hydrogel is presented in Figure 1A-Figure 1C middle.

[0231] 1.2 Production of fibrillar hydrogels using bovine pluripotent stem cells (PSCs)

[0232] Bovine pluripotent stem cells (PSC) are grown in 3D cultures. PSC is dissociated and resuspended in a serum-free growth medium containing activin A (20ng / ml) and CHIR-990215 (10μM) for a period of 4 days. Rho-associated kinase (ROCK) inhibitor (10μM) is introduced on the first day of growth. After 4 days of incubation, PSC is dissociated and seeded as a single cell in standard culture medium Dulbecco's modified Eagle medium (DMEM) and 10% fetal bovine serum (FBS). The culture medium is replaced once every 48 hours for 7 days. Next, ascorbic acid (150mg / L) is added to the standard culture medium to increase the induction of collagen synthesis. After an additional 7-day incubation period, the culture medium is aspirated and the cells are washed with Dulbecco's phosphate buffered saline (DPBS) without calcium and magnesium. Subsequently, preheated recombinant trypsin-ethylenediaminetetraacetic acid (EDTA) solution was added to the cells and the culture was incubated at 38.5°C for 5 minutes until a thin fibrillar hydrogel was observed. Trypsin activity was neutralized by adding an equal volume of DMEM. The resulting fibrillar hydrogel was washed three times with DPBS and then stored at 4°C.

[0233] Example 2: SDS-PAGE analysis of harvested fibril hydrogels

[0234] The fibrillar hydrogel (generated from bovine fibroblasts as described in Example 1 above) was resuspended in 8 M urea. The soluble fraction was mixed with reducing sample buffer, boiled at 95°C for 5 minutes, and loaded onto 8% SDS-PAGE. After the run, the gel was stained with Coomassie stain ( Figure 2 ).

[0235] Example 3: Anti-COL1 analysis of harvested fibril hydrogels

[0236] The fibrillar hydrogel (produced from bovine fibroblasts as described in Example 1 above) was resuspended in 8M urea. The soluble fraction was mixed with reducing sample buffer, boiled at 95°C for 5 minutes, and loaded on 8% SDS-PAGE. The protein was then transferred to a PVDF membrane and the membrane was analyzed for the presence of type I collagen using an anti-COL1 antibody. A major band of the expected size (130 / 139 kDa, Figure 3 ).

[0237] Example 4: Anti-COL3 Analysis of Harvested Fibrillar Hydrogels

[0238] The fibrillar hydrogel (produced from bovine fibroblasts as described in Example 1 above) was resuspended in 8M urea. The soluble fraction was mixed with reducing sample buffer, boiled at 95°C for 5 minutes, and loaded on 8% SDS-PAGE. The proteins were then transferred to a PVDF membrane and analyzed for the presence of type III collagen in membrane proteins using an anti-COL3A1 antibody. A band of the expected size (140 kDa, Figure 4 ).

[0239] Example 5: Anti-COL6 Analysis of Harvested Fibrillar Hydrogels

[0240] The fibrillar hydrogel (generated from bovine fibroblasts as described in Example 1 above) was resuspended in 8M urea. The soluble fraction was mixed with reducing sample buffer, boiled at 95°C for 5 minutes, and loaded on 8% SDS-PAGE. The protein was then transferred to a PVDF membrane and the presence of type VI collagen in the membrane proteins was analyzed using an anti-COL6A2 antibody. A band of the expected size (120-160 kDa, Figure 5 ).

[0241] Example 6: Anti-COL12 analysis of harvested fibril hydrogels

[0242] The fibrillar hydrogel (generated from bovine fibroblasts as described in Example 1 above) was resuspended in 8M urea. The soluble fraction was mixed with reducing sample buffer, boiled at 95°C for 5 minutes, and loaded on 8% SDS-PAGE. The proteins were then transferred to a PVDF membrane and analyzed for the presence of type XII collagen in membrane proteins using an anti-COL12A1 antibody. Several bands >185 kDa were observed (expected size: 333 kDa, Figure 6 ).

[0243] Example 7: Analysis of fibril hydrogel protein content

[0244] In order to analyze the protein content of the fibrillar hydrogels (fibroblasts from bovine origin and PSCs from bovine origin differentiated into collagen-producing cells) produced as described in Example 1, liquid chromatography-tandem mass spectrometry (LC-MS / MS) was applied to each preparation. The samples were digested by trypsin, analyzed by LC-MS / MS on Q-Exactive plus (Thermo), and identified by Discoverer software for the bovine proteome from the Uniprot database and the bait database (to determine the false discovery rate). All identified peptides were filtered with high confidence, and the identified proteins had a minimum of 2 peptides.

[0245] For LC-MS / MS, high confidence peptides have exceeded the 1% FDR threshold. (*FDR = False Discovery Rate, which is the estimated fraction of false positives in a peptide list). Proteins identified with a single unique peptide cannot be considered definitive identifications. The major proteins and protein classes are schematically presented in Figure 7 middle.

[0246] Example 8: MS-MS proteomic analysis of collagen 1 alpha chain 1 (COL1A1)

[0247] Samples of the fibrillar hydrogel (generated as described in Example 1 above) were analyzed by liquid chromatography-tandem mass spectrometry. The samples were prepared for MS-MS ((LC-MS) / MS) including trypsin digestion. The sequence and post-translational modifications (PTMs) were then compared with those of bovine COL1A1 (AAI05185.1) from the NCBI protein database ( Figure 8 ).

[0248] Example 9: MS-MS proteomic analysis of collagen 1 alpha chain 2 (COL1A2)

[0249] As described above in Example 8, after trypsin digestion, samples of the fibril hydrogels were analyzed by liquid chromatography-tandem mass spectrometry ((LC-MS) / MS). The sequences and PTMs were then compared with those of bovine COL1A2 (NP_776945.1) from the NCBI protein database ( Fig. 9 ).

[0250] Example 10: Yield stress analysis of fibrillar hydrogels produced by bovine fibroblasts and PSCs

[0251] The shear stress ( Fig.10 ). The fibrillar hydrogel samples (produced as described in Example 1, Section 1.2) (Batch 1, Batch 2 and Batch 3) were analyzed using a MCR 102e rheometer (Anton Paar) at a temperature of 25°C ( Fig.12 ).

[0252] Each sample was placed between two parallel plates, one of which was fixed and the other either rotated in one direction (shear flow) or oscillated back and forth (oscillatory shear). The rheological behavior of the hydrogels was studied under a range of stress conditions, and yield stress values ​​of 16.2 Pa and 4 Pa ​​were observed for the fibrillar hydrogel samples produced from bovine fibroblasts and PSCs, respectively.

[0253] Example 11: Oscillatory Amplitude Sweep Analysis of Fibrillar Hydrogels Produced by Bovine Fibroblasts and PSCs

[0254] A +Pro Kinexus Netzsch rheometer ( Fig.11) The viscoelastic behavior of the fibrillar hydrogel samples (generated as described in Example 1, Section 1.1 above) was analyzed using an oscillatory amplitude sweep protocol. A Modular Compact Rheometer (MCR) 102e (Anton Paar) ( Fig.13 ) The fibrillar hydrogel samples (generated as described in Example 1, Section 1.2) were analyzed using an oscillatory amplitude scanning protocol.

[0255] Each sample is placed between two plates, the upper plate oscillating back and forth in both directions at a constant frequency, but increasing the amplitude in each cycle, with a corresponding increase in shear stress. The experiments were carried out at 25°C.

[0256] The results of the oscillation amplitude sweep analysis showed that the fibril hydrogel samples exhibited a viscoelastic response with both elastic (G') and viscous (G") components. The elastic modulus (G') (blue) and viscous modulus (G") (red) in Pa were plotted against the Y* parameter. At lower Y* values, the hydrogels primarily exhibited solid-like behavior, as indicated by higher G' values ​​than G" values, and the phase angles (δ) were approximately 15° and 16.8° for the fibrous hydrogel samples produced by bovine fibroblasts and PSCs, respectively. This indicates that the fibril hydrogels are semi-solid materials that behave more like solids.

[0257] Example 12: pH Analysis of Fibrillar Hydrogels Produced Using Bovine Pluripotent Stem Cells (PSCs)

[0258] The pH of the fibrillar hydrogel (produced as described above in Example 1) was analyzed using a pH meter (Knick Portavo 907 Multi). The fibrillar hydrogel sample was placed in a 50 mL tube. The pH meter sensor was then inserted into the sample and the pH was measured to be 7.15.

[0259] The foregoing description of specific embodiments will so fully reveal the generality of the invention that others can easily modify and / or adapt such specific embodiments for a variety of applications by applying prior knowledge without undue experimentation and without departing from the general concepts, and therefore, such adjustments and modifications should and are intended to be included within the meaning and range of equivalents of the disclosed embodiments. It should be understood that the wording or terminology employed herein is for descriptive purposes and not for limiting purposes. The means, materials and steps for implementing the various disclosed functions may take a variety of alternative forms without departing from the present invention.

Claims

1. A fibrillar hydrogel comprising an aqueous solution and more than one fibril, the fibrils comprising collagen and at least one type of proteoglycan, wherein the pH of the hydrogel aqueous solution is at least about 6.00, and wherein at least a portion of the fibrils are orderly aligned within the hydrogel.

2. The fibrillar hydrogel according to claim 1, wherein the ordered alignment is selected from the group consisting of: biaxial alignment and multiaxial alignment.

3. The fibrillar hydrogel according to any one of claims 1-2, wherein the collagen molecules comprise hydroxylated proline and hydroxylated lysine residues of at least 40% of the total number of proline and lysine residues of the collagen molecules.

4. The fibrillar hydrogel according to any one of claims 1 to 3, wherein the aqueous solution is selected from the group consisting of: water, a buffer solution, and a cell culture medium.

5. The fibril hydrogel according to any one of claims 1 to 4, wherein the water content of the fibril hydrogel is at least 30% w / w of the total weight of the fibril hydrogel.

6. The fibril hydrogel according to any one of claims 1-5, wherein the water content of the fibril hydrogel is from about 90% w / w to about 99% w / w of the total weight of the fibril hydrogel.

7. The fibrillar hydrogel according to any one of claims 1-6, wherein the fibrils comprise at least one collagen type selected from the group consisting of collagen I, collagen VI, collagen XII, collagen V, collagen XIV, collagen II, collagen XI, collagen III, and any combination thereof.

8. A fibrillar hydrogel according to claim 7, wherein collagen I forms at least 60% of the total collagen content of the hydrogel.

9. The fibrillar hydrogel according to any one of claims 7-8, wherein at least 40% of the proline and lysine residues of the collagen I molecules are hydroxylated out of the total number of proline and lysine residues of the collagen I molecules.

10. The fibrillar hydrogel according to any one of claims 7 to 9, wherein at least 25% of the lysine residues of the collagen I molecules are hydroxylated out of the total number of lysine residues of the collagen I molecules.

11. The fibrillar hydrogel according to any one of claims 1-10, wherein the at least one type of proteoglycan is selected from the group consisting of decorin, biglycan, lumican, prolargin, asporin, fibromodulin, versican, and any combination thereof.

12. The fibrillar hydrogel according to any one of claims 1-11, wherein the proteoglycan comprises a core protein and at least one type of glycosaminoglycan (GAG) selected from the group consisting of chondroitin sulfate, dermatan sulfate, heparin, heparan sulfate, keratan sulfate, and any combination thereof.

13. The fibrillar hydrogel of any one of claims 1-12, wherein the fibrillar hydrogel comprises from about 20% w / w to about 90% w / w total collagen based on the total dry weight of the hydrogel.

14. The fibrillar hydrogel according to any one of claims 1-13, wherein the fibrillar hydrogel further comprises at least one additional protein selected from the group consisting of: glycoproteins, proteins of cytoskeletal filaments, cell membrane proteins, and any combination thereof.

15. The fibrillar hydrogel according to claim 14, wherein the glycoprotein is selected from the group consisting of: fibronectin, serpin H1, neuroblast differentiation-associated protein AHNAK (desmosome nexin), galectin-1, periostin, laminin, peroxiredoxin, EMILIN, fibrillin, tenascin C, secreted protein acidic and rich in cysteine ​​(SPARC), and any combination thereof.

16. The fibrillar hydrogel according to any one of claims 14-15, wherein the proteins of the cytoskeletal filaments are selected from the group consisting of actin, vimentin, myosin, filamin, tropomyosin, lamin, transcollin, tubulin, and any combination thereof.

17. The fibrillar hydrogel according to any one of claims 14 to 16, wherein the membrane protein is an annexin.

18. The fibrillar hydrogel according to any one of claims 1 to 17, wherein the fibrillar hydrogel further comprises hyaluronic acid.

19. The fibril hydrogel of any one of claims 1-18, wherein the fibril hydrogel comprising an aqueous solution content of about 90% to about 99% is characterized by a yield stress of about 1 Pa to about 50 Pa at a temperature in the range of about 22°C to about 26°C.

20. The fibril hydrogel of claim 20, wherein the fibril hydrogel is characterized by a yield stress in the range of about 2 Pa to about 20 Pa.

21. The fibril hydrogel of any one of claims 1-20, wherein at a temperature in the range of about 22°C to about 26°C, the fibril hydrogel comprising about 90% to about 99% aqueous solution content is characterized by a phase angle of about 1° to about 50°.

22. The fibril hydrogel of claim 21, wherein the fibril hydrogel is characterized by a phase angle of about 12° to about 20°.

23. The fibrillar hydrogel according to any one of claims 1 to 22, wherein the fibrillar hydrogel is produced by animal cells capable of producing collagen, and the animal cells are cultured in a culture medium under conditions capable of synthesizing collagen.

24. The fibril hydrogel according to claim 23, wherein the animal is selected from the group consisting of mammals, poultry, aquatic animals, invertebrates and reptiles.

25. The fibrillar hydrogel of claim 24, wherein the mammal is selected from the group consisting of humans and non-human mammals.

26. The fibrillar hydrogel of claim 25, wherein the non-human mammal is an ungulate selected from the group consisting of Bovinae, Buffalo, Ovis, Equine, Pig, Giraffe, Camel, Deer, Hippos, and Rhinoceros.

27. The fibrillar hydrogel of claim 26, wherein the ungulate is Bovinae.

28. The fibrillar hydrogel of claim 24, wherein the reptile is of the family Crocodylidae.

29. The fibril hydrogel of any one of claims 23-28, wherein the fibril hydrogel is substantially free of pyrogens.

30. The fibril hydrogel of claim 29, wherein the fibril hydrogel is substantially free of endotoxin.

31. Use of the fibril hydrogel according to any one of claims 1 to 30 in the preparation of medical products.

32. The use according to claim 31, wherein the medical product is selected from the group consisting of a wound dressing and a scaffold for tissue regeneration.

33. Use of the fibril hydrogel according to any one of claims 1 to 30 in the preparation of textiles.

34. Use according to claim 33, wherein the textile has skin-like or hide-like or leather-like characteristics.

35. Use of the fibril hydrogel of any one of claims 1-30 for preparing a food product.

36. The use according to claim 35, wherein the food product is cultured meat.

37. Use of the fibril hydrogel of any one of claims 1-30 for preparing at least one of a nutraceutical, a dietary supplement, and a functional food.

38. Use of the fibril hydrogel according to any one of claims 1 to 30 for the preparation of cosmetics.

39. A biomaterial comprising at least 10% of the fibrillar hydrogel according to any one of claims 1-30.

40. The biomaterial of claim 39, comprising about 20% to about 60% of the fibrillar hydrogel.

41. The biomaterial according to any one of claims 39-40, which is a textile having skin-like or hide-like or leather-like characteristics.

42. A food product comprising at least 0.5% of the fibrillar hydrogel according to any one of claims 1-30.

43. The food product of claim 42 comprising from about 1% to about 5% of the fibrillar hydrogel.

44. The food product of any one of claims 43-42, wherein the food product is cultured meat.

45. A cosmetic comprising at least 0.01% of the fibrillar hydrogel according to any one of claims 1-30.

46. ​​The cosmetic of claim 45, wherein the cosmetic comprises from about 0.1% to about 1.0% of the fibrillar hydrogel.

47. A medical product comprising at least 0.1% of the fibrillar hydrogel according to any one of claims 1-30.

48. The medical product of claim 47, wherein the medical product comprises from about 0.1% to about 1% of the fibrillar hydrogel.

49. A nutritional product comprising at least 1% of the fibrillar hydrogel according to any one of claims 1-30.

50. The nutritional product of claim 49, wherein the nutritional product comprises from about 1% to about 20% of the fibrillar hydrogel.

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