Method for biofabricating a composite material

Abstract

The present invention relates to a method for producing a composite material comprising a biofabricated material and a secondary component. The secondary component can be a porous material, such as a sheet of paper, cellulose or fabric that has been coated or otherwise contacted with the biofabricated material. The biofabricated material comprises a uniform network of cross-linked collagen fibrils and provides strength, elasticity and aesthetic appearance to the composite material.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 295,435, filed February 15, 2016, which is incorporated herein by reference in its entirety. This application relates to U.S. Patent Application No. 13 / 853,001, entitled “ENGINEERED LEATHER AND METHODS OF MANUFACTURE THEREOF,” filed March 28, 2013; U.S. Patent Application No. 14 / 967,173, entitled “ENGINEERED LEATHER AND METHODS OF MANUFACTURE THEREOF,” filed December 11, 2015; and PCT Patent Application No. PCT / US2015 / 058794, entitled “REINFORCED ENGINEERED BIOMATERIALS AND METHODS OF MANUFACTURE THEREOF,” filed November 3, 2015. Background of the Invention

[0003] Invention Field

[0004] This invention relates to biomanufactured leather materials composed of unbound and randomly oriented trimeric collagen fibrils. These biomanufactured leather materials exhibit superior strength, anisotropic properties, and uniformity compared to conventional leather products, while retaining the appearance, feel, and other aesthetic properties of natural leather. Unlike synthetic leather products composed of plastic resins, the biomanufactured leather of this invention is based on collagen, a natural component of leather.

[0005] Related technical descriptions.

[0006] Natural leather is typically a durable and supple material produced through the processing of animal hides and skins (such as cowhide). This processing usually involves three main parts: preparation, tanning, and retanning. Leather can also be surface-coated or embossed.

[0007] Various methods are known for preparing skin or raw hide and converting it into leather. These methods include salting or refrigerating raw hides or skin to preserve them; soaking or rehydrating raw hides in aqueous solutions containing surfactants or other chemicals to remove salt, dirt, debris, blood, and excess fat; removing flesh or subcutaneous material from raw hides; dehairing or plucking raw hides to remove most of the hair; sprinkling lime on raw hides to loosen the fibers and open the collagen bundles, allowing them to absorb chemicals; separating raw hides into two or more layers; deliming raw hides to remove alkali and lower their pH; softening raw hides to complete the deliming process and smooth the grain; degreasing to remove excess fat; curling; bleaching; acid washing by changing the pH; or deacidification.

[0008] Once the preparation stage is complete, the leather is tanned. Tanning increases the leather's durability compared to untreated raw hides. Tanning transforms the proteins in the hide or skin into stable substances that do not decay while allowing the leather material to remain soft. During tanning, the skin structure can be stabilized in an "open" form through the complexation reaction of some collagen with chromium or other tanning agents. Depending on the compounds used, the color and texture of the leather can vary.

[0009] Tanning is generally understood as the process of treating animal skin to produce leather. Tanning can be carried out in any number of well-known ways, including by contacting the skin or raw hide with vegetable tanning agents, chromium compounds, aldehydes, synthetic tanning agents, synthetic, semi-synthetic or natural resins or polymers and / or natural tanning oils or modified oils. Vegetable tanning agents include tanning agents based on pyrogallol or pyrocatechol, such as oak, mimosa, ten, tara, oak, pine, sumac, kaempferia, and chestnut tanning agents; chromium tanning agents include chromium salts such as chromium sulfate; aldehyde tanning agents include glutaraldehyde and oxazolidine compounds; synthetic tanning agents include aromatic polymers, polyacrylates, polymethacrylates, copolymers of maleic anhydride and styrene, condensation products of formaldehyde with melamine or dicyandiamide, lignin, and natural flour.

[0010] Chromium is the most commonly used tanning agent. The pH of the skin / raw hide can be adjusted (e.g., lowered to pH 2.8-3.2) to allow the tanning agent to penetrate; after penetration, the pH can be raised to fix the tanning agent ("alkalizing" to a slightly higher level, e.g., pH 3.8-4.2 for chromium).

[0011] After tanning, leather can be retanned. Retanning refers to post-tanning treatments that may include coloring, thinning, drying, or hydration. Examples of retanning techniques include: tanning, wetting (rehydration), wetting (drying), neutralization (adjusting the pH to a less acidic or alkaline state), coloring, fatliquoring, fixing unbound chemicals, setting, conditioning, softening, and polishing.

[0012] Tanned leather products can be finished mechanically or chemically. Mechanical finishing can polish leather to produce a glossy surface, iron and electroplate leather to have a flat, smooth surface, emboss leather to provide three-dimensional imprints or patterns, or tumble leather to provide a more pronounced grain and smooth surface. Chemical finishing can involve applying films, natural or synthetic coatings, or other leather treatments. These can be applied, for example, by spraying, spraying, or rolling.

[0013] Variations in the fibrous collagen structure of animal hides have been observed in animals of different ages or species. These differences affect the physical properties of the hides and the leather produced from them. Changes in collagen structure also occur throughout the entire thickness of the hide. The upper grain side of the hide consists of a network of fine collagen fibrils, while the deeper layers (dermis) consist of larger fiber bundles (Figure 2). The smaller fibrils in the grain layer produce a soft, smooth leather aesthetic, while the larger fiber bundles in the deeper regions produce a rougher, coarser leather aesthetic. The porous collagen fiber structure in the hide allows applied molecules to penetrate, stabilize, and lubricate the leather during tanning. The combination of the inherent collagen structure in the hide and the modifications achieved through tanning produces the desired strength, drape, and aesthetic properties of the leather.

[0014] The top grain surface of leather is generally considered the most desirable due to its smoothness and soft texture. This leather grain contains a highly porous network of organized collagen fibrils. Endogenous collagen fibrils are organized into cavitary and overlapping regions; see [link to related documentation]. Figure 1 The depiction shows the layered structure of collagen. The strength, microporosity, and density of fibrils in top-grain leather allow tanning agents or fatliquoring agents to penetrate it, thereby stabilizing and lubricating the collagen fibrils and producing the desired soft, smooth, and strong leather.

[0015] Raw hides can be separated to obtain leather that is predominantly top-grain. The separated hides can be further sanded to reduce the coarse-grained leather on the separated sides, but some residual leather and a associated rough appearance will always remain. To produce leather with a smooth grain on both sides, it is necessary to join two pieces of grain-side leather facing each other and sew them together or layer them using adhesive, with the smooth top-grain side facing outwards. There is a demand for leather products with a smooth top-grain-like surface on both sides, as this avoids the need to separate and sew or layer two separate pieces of leather together.

[0016] Control over the final properties of leather is limited by the natural variations in collagen structure between different animal hides. For example, the relative thickness from grain to dermis in goat hides is significantly greater than that in kangaroo hides. Additionally, the collagen fiber bundles in kangaroo hides are woven at angles more parallel to the hide surface, while in bovine hides, the fiber bundles are oriented both parallel and perpendicular to the hide surface. Furthermore, the density of fiber bundles varies within each hide depending on its anatomical location. Hides taken from the rump, abdomen, shoulder, and neck can have different compositions and properties. Animal age also affects the composition of its hides; for example, juvenile bovine hides contain fibers with smaller diameters than the larger fiber bundles found in adult bovine hides.

[0017] The final properties of leather can be controlled to a certain extent by incorporating stabilizing and lubricating molecules into the raw hide or skin during tanning and retanning; however, the choice of these molecules is limited by the need for a dense structure that penetrates the skin or raw hide. Particles with diameters of several micrometers have been incorporated into leather to enhance lubrication; however, the application of these particles is limited to raw hides with the largest pore sizes. Uniformly distributing particles throughout the raw hide presents many challenges.

[0018] Due to the size limitations of the substances that can uniformly penetrate raw hide, leather composites are typically laminates of leather and thin layers of other materials such as Kevlar or nylon for mechanical reinforcement, or thin layers of polyurethane and acrylic resins for aesthetically desired surfaces. The construction of leather with dispersed secondary material phases has not yet been achieved.

[0019] To address this limitation of natural leather, the inventors described the manufacture of leather composites in which a continuous phase of collagen fibrils can encapsulate dispersed fibers and three-dimensional materials. This technology enables the manufacture of a new class of leather materials with reinforcing properties.

[0020] While the fibrillation of soluble collagen and collagen-like proteins has been extensively explored for the production of collagen hydrogels for biomedical applications, the use of this phenomenon to create leather-like composites has not yet been reported. Starting with an aqueous mixture of collagen monomers or fibrils, almost any material can be readily added to the mixture and further encapsulated into biomanufactured leather. Furthermore, it is possible to achieve combinations of continuous collagen fibrillary phases with encapsulated fibrous phases, composites with granular aesthetics and a range of enhanced mechanical properties.

[0021] Many leather applications require durable products that won't crack or tear, even when the leather is sewn together. Typical products that include sewn leather and require durable leather include car steering wheel covers, car seats, furniture, sporting goods, athletic shoes, rubber-soled canvas shoes, watch straps, etc. There is a need to increase the durability of bio-manufactured leather to improve performance in these products.

[0022] The top grain surface of leather is generally considered the most desirable due to its soft texture and smooth surface. As previously discussed, the grain is a highly porous network of collagen fibrils. The collagen fibril strength, microporosity, and density in the top grain allow tanning agents to penetrate and stabilize and lubricate the fibrils, resulting in the desired soft, smooth, and stable material. While the aesthetic appeal of the grain is highly desirable, its strength and tear resistance are often limitations for the practical application of individual grains. Therefore, the grain is typically backed by the dermis (its naturally reinforced collagen layer) or can be artificially backed by a sheet layer of synthetic material. The collagen-reinforcing composites described herein allow for thick, homogeneous grain-like materials with tunable mechanical properties through control of the continuous and dispersed phases.

[0023] Beyond enhanced mechanical properties, this bottom-up manufacturing approach can also enable the encapsulation of materials for aesthetic functions. For example, photoluminescent materials can be encapsulated within bio-manufactured leather. In traditional tanning, smaller nanoparticles to single molecules, such as dyes, are used to produce uniform coloring and aesthetic appeal in leather. Because the incorporation of dyes and aesthetic features depends on these molecules penetrating into the raw hide or skin, patterned features with controlled spatial organization are currently not feasible for leather. Patterned photoluminescent features will provide leather with unique functionalities, including branding, personalization, aesthetically pleasing designs, and anti-counterfeiting technologies.

[0024] The materials described herein can be used to produce biomanufactured leather with patterned photoluminescent features. Methods for forming collagen fibrillary networks in front of or around a patterned substrate allow for the encapsulation of precisely controlled patterns of considerable size within the biomanufactured leather structure. Almost any photoluminescent material can be incorporated into or encapsulated in biomanufactured leather. For the pattern to be visualized, light emitted from the embedded photoluminescent molecules must penetrate the thickness of the leather. Current research shows that light penetrating into collagen-rich materials such as skin is highly wavelength-dependent and decreases exponentially with the thickness of the material. Therefore, variables such as the emission wavelength of the embedded photoluminescent material and the distance of the photoluminescent material from the surface of the biomanufactured leather need to be considered to produce photoluminescent features visible to the eye. Similarly, for features observable by readers other than the eye (such as, for example, light emission scanners), the intensity of the embedded photoluminescent material needs to be considered. Furthermore, three-dimensional objects can be encapsulated in biomanufactured leather to create unique surface textures and patterns. The surface patterning of conventional leather materials is limited by natural variations in the surface of animal skin or by the ability to emboss patterns onto the grain surface of the leather. To achieve unique patterns with intricate surface features, three-dimensional objects can be embedded into bio-manufactured leather. These textures and patterns offer distinctive aesthetic characteristics and can be used as identifiers for brand identity.

[0025] Collagen. Collagen is a component of leather. Skin or raw animal hide contains a large amount of collagen (fibrous protein). Collagen is a general term for a family of collagen with at least 28 different types; animal skin is typically composed of type I collagen, although other types, including type III collagen, can be used in the formation of leather. Collagen is characterized by repeating triplets of amino acids – (Gly-XY). n Furthermore, approximately one-third of the amino acid residues in collagen are glycine. X is usually proline and Y is usually hydroxyproline, although up to 400 possible Gly-XY triplets can exist. Different animals can produce different collagen amino acid compositions, which can cause different properties and variations in the resulting leather.

[0026] Collagen can be structured from three tangled peptide chains of varying lengths. Collagen triple helices (or monomers) can be generated from α-chains approximately 1,050 amino acids long, resulting in a rod-like form approximately 300 nm long with a diameter of approximately 1.5 nm. Triple helical monomers can be synthesized in the extracellular matrix produced by fibroblast skin cells, and these monomers can self-assemble into fibrous forms. These triple helices are held together by electrostatic interactions, including salt bridges, hydrogen bonds, van der Waals interactions, orientation forces, polarization forces, hydrophobic interactions, and / or covalent bonds. Triple helices can be bundled together to form protofibrils, and these protofibrils can further assemble to form fibers and fiber bundles. Figure 1 Fibrous fibers have a characteristic banded appearance due to the interlacing of collagen monomers. For type I collagen, the distance between bands is approximately 67 nm. Fibrous fibers and fibrous tissues typically branch throughout the skin layer and interact with each other. Variations in the organization or cross-linking of fibrils and fibrous tissues can provide strength to the material. Depending on the type of animal hide, the fibers can have a range of diameters. In addition to type I collagen, skin (raw hide) can also contain other types of collagen, including type III collagen (reticulin), type IV collagen, and type VII collagen.

[0027] Various types of collagen are present throughout the mammalian body. For example, in addition to being a major component of skin and animal hides, type I collagen is also found in cartilage, tendons, vascular ligaments, organs, muscles, and organic parts of bones. Successful efforts have been made to isolate collagen from various mammalian body regions other than animal skin or hides. Decades ago, researchers discovered that at neutral pH, acid-dissolved collagen self-assembles into fibrils composed of the same transverse striation pattern observed in natural tissues (Schmitt FOJ Cell. Comp Physiol. 1942;20:11). This has led to the use of collagen in tissue engineering and various biomedical applications. In recent years, recombinant technologies have been used to harvest collagen from bacteria and yeast.

[0028] Regardless of collagen type, all collagens are formed and stabilized through a combination of physical and chemical interactions, including electrostatic interactions such as salt bridges, hydrogen bonds, van der Waals interactions, orientation forces, polarization forces, hydrophobic interactions, and covalent bonds typically catalyzed by enzymatic reactions. For type I collagen fibrils, fibers, and fiber bundles, their complex assemblies are realized in vivo during development and are crucial for providing mechanical support to tissues while allowing cell movement and nutrient transport. Various different collagen types have been identified in vertebrates. These include bovine collagen, ovine collagen, porcine collagen, chicken collagen, and human collagen.

[0029] Typically, collagen types are numbered using Roman numerals, and the chains present in each collagen type are identified using Arabic numerals. Detailed descriptions of the structure and biological functions of various types of naturally occurring collagen are available in this field; see, for example, Ayad et al. (1998) The Extracellular Matrix Facts Book, Academic Press, San Diego, CA; Burgeson, R E. and Nimmi (1992) Clin. Orthop. "Collagen types: Molecular Structure and Tissue Distribution" 282:250-272; Kielty, CM et al. (1993) "The Collagen Family: Structure, Assembly And Organization In The Extracellular Matrix," Connective Tissue And Its Heritable Disorders, Molecular Genetics, And Medical Aspects, Royce, PM and B. Steinmann (eds.), Wiley-Liss, NY, pp. 103-147; and Prockop, DJ- and KIKivirikko (1995) "Collagens: Molecular Biology, Diseases, and Potentials for Therapy," Annu. Rev. Biochem., 64:403-434.

[0030] Type I collagen is the main fibrous collagen in bones and skin, accounting for approximately 80%-90% of total collagen in organisms. It is a major structural macromolecule present in the extracellular matrix of multicellular organisms and accounts for approximately 20% of total protein mass. Type I collagen is a heterotrimeric molecule containing two α1(I) chains and one α2(I) chain, encoded by the COL1A1 and COL1A2 genes, respectively. Other collagen types are less abundant than type I collagen and exhibit different distribution patterns. For example, type II collagen is the main collagen in cartilage and vitreous humor, while type III collagen is present at high levels in blood vessels and to a lesser extent in the skin.

[0031] Type II collagen is a homotrimeric collagen comprising three identical al(II) chains encoded by the COL2A1 gene. Purified type II collagen can be prepared from tissues using methods known in the art, such as those described in Miller and Rhodes (1982) Methods In Enzymology 82:33-64.

[0032] Type III collagen is the main fibrous collagen found in skin and vascular tissues. It is a homotrimeric collagen consisting of three identical α1(III) chains encoded by the COL3A1 gene. Methods for purifying type III collagen from tissues can be found, for example, by Byers et al. (1974) Biochemistry 13:5243-5248; and Miller and Rhodes, ibid.

[0033] Type IV collagen exists in the basement membrane in the form of sheets rather than fibrils. Most commonly, type IV collagen contains two α1(IV) chains and one α2(IV) chain. The specific chains containing type IV collagen are tissue-specific. Type IV collagen can be purified, for example, using the procedure described in Furuto and Miller (1987) Methods in Enzymology, 144:41-61, AcademicPress.

[0034] Type V collagen is a fibrous collagen primarily found in bones, tendons, cornea, skin, and blood vessels. Type V collagen exists in both homotrimer and heterotrimer forms. One form is a heterotrimer consisting of two α1(V) chains and one α2(V) chain. Another form is a heterotrimer consisting of α1(V), α2(V), and α3(V) chains. Yet another form is a homotrimer consisting of α1(V). Methods for isolating Type V collagen from natural sources can be found, for example, in Elstow and Weiss (1983) Collagen Rel. Res. 3:181-193, and Abedin et al. (1982) Biosci. Rep. 2:493-502.

[0035] Type VI collagen has a small triple helix region and two large non-collagenous portions. It is a heterotrimer comprising α1(VI) chains, α2(VI) chains, and α3(VI) chains. Type VI collagen is found in many connective tissues. Descriptions of how to purify type VI collagen from natural sources can be found, for example, in Wu et al. (1987) Biochem. J. 248:373-381, and Kielty et al. (1991) J. Cell Sci. 99:797-807.

[0036] Type VII collagen is a fibrous collagen found in specific epithelial tissues. Type VII collagen is a homotrimer molecule consisting of three α1(VII) chains. Descriptions of how to purify type VII collagen from tissues can be found, for example, in Lunstrum et al. (1986) J. Biol. Chem. 261:9042-9048, and Bentz et al. (1983) Proc. Natl. Acad. Sci. USA 80:3168-3172. Type VIII collagen can be found in the Descemet's membrane of the cornea. Type VIII collagen is a heterotrimer containing two α1(VIII) chains and one α2(VIII) chain, although other chain compositions have been reported. Methods for purifying type VIII collagen from nature can be found, for example, in Benya and Padilla (1986) J. Biol. Chem. 261:4160-4169, and Kapoor et al. (1986) Biochemistry 25:3930-3937.

[0037] Type IX collagen is a fibril-associated collagen found in cartilage and vitreous humor. It is a heterotrimeric molecule comprising α1(IX) chains, α2(IX) chains, and α3(IX) chains. Type IX collagen is classified as FACIT (fibril-associated collagen with interrupted triple helix) collagen, which has several triple helix domains separated by non-triple helix domains. Procedures for purifying type IX collagen can be found, for example, in Duance et al. (1984) Biochem. J. 221:885-889; Ayad et al. (1989) Biochem. J. 262:753-761; and Grant et al. (1988) The Control of Tissue Damage, eds. Glauert, AM, Elsevier Science Publishers, Amsterdam, pp. 3-28.

[0038] X-type collagen is a homotrimeric compound of the α1(X) chain. X-type collagen has been isolated from, for example, hypertrophic cartilage present in growth plates; see, for example, Apte et al. (1992) Eur J Biochem 206 (1):217-24.

[0039] Type XI collagen can be found in cartilage tissue associated with type II and IX collagen, and also in other locations throughout the body. Type XI collagen is a heterotrimeric molecule comprising α1(XI) chains, α2(XI) chains, and α3(XI) chains. Methods for purifying type XI collagen can be found, for example, by Grant et al., ibid.

[0040] Type XII collagen is FACIT collagen that mainly coexists with type I collagen. Type XII collagen is a homotrimeric molecule containing three α1(XII) chains. Methods for purifying type XII collagen and its variants can be found, for example, Dublet et al. (1989) J. Biol. Chem. 264:13150-13156; Lunstrum et al. (1992) J. Biol. Chem. 267:20087-20092; and Watt et al. (1992) J. Biol. Chem. 267:20093-20099.

[0041] Type XIII is non-fibrous collagen found, for example, in the skin, intestines, bones, cartilage, and skeletal muscle. A detailed description of type XIII collagen can be found, for example, Juvonen et al. (1992) J. Biol. Chem. 267: 24700-24707.

[0042] Type XIV collagen is characterized by a homotrimeric molecule containing an α1(XIV) chain, which is a form of FACIT collagen. Methods for isolating type XIV collagen can be found, for example, Aubert-Foucher et al. (1992) J. Biol. Chem. 267:15759-15764 and Watt et al., ibid.

[0043] XV type collagen is structurally homologous to XVIII type collagen. Information on the structure and isolation of natural XV type collagen can be found in, for example, Myers et al. (1992) Proc. Natl. Acad. Sci. USA 89:10144-10148; Huebner et al. (1992) Genomics 14:220-224; Kivirikko et al. (1994) J. Biol. Chem. 269:4773-4779; and Muragaki, J. (1994) Biol. Chem. 264:4042-4046.

[0044] Type XVI collagen is fibrillary-associated collagen found, for example, in skin, lung fibroblasts, and keratinocytes. Information on the structure of type XVI collagen and the genes encoding type XVI collagen can be found, for example, in Pan et al. (1992) Proc. Natl. Acad. Sci. USA 89:6565-6569; and Yamaguchi et al. (1992) J. Biochem. 112:856-863.

[0045] Type XVII collagen is a hemidesmosome transmembrane collagen known from bullous pemphigoid antigens. Information on the structure of type XVII collagen and the gene encoding type XVII collagen can be found, for example, Li et al. (1993) J. Biol. Chem. 268(12):8825-8834; and McGrath et al. (1995) Nat. Genet.11(1):83-86.

[0046] Type XVIII collagen is structurally similar to type XV collagen and can be isolated from the liver. Descriptions of the structure of type XVIII collagen and its isolation from natural sources can be found, for example, in Rehn and Pihlajaniemi (1994) Proc. Natl. Acad. Sci USA 91:4234-4238; Oh et al. (1994) Proc. Natl. Acad. Sci USA 91:4229-4233; Rehn et al. (1994) J. Biol. Chem. 269:13924-13935; and Oh et al. (1994) Genomics 19:494-499.

[0047] XIX type collagen is considered to be another member of the FACIT collagen family and has been found in mRNA isolated from rhabdomyosarcoma cells. Descriptions of the structure and isolation of XIX type collagen can be found, for example, in Inoguchi et al. (1995) J. Biochem. 117:137-146; Yoshioka et al. (1992) Genomics 13:884-886; and Myers et al., J. Biol. Chem. 289:18549-18557 (1994).

[0048] Type XX collagen is a newly discovered member of the FACIT collagen family and has been identified in chicken corneas. (See, for example, Gordon et al. (1999) FASEB Journal 13:A1119; and Gordon et al. (1998), IOVS 39:S1128.)

[0049] Any type of collagen, truncated collagen, unmodified or post-translational modified collagen, or amino acid sequence modified collagen that can be fibrillated and crosslinked using the methods described herein can be used to produce biomanufactured materials or biomanufactured leather. Biomanufactured leather may contain substantially homogeneous collagen, such as only type I collagen or type III collagen, or may contain a mixture of 2, 3, 4 or more different types of collagen.

[0050] Recombinant collagen.

[0051] Recombinant expression of collagen and collagen-like proteins is known and is incorporated herein by reference: Bell, EP 1232182B1. Bovine collagen and method for producing recombinant gelatin Olsen et al., U.S. Patent No. 6,428,978, Methods for the production of gelatin and full-length triple helical collagen in recombinant cells; VanHeerde et al., U.S. Patent No. 8,188,230 Method for recombinant microorganism expression and isolation of collagen-like polypeptides.This type of recombinant collagen has not yet been used in leather production.

[0052] Prokaryotic Expression. In prokaryotic systems such as bacterial systems, a number of expression vectors can be advantageously selected depending on the intended use of the expressed polypeptide. For example, when producing large quantities of the animal collagen and gelatin of the present invention, vectors that are easily purified and guide the expression of high-level fusion protein products, such as those for antibody production, are desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al. (1983) EMBO J. 2:1791), in which the coding sequence can be co-framed with the lacZ coding region in the vector to produce a hybridized AS-lacZ protein; pIN vectors (Inouye et al. (1985) Nucleic Acids Res. 13:3101-3109 and Van Heeke et al. (1989) J. Biol. Chem. 264:5503-5509), etc. pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). Typically, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vector is designed to include a thrombin or factor Xa protease cleavage site so that the cloning peptide of interest can be released from the GST moiety. Recombinant collagen may contain collagen molecules that have not been post-translational modified, such as unglycosylated or hydroxylated, or may contain one or more post-translational modifications, such as modifications that contribute to the fibrillation and formation of unbound and randomly oriented fibrils of collagen molecules. Recombinant collagen molecules may contain amino acid sequence fragments of native collagen molecules that can form trimer collagen fibrils, or have amino acid sequences similar to those of native collagen (or with its fibril-forming region or with a structure substantially containing [Gly-XY]). nModified or truncated collagen molecules having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity or similarity in amino acid sequences to segments of the protein, such as bovine collagen described by SEQ ID NO: 1, 2, or 3 and described by the amino acid sequences of Col1A1, Col1A2, and Col1A3, are eligible for accession through accession numbers NP_001029211.1 (https: / / www.ncbi.nlm.nih.gov / protein / 77404252, last accessed February 9, 2017), NP_776945.1 (https: / / www.ncbi.nlm.nih.gov / protein / 27806257, last accessed February 9, 2017), and NP_001070299.1 (https: / / www.ncbi.nlm.nih.gov / protein / 116003881, last accessed February 9, 2017). The references are described in accordance with the accessed February 9, 2017, and are incorporated by way of citation. (These links are undoed by including an underscore after a double forward slash).

[0053] Such recombined or modified collagen molecules will typically contain repeating -(Gly-XY) molecules as described herein. n -sequence.

[0054] BLASTN can be used to identify polynucleotide sequences that have at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, or 99% sequence identity with a reference polynucleotide, such as a polynucleotide encoding a collagen polypeptide or an amino acid sequence encoding SEQ ID NO: 1, 2, or 3. Optimized BLASTN settings for discovering highly similar sequences use an expected threshold of 10 and a word length of 28, a maximum match in the range query of 0, a match / mismatch score of 1 / -2, and a linear gap penalty. Low-complexity regions can be filtered or masked. The default settings for standard nucleotide BLAST are described at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome (last logged on January 27, 2017) and are incorporated herein by reference.

[0055] BLASTP can be used to identify amino acid sequences, such as collagen amino acid sequences, that have at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, or 99% sequence identity or similarity to a reference amino acid, using similarity matrices such as BLOSUM45, BLOSUM62, or BLOSUM80, where BLOSUM45 is used for closely related sequences, BLOSUM62 for intermediate-range sequences, and BLOSUM80 for more distantly related sequences. Unless otherwise specified, the similarity score will be based on the use of BLOSUM62. When using BLASTP, the similarity percentage is based on the BLASTP positive score, and the sequence identity percentage is based on the BLASTP identity score. BLASTP “Identity” shows the total number and score of residues in identical high-scoring sequence pairs; and BLASTP “Positives” shows the number and score of residues with positive alignment scores that are similar to each other. This disclosure conceives of and covers amino acid sequences that have these degrees of identity or similarity, or any intermediate degree of identity or similarity, with the amino acid sequences disclosed herein. A representative BLASTP setting uses a desired threshold of 10, a word length of 3, BLOSUM 62 as a matrix, and void penalties of 11 (presence) and 1 (expansion), along with conditional combination score matrix adjustment. Other default settings for BLASTP are described below and incorporated herein by reference: https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome (last logged on January 27, 2017).

[0056] Yeast expression. In one embodiment, collagen molecules are generated in a yeast expression system. In yeast, many vectors containing constitutive or inducible promoters known in the art can be used; Ausubel et al., ibid., Vol. 2, Chapter 13; Grant et al. (1987) Methods in Enzymology, Expression and Secretion Vectors for Yeast, edited by Wu & Grossman, Acad. Press, NY 153:516-544; Glover (1986) DNA Cloning, Vol. II, IRL Press, Wash., DC, Chapter 3; Bitter (1987) Methods in Enzymology, Heterologous Gene Expression in Yeast, edited by Berger & Kimmel, Acad. Press, NY 152:673-684; and The Molecular Biology of the Yeast Saccharomyces, edited by Strathen et al., Cold Spring Harbor Press, Vol. I and II (1982).

[0057] Collagen can be expressed using host cells, for example, from the yeast *Saccharomyces cerevisiae*. This particular yeast can be used with any large number of expression vectors. Commonly used expression vectors are shuttle vectors containing a 2P origin of replication for proliferation in yeast and a Col E1 origin for efficient transcription of foreign genes in *E. coli*. A typical example of such a 2P-based vector is pWYG4, which has a 2P ORI-STB element, a GAL1-10 promoter, and a 2P D gene terminator. In this vector, the Ncol cloning site is used to insert the gene for the polypeptide to be expressed and provides the ATG start codon. Another expression vector is pWYG7L, which has the complete 2αORI, STB, REP1, and REP2 promoters and a GAL1-10 promoter, and uses an FLP terminator. In this vector, the encoding polynucleotide is inserted into a multi-connector, with its 5' end at either the BamHI or Ncol site. The vector containing the inserted polynucleotide was transformed into Saccharomyces cerevisiae after the cell wall was removed to produce protoplasts, which then absorbed DNA by treatment with calcium and polyethylene glycol or by treating intact cells with lithium ions.

[0058] Alternatively, DNA can be introduced via electroporation. Transformants can be selected, for example, using host yeast cells that are auxotrophic (leucine, tryptophan, uracil, or histidine) and have selectable marker genes such as LEU2, TRP1, URA3, HIS3, or LEU2-D.

[0059] In one implementation, a polynucleotide encoding collagen is introduced from yeast. Pichia pastoris In the host cells of the genus. Non-brewing yeast Types such as Pichia pastoris It appears to have the particular advantage of producing high yields of recombinant proteins in scale-up processes. Additionally, Pichia pastoris The genus expression kit is available from Invitrogen (San Diego, CA).

[0060] Many methanol-responsive genes exist in methyltrophic yeasts such as Pichia pastoris, and the expression of each methanol-responsive gene is controlled by a methanol-responsive regulatory region (also known as a promoter). Any such methanol-responsive promoter is suitable for practicing this invention. Examples of specific regulatory regions include the AOX1 promoter, the AOX2 promoter, dihydroxyacetone synthase (DAS), the P40 promoter, and promoters from Pichia pastoris for the catalase gene, etc.

[0061] In other implementations, methyl-trophic yeast is used. Hansenula polymorpha Growth on methanol induces the production of important enzymes that metabolize methanol, such as MOX (methanol oxidase), DAS (dihydroxyacetone synthase), and FMHD (formate dehydrogenase). These enzymes can constitute up to 30-40% of total cellular proteins. Genes encoding the production of MOX, DAS, and FMHD are controlled by strong promoters that are induced by growth on methanol and repressed by growth on glucose. Any or all three of these promoters can be used in... Hansenula polymorpha High levels of heterologous gene expression are obtained. Therefore, on one hand, under the control of the inducible Hansenula polymorpha promoter, a polynucleotide encoding animal collagen, or a fragment or variant thereof, is cloned into an expression vector. If a secretory product is desired, then a polynucleotide encoding a signal sequence for secretion in yeast is fused in-frame with said polynucleotide. In yet another embodiment, the expression vector preferably contains a auxotrophic marker gene, such as URA3 or LEU2, that can be used to compensate for deficiencies in a auxotrophic host.

[0062] The expression vector was then used for technology transfer known to those skilled in the art. Polymorphic Hansenula Mother host cell. Hansenula polymorphaA useful characteristic of the transformation is the spontaneous integration of up to 100 copies of the expression vector into the genome. In most cases, the integrated polynucleotides form multimers arranged head-to-tail. The integrated foreign polynucleotides have shown mitotic stability in several recombinant strains, even under non-selective conditions. This phenomenon of high-copy integration further contributes to the system's high productivity potential.

[0063] Fungal expression. Filamentous fungi can also be used to produce the polypeptides of the present invention. Vectors for expressing and / or secreting recombinant proteins in filamentous fungi are well known, and those skilled in the art can use these vectors to express the recombinant animal collagen of the present invention.

[0064] Plant expression. On the one hand, animal collagen is produced in plants or plant cells. In the case where a plant expression vector is used, the expression of the sequence encoding the collagen of the present invention can be driven by any number of promoters. For example, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al. (1984) Nature 310:511-514) or the coat protein promoter of TMV (Takamatsu et al. (1987) EMBO J. 6:307-311) can be used; or plant promoters such as the smaller subunit of RUBISCO (Coruzzi et al. (1984) EMBO J. 3:1671-1680; Broglie et al. (1984) Science 224:838-843) or heat shock promoters, such as soybean hsp17.5-E or hsp17.3-B (Gurley et al. (1986) Mol. Cell. Biol. 6:559-565), can be introduced into plant cells by various methods known to those skilled in the art, such as using Ti plasmids, Ri plasmids, plant viral vectors, direct DNA transformation, microinjection, electroporation, etc. For reviews of such technologies, see, for example, Weissbach & Weissbach, Methods for Plant Molecular Biology, Academic Press, NY, Part VIII, pp. 421-463 (1988); Grierson & Corey, Plant Molecular Biology, 2nd ed., Blackie, London, Chapters 7-9 (1988); Transgenic Plants: A Production System for Industrial and Pharmaceutical Proteins, eds. Owen and Pen, John Wiliey & Sons, 1996; Transgenic Plants, eds. Galun and Breiman, Imperial College Press, 1997; and Applied Plant Biotechnology, eds. Chopra, Malik and Bhat, Science Publishers, Inc., 1999.

[0065] Plant cells do not naturally produce sufficient amounts of post-translational enzymes to efficiently produce stable collagen. Therefore, in cases where hydroxylation is desired, plant cells used to express animal collagen are supplemented with the necessary post-translational enzymes to adequately produce stable collagen. In a preferred embodiment of the invention, the post-translational enzyme is prolyl 4-hydroxylase.

[0066] The method for producing the animal collagen of the present invention in a plant system can be achieved by providing biomass from a plant or plant cell, wherein the plant or plant cell contains at least one coding sequence operable to a promoter to enable polypeptide expression, and then extracting the polypeptide from the biomass. Alternatively, the polypeptide can be unextracted, for example, expressed in the endosperm.

[0067] Plant expression vectors and reporter genes are generally known in the art; see, for example, Gruber et al. (1993). Methods of Plant Molecular Biology and Biotechnology (China, CRC Press) Typically, expression vectors contain, for example, recombinant or synthetically produced nucleic acid constructs and include a promoter that functions in plant cells, wherein such promoter is operatively linked to a nucleic acid sequence encoding animal collagen or a fragment or variant thereof, or a post-translational enzyme important for collagen biosynthesis.

[0068] Promoters drive protein expression levels in plants. To produce desired levels of protein expression in plants, expression can be guided by plant promoters. Promoters suitable for use according to the invention are generally available in the art; see, for example, PCT Publication No. WO 91 / 19806. Examples of promoters that can be used according to the invention include non-constitutive promoters or constitutive promoters. These promoters include, but are not limited to, promoters for the smaller subunit of ribulose-1,5-bisphosphate carboxylase; promoters of tumor-inducing plasmids from Agrobacterium tumefaciens, such as the RUBISCO carmine synthase (NOS) and octopus carmine synthase promoters; bacterial T-DNA promoters, such as the mas and ocs promoters; and viral promoters, such as the cauliflower mosaic virus (CaMV) 19S and 35S promoters or the Scrophularia mosaic virus 35S promoter.

[0069] The polynucleotide sequence of this invention can be placed under the transcriptional control of a constitutive promoter, thereby guiding the expression of collagen or post-translational enzymes in most plant tissues. In one embodiment, the polynucleotide sequence is under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The double-stranded cauliflower mosaic virus family provides the most important promoters for transgenic expression in plants, particularly the 35S promoter; see, for example, Kay et al. (1987) Science 236:1299. Other promoters from this family, such as the Scrophularia mosaic virus promoter, have been described in the art and are also available for use; see, for example, Sanger et al. (1990) Plant Mol. Biol. 14:433-443; Medberry et al. (1992) Plant Cell 4:195-192; and Yin and Beachy (1995) Plant J. 7:969-980.

[0070] Promoters used in polynucleotide constructs for collagen expression can be modified (if desired) to affect their control characteristics. For example, the CaMV promoter can be linked to a portion of the RUBISCO gene that represses RUBISCO expression in the absence of light, to create a promoter that is active in leaves but inactive in roots. The resulting chimeric promoter can be used as described herein.

[0071] Constitutive plant promoters with general expression properties known in the art can be used with the expression vectors of the present invention. These promoters are abundantly expressed in most plant tissues and include, for example, actin promoters and ubiquitin promoters; see, for example, McElroy et al. (1990) Plant Cell 2:163-171; and Christensen et al. (1992) Plant Mol. Biol. 18:675-689.

[0072] Alternatively, the polypeptides of the present invention can be expressed in specific tissues, cell types, or under more precise environmental conditions or developmental control. In these cases, the promoters that guide expression are considered inducible promoters. Where tissue-specific promoters are used, protein expression is particularly high in the tissue from which the protein is desired. Depending on the desired tissue, expression can be targeted to the endosperm, aleurone layer, embryo (or its portions as scutes and cotyledons), pericarp, stem, leaf, tuber, root, etc. Examples of known tissue-specific promoters include tuber-guided class I potato glycoprotein promoters, promoters associated with the potato tuber ADPGPP gene, soybean promoters that drive seed-guided transcription of β-conglycinin (7S protein), and seed-guided promoters of the zein gene from maize endosperm; see, for example, Bevan et al. (1986) Nucleic Acids Res. 14: 4625-38; Muller et al. (1990) Mol. Gen. Genet. 224: 136-46; Bray (1987) Planta 172: 364-370; and Pedersen et al. (1982) Cell 29: 1015-26.

[0073] Collagen peptides can be produced in seeds using seed-based production techniques, such as rapeseed, corn, soybean, rice, and barley seeds. In such a method, the product is recovered, for example, during seed germination; see, for example, PCT Publications WO 9940210; WO 9916890; WO 9907206; U.S. Patent Nos. 5,866,121; and 5,792,933; and all references cited therein. Promoters that can be used to guide peptide expression can be heterologous or non-heterologous. These promoters can also be used to drive the expression of antisense nucleic acids to reduce, increase, or alter the concentration and composition of the animal collagen of the present invention in desired tissues.

[0074] Other modifications that can be made to increase and / or maximize the transcription of the polypeptides of the present invention in plants or plant cells are standard and known to those skilled in the art. For example, vectors containing a polynucleotide sequence encoding recombinant animal collagen or fragments or variants thereof, operably linked to a promoter, may also contain at least one factor that alters the transcription rate of collagen or related post-translational enzymes, including but not limited to peptide output signal sequences, codon usage, introns, polyadenylation, and transcription termination sites. Methods for modifying constructs to increase expression levels in plants are generally known in the art; see, for example, Rogers et al. (1985) J. Biol. Chem. 260:3731; and Cornejo et al. (1993) Plant Mol Biol 23:567-58. In engineered plant systems that affect the transcription rate of the collagen and related post-translational enzymes of the present invention, various factors known in the art, including regulatory sequences such as positive or negative action sequences, enhancers and silencers, and chromatin structure, can influence the transcription rate in plants. When expressing recombinant animal collagen (including but not limited to the collagen types described above), at least one of these factors may be used.

[0075] Vectors containing the polynucleotides of this invention will typically contain marker genes that confer selectable phenotypes to plant cells. Typically, the selectable marker genes will encode antibiotic resistance using suitable genes, including at least one set of genes encoding resistance to the antibiotic spectinomycin, a streptomycin phosphotransferase (SPT) gene encoding resistance to streptomycin, a neomycin phosphotransferase (NPTH) gene encoding resistance to kanamycin or genimycin, resistance to hygromycin, and genes encoding resistance to herbicides (especially sulfonylurea-type herbicides) that inhibit acetyllactone synthase (ALS) activity; for example, containing acetyllactone synthase (ALS) genes containing mutations that cause such resistance, particularly S4 and / or Hra mutations, genes encoding resistance to herbicides (such as phosphinic acid or basta) that inhibit glutamyl synthase activity, such as the bar gene, or other similar genes known in the art. The bar gene encodes resistance to the herbicide basta, the nptII gene encodes resistance to the antibiotics kanamycin and genimycin, and the ALS gene encodes resistance to the herbicide chlorsulfuron.

[0076] Common vectors used to express foreign genes in plants are well known in the art, including, but not limited to, vectors derived from tumor-inducing (Ti) plasmids of Agrobacterium tumefaciens. These vectors are plant integration vectors that integrate a portion of DNA into the genome of the host plant during transformation; see, for example, Rogers et al. (1987) Meth In Enzymol. 153:253-277; Schardl et al. (1987) Gene 61:1-11; and Berger et al., Proc. Natl. Acad. Sci. USA 86:8402-8406.

[0077] Vectors containing sequences encoding the polypeptides of the present invention and vectors containing post-translational enzymes or their subunits can be co-introduced into desired plants. Procedures for transforming plant cells are available in the art, such as direct gene transfer, in vitro protoplast transformation, plant virus-mediated transformation, liposome-mediated transformation, microinjection, electroporation, etc. Agrobacterium Mediated transformation and gene gun method; see, for example, Paszkowski et al. (1984) EMBO J. 3:2717-2722; U.S. Patent No. 4,684,611; European Application No. 0 67 553; U.S. Patent No. 4,407,956; U.S. Patent No. 4,536,475; Crossway et al. (1986) Biotechniques 4:320-334; Riggs et al. (1986) Proc. Natl. Acad. Sci USA 83:5602-5606; Hinchee et al. (1988) Biotechnology 6:915-921; and U.S. Patent No. 4,945,050. Standard methods for converting, for example, rice, wheat, corn, sorghum, and barley are described in the art; see, for example, Christou et al. (1992) Trends in Biotechnology 10: 239 and Lee et al. (1991) Proc. Nat'l Acad. Sci. USA 88:6389. Wheat can be converted using techniques similar to those used for converting corn or rice. Furthermore, Casas et al. (1993) Proc. Nat'l Acad. Sci. USA 90:11212 describes methods for converting sorghum, while Wan et al. (1994) Plant Physiol. 104: 37 teaches methods for converting barley. Suitable methods for converting corn are provided by Fromm et al. (1990) Bio / Technology 8:833 and Gordon-Kamm et al., ibid.

[0078] Other methods have been developed in the art for producing plants used to manufacture the animal collagen of the present invention; see, for example, U.S. Patent Nos. 5,959,091; 5,859,347; 5,763,241; 5,659,122; 5,593,874; 5,495,071; 5,424,412; 5,362,865; 5,229,112; 5,981,841; 5,959,179; 5,932,439; 5,869,720; and 5,800. 4,425; US Patent No. 5,763,245; US Patent No. 5,716,837; US Patent No. 5,689,052; US Patent No. 5,633,435; US Patent No. 5,631,152; US Patent No. 5,627,061; US ​​Patent No. 5,602,321; US ​​Patent No. 5,589,612; US Patent No. 5,510,253; US Patent No. 5,503,999; US Patent No. 5,378,619; US Patent No. 5,349,124; US Patent No. 5,304,730; US Patent No. 5,185,253; US Patent No. 4,970,168; European Publication No. EPA 00709462; European Publication No. EPA 00578627; European Publication No. EPA 00531273; European Publication No. EPA 00426641; PCT Publication No. WO99 / 31248; PCT Publication No. WO 98 / 58069; PCT Publication No. WO 98 / 45457; PCT Publication No. WO 98 / 31812; PCT Publication No. WO 98 / 08962; PCT Publication No. WO 97 / 48814; PCT Publication No. WO 97 / 30582; and PCT Publication No. WO9717459.

[0079] Insect Expression. Another alternative expression system for collagen is the insect system. Baculoviruses are highly efficient expression vectors for the large-scale production of various recombinant proteins in insect cells. Expression vectors containing the collagen-coding sequence for the collagen of this invention and appropriate transcription / translation control signals can be constructed using methods such as those described in Luckow et al. (1989) Virology 170:31-39 and Gruenwald, S. and Heitz, J. (1993) Baculovirus Expression Vector System: Procedures & Methods Manual, Pharmingen, San Diego, CA. For example, recombinant protein production can be achieved in insect cells by infecting baculovirus vectors encoding polypeptides. The production of recombinant collagen, collagen-like collagen, or collagen polypeptides with stable triple helices can involve co-infecting insect cells with three baculoviruses, one encoding the animal collagen to be expressed and each encoding the α and β subunits of prolyl 4-hydroxylase. This insect cell system allows for the large-scale production of recombinant proteins. In one such system, Alfalfa Silver-striped Noctuid Moth Nucleopolyhedrovirus (AcNPV) is used as a vector for expressing foreign genes. This virus grows in fall armyworm cells. The coding sequence for collagen or collagen-like polypeptides is cloned into a non-essential region of the virus (e.g., a polyhedrosis protein gene) and placed under the control of an AcNPV promoter (e.g., a polyhedrosis protein promoter). Successful insertion of the coding sequence inactivates the polyhedrosis protein gene and produces a nucleus-free recombinant virus; for example, a virus lacking the protein coat encoded by the polyhedrosis protein gene. These recombinant viruses are then used to infect cells expressing the inserted gene. fall armyworm Cells; see, for example, Smith et al. (1983) J. Virol. 46:584; and U.S. Patent No. 4,215,051. Further examples of such expression systems can be found, for example, Ausubel et al. above.

[0080] Animal Expression. Many expression systems can be used in animal host cells. In cases where adenovirus is used as the expression vector, a multinucleotide sequence encoding collagen or a collagen-like polypeptide can be linked to the adenoviral transcription / translation control complex, such as the late promoter and triplet leader sequence. This chimeric gene can then be inserted into the adenoviral genome via in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., region E1 or E3) will produce a recombinant virus that is viable and capable of expressing the encoded polypeptide in an infected host; see, for example, Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:3655-3659 (1984). Alternatively, the vaccinia 7.5 K promoter can be used; see, for example, Maskett et al. (1982) Proc. Natl. Acad. Sci. USA 79:7415-7419; Maskett et al. (1982) J. Virol. 49:857-864; and Panicali et al. (1982) Proc. Natl. Acad. Sci. USA 79:4927-4931.

[0081] The preferred expression system in mammalian host cells is Semliki forest virus. Infection of mammalian host cells, such as young hamster kidney (BHK) cells and Chinese hamster ovary (CHO) cells, yields very high levels of recombinant expression. Semliki forest virus is the preferred expression system because its broad host range makes it possible to infect mammalian cell lines. More specifically, Semliki forest virus can be used in a wide range of hosts because the system is not based on chromosomal integration, and therefore provides a simpler method for obtaining recombinant animal collagen modifications in studies aimed at identifying structure-function relationships and testing the roles of various hybrid molecules. Methods for constructing Semliki forest virus for expressing exogenous proteins in mammalian host cells are described, for example, in Olkkonen et al. (1994) Methods Cell Biol 43:43-53.

[0082] Non-human transgenic animals can also be used to express the polypeptides of the present invention. Such systems can be constructed by operatively linking the polynucleotides of the present invention to a promoter, along with other desired or optional regulatory sequences that enable expression in mammalian glands. Similarly, desired or optional post-translational enzymes can be simultaneously generated in target cells using a suitable expression system. Methods for recombinantly producing proteins using non-human transgenic animals are known in the art; see, for example, U.S. Patent Nos. 4,736,866; 5,824,838; 5,487,992; and 5,614,396.

[0083] References cited in the above sections describing the production of recombinant collagen are incorporated herein by reference.

[0084] Composite collagen fiber sheets. (Example) Figure 1 As shown, triple-helix collagen molecules associate to form fibrils, which assemble into larger bundles of fibrils or collagen fibers in animal skin. Existing methods for manufacturing collagen sheets use a mixture of ground animal skin or leather scraps with dissolved or suspended collagen. Such collagen-containing products are described in U.S. Patent Nos. 2,934,446; 3,073,714; 3,122,599; and 3,136,682. U.S. Patent No. 2,934,446, by Highberger et al., describes a method of using a meat grinder to produce a slurry of calfskin or dermis, which is shaped into sheets, tanned, and used to form linked collagen fiber blocks by pulverizing and dispersing animal skin in an acidic aqueous solution at 5°C, then increasing the pH and temperature to precipitate the collagen fibers, thereby forming a subsequently dried gel. These sheets of collagen fiber blocks utilize leather scraps and form similar sheet-like leather. Highberger did not indicate that these leather sheets are suitable for commercial use. U.S. Patent No. 3,073,714, authored by Tu et al., discloses the production of sheets from calfskin paste containing 25% solids, said calfskin paste being tanned with a vegetable tanning solution and treated with glycerin and oleic acid. These collagen fiber sheets are described as reproducing the internal arrangement of collagen fibers in natural skin and raw hides. Tu does not indicate that the leather sheets are compositionally or aesthetically suitable for use in consumer products. U.S. Patent No. 3,122,599, authored by Tu et al., describes leather sample sheets manufactured from milled animal skin or leather containing collagen fibers and soluble collagen, as well as other components derived from animal skin. Tu discloses treating this mixture with chromium, dehydrating it with acetone, and treating it with oleic acid to produce a leather sample product containing collagen fiber blocks. Tu does not indicate that said sheets are compositionally, physically, or aesthetically suitable for use in consumer products. U.S. Patent No. 3,136,682, authored by Tu et al., describes a method of manufacturing leather sample materials containing a mixture of collagen fibers and a binder of a water-soluble protein material derived from animal skin. The U.S. patent also describes the use of chromium tanning agents and treatment with oleic acid. Tu describes, but does not show, its suitable form of tablets for incorporation into consumer products that have a good appearance and feel. These products incorporate coarse, abrasive, or digested collagen fibers.

[0085] Cultured leather products. These products typically comprise multiple layers containing collagen produced from in vitro cultured cells as described by Forgacs et al., US 2016 / 0097109 A1 and Greene, US Patent No. 9,428,817 B2. These products are produced in vitro via cultured cell explants or cultured collagen-producing cells. Such cells produce collagen and process it into four bundles of collagen fibrils, and do not possess the random, anisotropic structure of the collagen fibrils of this invention. Forgacs describes engineered animal skin that can be shaped to produce leather products. Greene describes various products such as footwear, clothing, and luggage that can be incorporated into in vitro cultured leather. US 2013 / 0255003 describes the production of collagen for leather-like products by growing bovine skin cells in a culture. Other types of host cells have been used to produce collagen for medical implants or to produce gelatin. For example, US patent application US 2004 / 0018592 describes a method for producing gelatin by recombinantly expressing bovine gelatin in host cells such as yeast.

[0086] Medical Products. Collagen networks have been generated in vitro as materials for biomedical applications. In those applications, monomers of the collagen triple helix are extracted from animal tissues such as bovine dermis and dissolved from the tissue by acid treatment or treatment with protein-degrading enzymes such as pepsin. Once purified, these dissolved collagens (typically a mixture of collagen triple helix monomers, dimers, and trimers) can be fibrillated into fibrils by pH changes in an aqueous buffer. Under the correct conditions, the collagen monomers self-assemble into fibrils, and depending on their source and how they are separated, the fibrils can be physically cross-linked to form a solid hydrogel. Additionally, recombinant collagen and collagen-like proteins have shown similar in vitro fibrillation through pH and salt concentration regulation. Examples of such products for medical applications include biodegradable collagen matrices manufactured from collagen slurries that self-assemble into macroscopic collagen fibers (US Patent No. 9,539,363), and organized arrays of collagen fibrils generated by using externally guided structures or internal templates and applying tension (US Patent No. 9,518,106). Collagen products used in medicine, such as those for tissue engineering or transplantation, typically aim to provide a form that resembles the collagen in the specific tissue being engineered or repaired. While the fibrillation of soluble collagen and collagen-like proteins has been explored to produce collagen hydrogels for biomedical applications, this technology has not yet been successfully applied to produce materials with the strength and aesthetic properties of natural leather.

[0087] Synthetic, plastic-based leather. Attempts to create synthetic leather require addressing the challenge of replicating the unique functional and aesthetic properties of leather. Examples of synthetic leather materials include Clarino, Naugahyde®, Corfam, and Alcantara. They consist of various chemical and polymeric components, including polyvinyl chloride, polyurethane, nitrocellulose-coated cotton, polyester, or other natural fabrics or fibers coated with synthetic polymers. These materials are assembled using a variety of techniques typically derived from chemical and textile production methods, including nonwovens and advanced spin methods. While many of these materials are suitable for footwear, furniture, and apparel applications, they are not suitable for luxury applications because they do not match the breathability, performance, feel, or aesthetic properties that make leather so unique and desirable. To date, no alternative commercially available leather-like material has been created from a homogeneous network of collagen or collagen-like proteins. Synthetic plastic materials lack the chemical composition and structure of the collagen network that produces an acceptable leather aesthetic. Unlike synthetic compounds, the chemical composition of the amino acid side groups along the collagen polypeptide chain, along with its structure, enters the strong and porous fibrous structure, allowing the fibrillary network to be stabilized and functionalized through cross-linking methods in order to produce the desired strength, softness, and aesthetics of leather.

[0088] Although the fibrillation of soluble collagen and collagen-like proteins has been explored to bind milled or pulverized leather scraps together or to produce collagen hydrogels for biomedical applications, the production of commercially available and acceptable leather-like materials using this phenomenon has not yet been achieved.

[0089] In view of the problems of existing natural leather and composite, cultured and synthetic plastic-based leather products, the inventors have strived to provide a method for biomanufacturing leather with superior strength and uniformity as well as anisotropic properties, said biomanufacturing leather incorporating natural components present in leather.

[0090] This article describes materials composed of in vitro fibrillated collagen fibrils, which are endowed with leather-like properties through crosslinking, dehydration, and lubrication. Compared to tanned and fattened animal hides, these biomanufactured materials can possess uniformity in structure, composition, and function, such as advantageous substantially anisotropic strength and other mechanical properties, as well as a grain-like aesthetic on their upper and lower surfaces. Invention Overview

[0092] This invention relates to composite materials incorporating biomanufacturing materials described herein. The composites of this invention include those in which (i) one or more secondary components, such as particles, filaments, fabrics, or three-dimensional objects, are incorporated into or embedded in a collagen fibrillary network; (ii) the biomanufacturing material is coated or deposited, for example by filtration, on one side of one or more secondary components, such as woven or nonwoven fabrics (e.g., textiles, paper, or regenerated cellulose); (iii) the biomanufacturing component is coated or deposited on both sides of one or more secondary materials having an upper and lower side or an inner and outer side; or (iv) the biomanufacturing material component and one or more secondary components are adhered, attached, or laminated to each other, for example by direct lamination with or without an adhesive.

[0093] The composite of the present invention contains a biomanufacturing material component. This component consists of a network of cross-linked and lubricated collagen fibrils. It can be produced from collagen isolated from animal sources or recombinant collagen. It can be produced from collagen that is substantially free of residues. Preferably, it is substantially free of large bundles of collagen fibers or other non-hydroxylysine or non-3-hydroxyproline collagen components of leather, such as elastin. This material consists of collagen, which is also a major component of natural leather, and is produced by fibrillating collagen molecules into fibrils, cross-linking the fibrils, and lubricating the cross-linked fibrils. Unlike natural leather, this biomanufacturing material exhibits anisotropic (non-direction-dependent) physical properties; for example, a biomanufacturing material sheet can have substantially the same elasticity or tensile strength when measured in different directions. Unlike natural leather, the biomanufacturing material has a uniform texture that facilitates the uniform absorption of dyes and coatings. Aesthetically, the biomanufacturing material produces a uniform and consistent grain that is easy to manufacture. Unlike natural leather, composite materials incorporating this bio-manufactured material can have substantially the same grain, texture and other aesthetic properties on both sides, where the grain increases from one side (e.g., the distal surface) to the other side (the proximal inner layer). Brief description of the attached diagram

[0095] Figure 1 A diagram showing the composition of collagen in a layered manner. Reference characters (1) show each triple helix collagen monomer and how they assemble relative to adjacent collagen monomers; (2) show the collagen assembly that makes up the ribbon-like collagen fibrils; (3) show the collagen fibrils at a larger scale; (4) show the collagen fibrils arranged into fibers; and (5) show the collagen fiber bundles.

[0096] Figure 2AAn image illustrating the composition of buffalo hide. The upper grain layer and the underlying dermis are shown, with the relative degree of higher tissue from collagen fibrils to collagen fiber bundles indicated. The upper grain layer is typically composed primarily of fine collagen fibrils, while the dermis is typically composed primarily of coarser collagen fibers and fiber bundles.

[0097] Figure 2B and Figure 2C The texture and grain of the outer and inner surfaces of the leather were compared, depicting the fine grain on one side and the rough leather on the other.

[0098] Figure 3A A scanning electron micrograph of a fibrillated collagen hydrogel showing a network of fine collagen fibrils.

[0099] Figure 3B A scanning electron micrograph showing bovine dermis with relatively coarse fiber bundles.

[0100] Figure 4 Transmission electron micrographs showing fibrillated collagen networks or hydrogels bound together with fibrils. Invention Details

[0102] As used herein, “biomanufactured material” or “biomanufactured leather” refers to a material derived from collagen or collagen-like proteins. It can be produced from non-human collagen such as bovine, buffalo, bull, deer, sheep, goat, or porcine collagen, which can be isolated from natural sources like animal hides, recombinantly produced through in vitro culture of mammalian or animal cells, or chemically synthesized. It is not a conventional material or leather derived from animal skin. Methods for producing such biomanufactured material or biomanufactured leather are disclosed herein, and generally involve the preparation of isolated or purified solutions or suspensions of fibrillated collagen molecules to produce collagen fibrils, cross-linking the fibrils, dehydrating the fibrils, and lubricating the fibrils.

[0103] Compared to natural leather, which exhibits a heterogeneous internal collagen structure, biomanufactured materials or biomanufactured leather can exhibit a substantially uniform internal structure, characterized by unbound and randomly oriented collagen fibrils throughout its volume.

[0104] Biomanufactured materials can be used in any way that uses natural leather and can be very similar to genuine leather in appearance and feel, while possessing compositional, functional, or aesthetic characteristics that distinguish them from ordinary leather. For example, unlike natural leather, biomanufactured leather does not need to contain potentially allergenic non-collagenous proteins or components present in natural leather. Biomanufactured leather can exhibit similar flexibility and strength (anisotropy) in all directions due to the essentially unarranged nature of its collagen fibrils and aesthetically can have a smooth grain texture on both sides. Biomanufactured leather can exhibit properties of uniformity, including uniform thickness and consistency, uniform distribution of lubricants, crosslinking agents, and dyes, and uniform anisotropic strength, stretch, flexibility, and flexural strength (or the tendency of natural leather to separate or split parallel to the sheet plane). By selecting collagen content and processing conditions, biomanufactured leather can be "tuned" to a specific thickness, consistency, flexibility, softness, wrinkled surface texture, or other functions. Laminated, layered, or composite products can contain biomanufactured leather.

[0105] A “complex” is a combination of a biomanufacturing material or biomanufacturing leather component and a secondary material. The secondary component may be incorporated into the biomanufacturing material; the biomanufacturing material may be at least partially incorporated into the secondary material, or coated, laminated, or pressed onto the secondary material. Examples of complexes include biomanufacturing materials encapsulating secondary materials, secondary materials coated with biomanufacturing material on one side, secondary materials coated with biomanufacturing material on both outer sides, and one or more layers of secondary material laminated to one or more layers of biomanufacturing material. This term encompasses all forms and combinations of biomanufacturing materials and one or more secondary materials.

[0106] The term "collagen" refers to any known type of collagen, including type I through type XX collagen, as well as any other collagen, whether natural, synthetic, semi-synthetic, or recombinant. It includes all collagens, modified collagens, and collagen-like proteins described herein. The term also encompasses procollagen and collagen-like proteins or collagen proteins containing the motif (Gly-XY)n, where n is an integer. It covers molecules of collagen and collagen-like proteins, trimers of collagen molecules, collagen fibrils, and collagen fibrils. It also refers to chemically, enzymatically, or recombinantly modified collagen or collagen-like molecules that can be fibrillated, as well as fragments of collagen and collagen-like molecules and collagen molecules capable of assembling into nanofibers.

[0107] In some embodiments, amino acid residues such as lysine and proline in collagen or collagen-like proteins may lack hydroxylation or have a lower or higher degree of hydroxylation compared to the corresponding native or unmodified collagen or collagen-like proteins. In other embodiments, amino acid residues in collagen or collagen-like proteins may lack glycosylation or have a lower or higher degree of glycosylation compared to the corresponding native or unmodified collagen or collagen-like proteins.

[0108] The collagen in a collagen composition may homogeneously contain a single type of collagen molecules, such as 100% type I bovine collagen or 100% type III bovine collagen, or may contain a mixture of different types of collagen molecules or collagen-like molecules, such as a mixture of bovine type I and type III molecules. Such mixtures may contain >0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or <100% of individual collagen or collagen-like protein components. This range includes all intermediate values. For example, a collagen composition may contain 30% type I collagen and 70% type III collagen, or it may contain 33.3% type I collagen, 33.3% type II collagen, and 33.3% type III collagen, wherein the percentage of collagen is based on the total mass of collagen in the composition or on the molecular percentage of collagen molecules.

[0109] "Collagen fibrils" are nanofibers composed of procollagen (triple helixes of collagen molecules). Procollagen also includes procollagen-like structures exhibiting a triple helix structure. The collagen fibrils of the present invention can have diameters in the range of 1 nm and 1 µm. For example, the collagen fibrils of the present invention can have average or individual fibril diameters in the range of 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 nm (1 µm). This range includes all intermediate values ​​and sub-ranges. In some embodiments of the present invention, the collagen fibrils will be formed, for example, as shown in Figure 3 and Figure 4 The network depicted. Collagen fibrils can associate to exhibit characteristics such as Figure 1 The illustrated fibrils are strip-shaped fibers, and these fibrils can associate into larger fibril aggregates. In some embodiments, collagen or collagen-like fibrils will have a diameter and orientation similar to those in the grain or surface layer of cowhide or other conventional leather. In other embodiments, collagen fibrils may have diameters including those in the grain and dermal layers of conventional leather.

[0110] "Collagen fibers" are composed of collagen fibrils that are tightly compressed and, as... Figure 1The fibers shown exhibit a high degree of alignment. Their diameter can vary from greater than 1 µm to greater than 10 μm, for example, >1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 μm or larger. Some embodiments of the collagen fibril network of the present invention do not contain a large number of collagen fibers with a diameter greater than 5 μm. As shown in Figure 2, the grain surface composition of leather can differ from its more internal portions, such as dermis containing coarser fiber bundles.

[0111] "Fibrinolysis" refers to the process of producing collagen fibrils. It can be achieved by increasing the pH or by adjusting the salt concentration of the collagen solution or suspension. In fibrinoly fused collagen, the collagen can be incubated to form fibrils for any suitable length of time, including between 1 minute and 24 hours and all intermediate values.

[0112] The fibrillated collagen described herein can generally be formed in any suitable shape and / or thickness, including flat sheets, curved shapes / sheets, cylinders, threads, and composite shapes. These sheets and other forms can have virtually any linear dimensions, including thicknesses, widths, or heights greater than 10, 20, 30, 40, 50, 60, 70, 80, 90 mm; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 200, 500, 1,000, 1,500, 2,000 cm or greater.

[0113] The fibrillated collagen in biomanufactured leather may lack any amount or any large quantity of higher-order structures. In a preferred embodiment, the collagen fibrils in the biomanufactured leather will be unbound and will not form the larger collagen fibers present in animal skin, thus providing the biomanufactured leather with a strong and uniform anisotropic structure.

[0114] In other embodiments, some collagen fibrils may be bundled or arranged into a more advanced structure. Collagen fibrils in biomanufactured leather may exhibit an orientation index ranging from 0, >0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, <1.0, or 1.0, where an orientation index of 0 describes collagen fibrils lacking alignment with other fibrils, and an orientation index of 1.0 describes fully aligned collagen fibrils. This range includes all intermediate values ​​and subranges. The orientation index is familiar to those skilled in the art and is also incorporated by reference to: Sizeland et al., J. Agric. Food Chem. 61: 887-892 (2013) or Basil-Jones et al., J. Agric. Food Chem. 59:9972-9979 (2011).

[0115] The methods disclosed herein enable the production of biomanufactured leather comprising collagen fibrils of different diameters than those produced by animals expressing the same type of collagen. Characteristics of natural collagen, such as fibril diameter and the degree of cross-linking between fibrils, are influenced by genetic and environmental factors (such as animal species or breed), and by animal condition (e.g., fat content, type of feed (e.g., grain, straw), and level of exercise).

[0116] Biomanufactured leather can be fibrillated and processed to contain collagen fibrils that resemble or mimic the properties of collagen fibrils produced by a particular animal species or breed or by animals fed under specific conditions.

[0117] Alternatively, fibrillation and processing conditions can be selected to provide collagen fibrils that differ from those found in nature, such as by reducing or increasing the diameter, arrangement, or cross-linking of fibrils compared to those in natural leather.

[0118] A collagen cross-linked network (sometimes called a hydrogel) can be formed from fibrillated collagen, or it can form a network after fibrillation; in some variants, the fibrillation of collagen also forms a gel-like network. Once formed, the fibrillated collagen network can be further stabilized by incorporating molecules with bifunctional, trifunctional, or multifunctional reactive groups, including chromium, amines, carboxylic acids, sulfuric acid, sulfurous acid, sulfonic acid, aldehydes, hydrazides, mercapto, diaziridine, aryl-, azides, acrylates, epoxides, or phenols.

[0119] The fibrillated collagen network can also be polymerized with other agents (e.g., polymerizable polymers or other suitable fibers) that can be used to further stabilize the matrix and provide the desired end structure. Hydrogels based on acrylamide, acrylic acid, and their salts can be prepared using reverse suspension polymerization. The hydrogels described herein can be prepared from polar monomers. The hydrogels used can be natural polymer hydrogels, synthetic polymer hydrogels, or a combination of both. The hydrogels used can be obtained using graft polymerization, crosslinking polymerization, networks formed from water-soluble polymers, radiation crosslinking, etc. A small amount of crosslinking agent can be added to the hydrogel composition to enhance polymerization.

[0120] Throughout the thickness of the bio-manufactured leather, the average or individual collagen fibrillary lengths range from 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 (1 µm); to 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 µm (1 mm). These ranges include all intermediate values ​​and subranges.

[0121] The fibrils may be arranged above other fibrils at lengths of 50, 100, 200, 300, 400, 500 µm or greater, or may exhibit less or no arrangement. In other embodiments, some collagen fibrils may be bundled or arranged into a more advanced structure.

[0122] Collagen fibrils in biomanufactured leather can exhibit an orientation index ranging from 0, >0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, <1.0, or 1.0, where an orientation index of 0 describes collagen fibrils lacking alignment with other fibrils, and an orientation index of 1.0 describes fully aligned collagen fibrils. This range includes all intermediate values ​​and subranges. The orientation index is familiar to those skilled in the art and is also incorporated by reference to: Sizeland et al., J. Agric. Food Chem. 61: 887-892 (2013) or Basil-Jones et al., J. Agric. Food Chem. 59: 9972-9979 (2011).

[0123] The collagen fibrillation density of bio-manufactured leather can range from about 1 to 1,000 mg / cc, preferably from 5 to 500 mg / cc, including all intermediate values ​​such as 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 and 1,000 mg / cc.

[0124] Collagen fibrils in biomanufactured leather can exhibit unimodal, bimodal, trimodal, or multimodal distributions. For example, biomanufactured leather can be composed of two different fibril formulations, each having a different range of fibril diameters arranged around one of two different modalities. Such mixtures can be selected to impart additive, synergistic, or balanced physical properties to biomanufactured leather obtained from fibrils of different diameters.

[0125] Based on the weight of the leather product, natural leather products may contain 150-300 mg / cc of collagen. Based on the weight of biomanufactured leather, biomanufactured leather may contain similar amounts of collagen or collagen fibrils as conventional leather, such as collagen concentrations of 100, 150, 200, 250, 300, or 350 mg / cc.

[0126] Fibrous collagen (sometimes called hydrogel) can have a thickness chosen based on its end use. Thicker or more concentrated formulations of fibrillated collagen typically produce thicker biomanufactured leather. The final thickness of biomanufactured leather can be only 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the fibrillated formulation before shrinkage caused by crosslinking, dehydration, and lubrication.

[0127] "Cross-linking" refers to the formation (or reforming) of chemical bonds within collagen molecules. Cross-linking reactions stabilize the collagen structure and, in some cases, form a network between collagen molecules. Any suitable cross-linking agent known in the art can be used, including but not limited to inorganic salts such as chromium-based ones, formaldehyde, hexamethylene diisocyanate, glutaraldehyde, polyepoxides, gamma radiation targeting riboflavin, and ultraviolet radiation. Crosslinking can be performed by any known method; see, for example, Bailey et al., Radiat. Res. 22:606-621 (1964); Housley et al., Biochem. Biophys. Res. Commun. 67:824-830 (1975); Siegel, Proc. Natl. Acad. Sci. USA 71:4826-4830 (1974); Mechanic et al., Biochem. Biophys. Res. Commun. 45:644-653 (1971); Mechanic et al., Biochem. Biophys. Res. Commun. 41:1597-1604 (1970); and Shoshan et al., Biochim. Biophys. Acta 154:261-263 (1968), each of which is incorporated herein by reference.

[0128] Crosslinking agents include isocyanates, carbodiimides, poly(aldehyde), poly(acrididine), inorganic salts, poly(epoxides), enzymes, thiopropylcyclohexane, phenolic resins, phenolic varnishes, soluble phenolic resins, and other compounds having chemical properties that react with amino acid side chains, such as lysine, arginine, aspartic acid, glutamic acid, hydroxyproline, or hydroxylysine.

[0129] Collagen or collagen-like proteins can be chemically modified to promote chemical and / or physical cross-linking between collagen fibrils. Chemical cross-linking is possible because reactive groups on collagen molecules, such as lysine, glutamic acid, and hydroxyl groups, extend from the rod-like fibril structure of collagen. Cross-linking involving these groups prevents collagen molecules from sliding past each other under stress and thus increases the mechanical strength of collagen fibers. Examples of chemical cross-linking reactions include, but are not limited to, reactions with lysine. Reacting with amino groups or carboxyl groups in collagen molecules. Enzymes such as transglutaminase can also be used to create cross-links between glutamate and lysine to form stable compounds. -Glutamicyl-lysine crosslinking. Inducing crosslinking between functional groups of adjacent collagen molecules is known in the art. Crosslinking is another step that can be performed here to modulate the physical properties obtained from materials derived from fibrillated collagen hydrogels.

[0130] Collagen that is still fibrillating or has undergone fibrillation can be crosslinked or lubricated. Collagen fibrils can be treated with a compound containing chromium or at least one aldehyde group, or with a vegetable tanning agent before, during, or after network formation. Crosslinking further stabilizes fibrillated collagen leather. For example, collagen fibrils pretreated with an acrylic polymer and then treated with a vegetable tanning agent such as hygroscopic oleoresin can exhibit increased hydrothermal stability. In other embodiments, glyceraldehyde can be used as a crosslinking agent to increase the thermal stability, protein hydrolysis resistance, and mechanical properties of fibrillated collagen, such as Young's modulus and tensile stress.

[0131] Biomanufactured materials containing collagen fibrillary networks may contain 0, >0, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or more crosslinking agents, including tanning agents used for conventional leather. The crosslinking agents may be covalently bonded to collagen fibrils or other components of the biomanufactured material or non-covalently associated with them. Preferably, the biomanufactured leather will contain no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% crosslinking agents.

[0132] "Lubrication" describes a method of applying a lubricant to leather or to a biomanufactured product containing collagen, such as fats or other hydrophobic compounds or any material that modulates or controls fibril-fibril bonding during dehydration. A desirable aesthetic characteristic of leather is the material's rigidity or feel. To achieve this property, a lubricant is used to confine water-mediated hydrogen bonds between fibrils and / or fibers within the leather. Examples of lubricants include fats, bio-oils, mineral or synthetic oils, cod oil, sulfonated oils, polymers, organofunctional siloxanes, and other hydrophobic compounds or agents used for fatliquoring conventional leather, as well as mixtures thereof. While lubrication is performed in some ways similar to fatliquoring natural leather, biomanufactured products can be treated more uniformly with lubricants due to their manufacturing methods, more homogeneous compositions, and less complex formulations.

[0133] Other lubricants include surfactants, anionic surfactants, cationic surfactants, cationic polymeric surfactants, anionic polymeric surfactants, amphoteric polymers, fatty acids, modified fatty acids, nonionic hydrophilic polymers, nonionic hydrophobic polymers, polyacrylic acid, polymethacrylic acid, acrylic acid, natural rubber, synthetic rubber, resins, amphoteric anionic polymers and copolymers, amphoteric cationic polymers and copolymers and mixtures thereof, as well as emulsions or suspensions of these in water, alcohols, ketones and other solvents.

[0134] Lubricants can be added to biomanufactured materials containing collagen fibrils. Any amount that can facilitate fibril movement or impart leather-like properties such as flexibility, reduced brittleness, durability, or water resistance can be incorporated into the lubricant. Lubricant content can range from approximately 0.1%, 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, and 60% by weight of the biomanufactured leather.

[0135] "Dehydration" or "water removal" describes a method for removing water from a mixture containing collagen fibrils and water, such as an aqueous solution, suspension, gel, or hydrogel containing fibrillated collagen. Water can be removed by filtration, evaporation, freeze-drying, solvent exchange, vacuum-drying, convection-drying, heating, radiation, or microwaves, or by other known methods for water removal. Additionally, chemical cross-linking of collagen is known to remove bound water from collagen by consuming hydrophilic amino acid residues such as lysine, arginine, and hydroxylysine. The inventors have found that acetone can rapidly dehydrate collagen fibrils and also remove water bound to hydrated collagen molecules. The water content of the dehydrated biomanufactured material or leather is preferably no more than 60% by weight, for example, no more than 5%, 10%, 15%, 20%, 30%, 35%, 40%, 50%, or 60% by weight of the biomanufactured leather. This range includes all intermediate values. The water content is measured by equilibration at 65% relative humidity, 25°C, and 1 atmosphere.

[0136] "Grain texture" describes a leather-like texture that is aesthetically or texturally similar to that of full-grain leather, top-grain leather, modified grain leather (where artificial grain has been applied), or a coarser, split grain leather. Advantageously, the bio-manufacturing materials of the present invention can be modified to provide a similar texture to those found in leathers such as those made from… Figure 2A , Figure 2B and Figure 2C The fine grain of the surface of the leather is depicted.

[0137] "Biomanufactured leather products" include products containing at least one component of biomanufactured leather, such as work shoes, clothing, gloves, furniture or automotive trim, and other leather goods and products. It includes, but is not limited to, clothing such as coats, jackets, shirts, trousers, underpants, shorts, swimwear, underwear, uniforms, badges or lettering, theatrical costumes, bow ties, skirts, dresses, blouses, leggings, gloves, baseball gloves, work shoes, footwear, shoe components (such as soles, quarters, tongues, collars, welts, and heel counters), fashion shoes, athletic shoes, running shoes, casual shoes, athletic, running, or casual shoe components (such as toe caps, pullovers, outsoles, insoles, uppers, laces, eyelets, collars, linings, Achilles' heels, and heel counters), fashion or women's shoes and their components (such as uppers, outsoles, toe caps). Shoes with upturned toes, embellishments, uppers, linings, insoles, midsoles, platform soles, boots, and heels; or high heels, boots, sandals, buttons, hats, masks, headwear, headbands, backbands, and belts; jewelry such as bracelets, watch straps, and necklaces; gloves, umbrellas, canes, wallets, mobile phone or wearable computer covers, handbags, backpacks, briefcases, purses, registers, folders, boxes, and other personal items; sports, athletic, hunting, or recreational equipment such as horse harnesses, reins, ropes, bits, belts, gloves, tennis rackets, golf clubs, water polo, hockey or hockey equipment, chessboards and game boards, medicine balls, and kick balls. Balls, baseballs and other types of balls and toys; book covers, book covers, picture frames or works of art; furniture and home furnishings, office or other indoor or outdoor furniture, including chairs, sofas, doors, seats, footstools, partitions, coasters, mouse pads, desktop notebooks or other mats, tables, beds, floors, wall or ceiling coverings, flooring materials; automotive, marine, aircraft and other vehicle products, including seats, headrests, furniture, paneling, steering wheels, joysticks or control covers and other packaging or coverings.

[0138] Many leather product applications require durable materials that will not crack or tear, even when the leather is sewn together. Common products that include sewn leather and require durable leather include car steering wheel covers, car seats, furniture, sporting goods, athletic shoes, rubber-soled canvas shoes, watch straps, etc. There is a need to increase the durability of bio-manufactured leather to improve the performance of these products. The bio-manufactured leather according to the present invention can be used to prepare any of these products.

[0139] The physical properties of biomanufactured collagen fibrils or biomanufactured leather can be selected or adjusted by choosing the type of collagen, the concentration and amount of fibrillated collagen, and the degree of fibrillation, cross-linking, dehydration, and lubrication. Many advantageous properties are associated with the network structure of collagen fibrils, which can provide the resulting biomanufactured material or leather with strong, soft, and substantially uniform properties. Preferred physical properties of the bio-manufactured leather according to the invention include tensile strength in the range of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or greater MPa; flexibility determined by elongation at break in the range of 1%, 5%, 10%, 15%, 20%, 25%, 30% or greater; softness as defined by ISO 17235 having 4, 5, 6, 7, 8 mm or greater; and a tensile strength in the range of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 MPa. Thicknesses in the range of mm or greater and collagen densities (collagen fibrillary densities) of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 mg / cc or greater, preferably 100-500 mg / cc. The above ranges include all subranges and intermediate values.

[0140] Thickness. Depending on its end application, biomanufactured materials or leather can have any thickness. Its thickness is preferably in the range of about 0.05 mm to 20 mm, and any intermediate values ​​within this range, such as 0.05, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50 mm or greater. The thickness of biomanufactured leather can be controlled by adjusting the collagen content.

[0141] Elastic modulus. The elastic modulus (also known as Young's modulus) is a numerical measure of an object's resistance to elastic deformation (i.e., non-permanent) when a force is applied to it. The elastic modulus of an object is defined as the slope of its stress-strain curve in the region of elastic deformation. Stiffer materials will have a higher elastic modulus. The elastic modulus can be measured using a texture analyzer.

[0142] Biomanufactured leather can have an elastic modulus of at least 100 kPa. It can be in the range of 100 kPa to 1,000 MPa, and any intermediate values ​​within this range, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 MPa. Biomanufactured leather can be stretched from its relaxed length to 300%, for example, >0, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, or 300% of its relaxed length.

[0143] Tensile strength (also known as ultimate tensile strength) is the ability of a material or structure to withstand a load that tends to elongate, whereas compressive strength resists a load that tends to shrink. Tensile strength resists being pulled apart or stretched, while compressive strength resists being compressed or pushed together.

[0144] The tensile strength of samples of biomanufactured materials can be tested using an Instron machine. A clamp is attached to the end of the sample and pulled in opposite directions until failure. A sample with a tensile strength of at least 1 MPa demonstrates good strength. Biomanufactured leather can have a tensile strength of at least 1 kPa. It can be in the range of 1 kPa to 100 MPa and any intermediate values ​​within this range, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 200, 300, 400, 500 kPa; 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 MPa.

[0145] Tear strength (also known as tear resistance) is a measure of how well a material can withstand tearing. More precisely, while it is how well a material (typically rubber) resists any cut growth under tension, it is usually measured in kN / m. Tear resistance can be measured using the ASTM D 412 method (the same method used to measure tensile strength, modulus, and elongation). ASTM D 624 can be used to measure resistance to tear initiation (tear onset) and resistance to tear propagation (tear spread). Regardless of which one is being measured, the sample is held between two clamps and a uniform tensile force is applied until the aforementioned deformation occurs. Tear resistance is then calculated by dividing the applied force by the thickness of the material. Bio-manufactured leather can exhibit at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 150%, or 200% greater tear resistance than other leathers of the same thickness or containing the same type of collagen (e.g., bovine type I or III collagen) and processed with the same crosslinking agent or lubricant. Biomanufactured materials can have tear strengths in the range of about 1 to 500 N, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 or 500, and any intermediate tear strengths within this range.

[0146] Softness. ISO 17235:2015 describes a non-destructive method for determining the softness of leather. It can be applied to all non-rigid leathers, such as upper leather, decorative leather, leather goods leather, and garment leather. Biomanufactured leathers can have a softness of 2, 3, 4, 5, 6, 7, 8, 10, 11, 12 mm or greater as determined by ISO 17235.

[0147] Grain. The top grain surface of leather is generally considered the most desirable due to its soft texture and smooth surface. The top grain is a highly porous network of collagen fibrils. The strength and tear resistance of the grain usually limit the practical application of a single top grain, and regular leather products typically feature genuine leather with a coarser grain on the back. Figure 2A , Figure 2B and Figure 2C Grained and genuine leather surfaces were compared. Bio-manufacturing materials, as disclosed herein, can produce products with strong and uniform physical properties or increased thickness, and can be used to provide grained sample products without requiring a genuine leather backing.

[0148] Content of other components. In some embodiments, the collagen does not contain other leather components such as elastin or non-structural animal proteins. However, in some embodiments, the content of actin, keratin, elastin, fibrin, albumin, globulin, mucin, mucinoids, non-collagen structural proteins and / or non-collagen non-structural proteins in the biomanufactured leather may range from 0, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% to 10% by weight of the biomanufactured leather. In other embodiments, the content of actin, keratin, elastin, fibrin, albumin, globulin, mucin, mucinoids, non-collagen structural proteins and / or non-collagen non-structural proteins may be incorporated into the biomanufactured leather in amounts ranging from >0, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% or greater by weight of the biomanufactured leather. Such components can be introduced during or after fibrillation, crosslinking, dehydration, or lubrication.

[0149] "Leather dyes" refer to dyes that can be used to dye leather or biomanufacture leather. These include acid dyes, direct dyes, lake pigments, sulfur dyes, basic dyes, and reactive dyes. Dyes and pigments can also be incorporated into precursors for biomanufactured leather, such as into suspensions or network gels containing collagen fibrils during the production of biomanufactured leather.

[0150] "Fillers." In some embodiments, biomanufactured leather may include fillers, such as microspheres, in addition to leather components. One method of controlling the organization of the dehydrated fibril network is to include filler materials that keep the fibrils separated during the dehydration process. These filler materials include nanoparticles, microparticles, or various polymers such as synthetic tanning agents commonly used in the tanning industry. These filler materials may be part of the final dehydrated leather material, or they may be sacrificial, i.e., they are degraded or dissolved to leave open spaces for a more porous fibril network. The shape and size of these fillers can also be used to control the orientation of the dehydrated fibril network.

[0151] In some embodiments, the filler or secondary component may include polymer microspheres, beads, fibers, filaments, or organic salts. Other materials may also be embedded or otherwise incorporated into the biomanufactured leather or into the collagen fibrillary network according to the invention. These include, but are not limited to, fibers, including woven and nonwoven fibers, as well as cotton, wool, cashmere, Angora goat hair, flax, bamboo, bast, hemp, soybean, seaweed fiber, fibers derived from milk or milk proteins, silk, spider silk, other peptides or polypeptides (including recombinant peptides or polypeptides), chitosan, mycelium, cellulose (including bacterial cellulose), wood (including wood fibers), rayon, lyocell, fiber glue, antimicrobial yarn (AMY), Sorbtek, nylon, polyester, elastomers such as lycra®, spandex elastic fibers or other polyester-polyurethane copolymers, polyaromatic amides, carbon (including carbon fibers and fullerenes), glass (including glass fibers and nonwovens), silicon and silicon-containing compounds, minerals (including mineral particles and mineral fibers), and metals or metal alloys, including those containing iron, steel, lead, gold, silver, platinum, copper, zinc, and titanium, which may be in the form of particles, fibers, threads, or other forms suitable for incorporation into bio-manufactured leather. Such fillers may include conductive materials, magnetic materials, fluorescent materials, bioluminescent materials, phosphorescent materials, or other photoluminescent materials, or combinations thereof. Mixtures or blends of these components may also be embedded or incorporated into biomanufactured leather, for example, to modify the chemical and physical properties disclosed herein.

[0152] A method for manufacturing biomaterial components of a complex.

[0153] Methods for forming biomanufacturing material components from collagen used in composites include steps of fibrillation, crosslinking, dehydration / removal, and lubrication in any order. For example, a collagen solution may be fibrillated, the fibrils may be crosslinked with a reagent such as glutaraldehyde, then coated with a lubricant such as sulfite oil, followed by dehydration through filtration to form fibrillated collagen leather. However, manufacturing methods are not limited to this specific sequence of steps.

[0154] Alternatively, after the fibrils are cross-linked, they can be dehydrated by acetone solvent exchange, followed by fatliquoring with sulfite oil before the dissolution evaporates to form fibrillated collagen leather. Furthermore, the incorporation of chemical or physical cross-links between the fibrils (to apply material strength) can be done at any point in the process. For example, solid fibrillated collagen (sometimes called a hydrogel) can be formed, and this fibril network can then be dehydrated by acetone solvent exchange, followed by fatliquoring with sulfite oil. Additionally, collagen fibrils can be cross-linked into a network by incorporating other polymers, such as those commonly used in resin formulations.

[0155] Materials such as lubricants, humectants, dyes, and other treatment agents can be uniformly distributed throughout the biomanufacturing leather product during the biomanufacturing process. This is an advantage over conventional leather tanning and fatliquoring, which typically cannot achieve uniform treatment due to their structural heterogeneity. Furthermore, since chemicals can be incorporated before network formation, fewer treatment chemicals are required due to reduced chemical loss by not having to permeate the collagen network from a suspension containing treatment chemicals. Unlike the high temperatures typically used to treat natural leather, biomanufacturing leather can be heated at ambient temperature or no higher than 37°C during processing, before the solvent evaporates, to form fibrillated collagen leather. Alternatively, suspended collagen fibrils can be crosslinked and lubricated before network formation between fibrils during dehydration, or by adding binders to the suspension or dehydrated material.

[0156] Methods for forming biomanufactured leather materials may include inducing fibrillation of collagen in solution; cross-linking (e.g., tanning) and dehydrating the fibrillated collagen, which may be in the form of a hydrogel to obtain fibrillated collagen sheets or other products; and incorporating at least one humectant or lubricant, such as fat or oil, into the fibrillated collagen sheets or products to obtain flexible biomanufactured leather.

[0157] Methods for biomanufacturing leather from fibrils may include inducing fibrillation of collagen or collagen-like proteins in solution to obtain fibrillated collagen hydrogels; crosslinking the fibrillated collagen hydrogels to obtain fibrillated collagen hydrogel leather; and incorporating at least one lubricant into the fibrillated collagen hydrogel leather.

[0158] In the methods described herein for producing biomanufactured leather, the order of steps for forming the biomanufactured leather may be altered or two or more steps may be performed simultaneously. For example, fibrillation and crosslinking may be performed simultaneously, or one or more reagents, or crosslinking agents and lubricants, may be incorporated into the solution prior to fibrillating collagen, etc.

[0159] Collagen or collagen-like proteins can be obtained from animal sources such as, but not limited to, bovine raw hide or tendon collagen extracts. Alternatively, collagen or collagen-like proteins can be obtained from non-animal sources, for example, through recombinant DNA technology, cell culture technology, or chemical peptide synthesis.

[0160] Any of these methods may include polymerizing collagen or collagen-like proteins into dimers, trimers, and higher oligomers prior to fibrillation and / or chemical modification of collagen or collagen-like proteins to promote cross-linking between collagen or collagen-like proteins.

[0161] Any of these methods may include functionalizing collagen or collagen-like proteins with one or a combination of the following: chromium, amine, carboxylic acid, sulfate, sulfite, sulfonate, aldehyde, acylhydrazine, mercapto, diaziridine, aryl, azide, acrylate, epoxide, or phenolic groups.

[0162] Inducing fibrillation may involve adding salts or combinations of salts, such as Na3PO4, K3PO4, KCl, and NaCl, with the concentration of each salt ranging from 10 mM to 5 M.

[0163] Typically, inducing fibrillation may involve adjusting the pH with an acid or base and adding a nucleating agent such as branched-chain collagen microgels, wherein the nucleating agent has a concentration between 1 mM and 100 mM.

[0164] Fibrous collagen can be stabilized using chromium compounds, aldehyde compounds, vegetable tanning agents, or any other cross-linking agents. For example, chromium compounds, aldehyde compounds, or vegetable tanning agents can be used to stabilize fibrillated collagen, wherein the chromium, aldehyde, or vegetable tanning agent compound has a concentration between 1 mM and 100 mM.

[0165] Any of these methods may include adjusting the moisture content of fibrillated collagen to 5%, 10%, 20%, 25%, 30%, 40%, 50%, or 60% by weight or less to obtain fibrillated collagen hydrogel leather. For example, the fibrillated collagen raw material may be dehydrated. Any of these methods may also include dyeing and / or applying surface finishing to the fibrillated collagen leather.

[0166] The selection of collagen-starting materials for the biomanufacturing of the engineered leather materials described herein can be controlled, resulting in products with varying physical and aesthetic properties for different end uses, such as features suitable for work shoes and features suitable for clothing. Typically, the biomanufactured fibrillated collagen hydrogel-derived leather described herein is formed from collagen solutions induced to self-assemble into collagen fibrils.

[0167] Unlike endogenous collagen fibrils, collagen fibrils cannot assemble into any higher-order structure (e.g., fiber bundles), but remain somewhat disordered, more specifically, unbound fibrils. When assembled in vivo, collagen fibrils typically align laterally to form bundles with higher-order structures and constitute, for example, the tough, micron-sized collagen fibers present in the skin. A characteristic feature of natural collagen fibrils is their banded structure. The diameter of natural collagen fibrils varies slightly along their length, with highly reproducible D-bands repeating at approximately 67 nm. In some of the methods described herein, collagen fibrils may be non-banded and unbound, or may be banded and unbound, or may have D-bands with varying intervals in the range of 1 to 100 nm and all intermediate values ​​within this range. Collagen fibrils may be randomly oriented (e.g., unoriented or not oriented in any particular direction or axis).

[0168] Starting materials used to form the biomanufactured leather materials described herein may include any suitable non-human collagen source or modified or engineered collagen that can be fibrillated.

[0169] Various forms of collagen exist throughout the animal kingdom. The collagen used in this article can be obtained from animal sources (including vertebrates and invertebrates) or from synthetic sources. Collagen can also be derived from byproducts of existing animal processing. Collagen obtained from animal sources can be isolated using standard laboratory techniques known in the art, such as Silva et al., Marine Origin Collagens and its Potential Applications, Mar. Drugs, December 2014, 12(12);5881-5901).

[0170] A key benefit of the biomanufactured leather materials described herein and the methods for forming said biomanufactured leather materials is that collagen can be obtained from sources that do not require killing animals.

[0171] The collagen described in this article can also be obtained through cell culture techniques, including cells grown from bioreactors.

[0172] Collagen can also be obtained through recombinant DNA technology. Constructs encoding non-human collagen can be introduced into host organisms to produce non-human collagen. For example, collagen can also be produced using yeasts such as *Hansenula polymorpha*, *Saccharomyces cerevisiae*, and *Pichia pastoris* as hosts. Furthermore, in recent years, bacterial genomes providing marker (Gly-Xaa-Yaa)n repeating amino acid sequences characteristic of triple-helical collagen have been identified. For example, the Gram-positive bacterium *Streptococcus pyogenes* contains two collagen-like proteins, Scl1 and Scl2, which now possess well-characterized structural and functional properties. Therefore, it will be possible to obtain constructs in recombinant *Escherichia coli* systems with various sequence modifications of Scl1 or Scl2 for the establishment of large-scale production methods. Collagen can also be obtained through standard peptide synthesis techniques. Collagen obtained from any of the aforementioned techniques can be further polymerized. Collagen dimers and trimers are formed by the self-association of collagen monomers in solution.

[0173] As the initial step in forming the collagen raw material described herein, the starting collagen raw material can be placed in solution and fibrillated. Collagen fibrillation can be induced by introducing a salt into the collagen solution. Adding a salt or combination of salts, such as phosphates, potassium phosphates, potassium chloride, and sodium chloride, to the collagen solution alters the ionic strength of the collagen solution. Collagen fibrillation can occur due to increased electrostatic interactions, through greater hydrogen bonding, van der Waals interactions, and covalent bonding. Suitable salt concentrations can be, for example, in the range of approximately 10 mM, 50 mM, 100 mM, 500 mM, 1 M, 2 M, 3 M, 4 M to 5 M, and any intermediate values ​​within this range.

[0174] Collagen fibrillation can also be induced or enhanced using nucleating agents other than salts. Nucleating agents provide a surface on which collagen monomers can come into close contact with each other to initiate fibrillation, or they can act as branching points where multiple fibrils connect via the nucleating agent. Examples of suitable nucleating agents include, but are not limited to: collagen-containing microgels, collagen microparticles or nanoparticles, metal particles, or naturally or synthetically derived fibers. Suitable nucleating agent concentrations can range from approximately 1 mM to 100 mM.

[0175] Collagen networks are also highly sensitive to pH. During the fibrillation step, pH can be adjusted to control fibril dimensions such as diameter and length. The overall size and structure of the collagen fibrils will affect the toughness, tensile strength, and breathability of the resulting fibrillated collagen-derived material. This can be used to manufacture fibrillated collagen-derived leather for a variety of applications that may require different levels of toughness, flexibility, and breathability. pH adjustment can be used during fibrillation with or without changes in salt concentration.

[0176] One approach to controlling the organization of dehydrated fibril networks is to include filler materials that maintain the separation of fibrils during the drying process. These filler materials can include nanoparticles, microparticles, or various polymers such as synthetic tanning agents commonly used in the tanning industry. These filler materials can be part of the final dehydrated leather material, or they can be sacrificed, i.e., they are degraded or dissolved to leave open spaces for a more porous fibril network.

[0177] Collagen or collagen-like proteins can be chemically modified to promote chemical and physical cross-linking between collagen fibrils. Chemical cross-linking is possible because reactive groups on collagen molecules, such as lysine, glutamic acid, and hydroxyl groups, extend from the rod-like fibril structure of collagen. Cross-linking involving these groups prevents collagen molecules from sliding past each other under stress and thus increases the mechanical strength of collagen fibers. Examples of chemical cross-linking reactions include, but are not limited to, reactions with lysine. Reacting with amino groups or carboxyl groups in collagen molecules. Enzymes such as transglutaminase can also be used to create cross-links between glutamate and lysine to form stable compounds. -Glutamicyl-lysine crosslinking. Inducing crosslinking between functional groups of adjacent collagen molecules is known in the art. Crosslinking is another step that can be performed here to modulate the physical properties obtained from materials derived from fibrillated collagen hydrogels.

[0178] Once formed, the fibrillated collagen network can be further stabilized by incorporating molecules with bifunctional, trifunctional, or multifunctional reactive groups, including chromium, amines, carboxylic acids, sulfuric acid, sulfurous acid, sulfonic acid, aldehydes, hydrazides, mercapto, diaziridine, aryl-, azides, acrylates, epoxides, or phenols.

[0179] The fibrillated collagen network can also be polymerized with other agents that form hydrogels or have fibrous qualities (e.g., polymerizable polymers or other suitable fibers), which can be used to further stabilize the matrix and provide the desired end structure. Hydrogels based on acrylamide, acrylic acid, and their salts can be prepared using reverse suspension polymerization. The hydrogels described herein can be prepared from polar monomers. The hydrogels used can be natural polymer hydrogels, synthetic polymer hydrogels, or a combination of both. The hydrogels used can be obtained using graft polymerization, crosslinking polymerization, networks formed from water-soluble polymers, radiation crosslinking, etc. A small amount of crosslinking agent can be added to the hydrogel composition to enhance polymerization.

[0180] Fibrillated collagen hydrogels of any suitable thickness can be prepared as described herein. Because the final thickness will be smaller than the hydrogel thickness (e.g., between 10% and 90% thinner), the initial hydrogel thickness may depend on the desired thickness of the final product, assuming that thickness variations (or total volume) include shrinkage during crosslinking, dehydration, and / or the addition of one or more oils or other lubricants as described herein.

[0181] The hydrogel thickness can be between 0.1 mm and 50 cm or any intermediate value within this range. In the formation of fibrillated hydrogels, the hydrogel can be incubated to form a thickness for any suitable duration, including between 1 minute and 24 hours.

[0182] The fibrillated collagen hydrogels described herein can typically be formed in any suitable shape and / or thickness, including flat sheets, curved shapes / sheets, cylinders, threads, and composite shapes. Furthermore, they can be shaped into virtually any linear size. For example, any of these hydrogels can be formed to have the stated thickness and a length greater than 10 mm (e.g., greater than 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1500 cm, etc.) and a width greater than 10 mm (such as greater than 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1500 cm). (cm, etc.) of film.

[0183] Once collagen fibrils characterized as hydrogels have formed or are forming, they can be crosslinked. For example, fibrillated collagen hydrogels can be treated with compounds containing chromium or at least one aldehyde group, or with vegetable tanning agents, before, during, or after gel formation to further stabilize the fibrillated collagen hydrogels. For example, collagen fibrils pretreated with an acrylic polymer followed by treatment with a vegetable tanning agent (e.g., hyaluronic acid) can exhibit increased hydrothermal stability. In other instances, glyceraldehyde can be used as a crosslinking agent to increase the thermal stability, protein hydrolysis resistance, and mechanical properties (e.g., Young's modulus and tensile stress) of fibrillated collagen hydrogels.

[0184] Depending on the temperature and volume of the starting material, fibrillation and hydrogel formation can occur relatively rapidly after induction and are essentially complete after one and a half hours, as indicated by the decrease in absorbance levels after 70 minutes. Following induction, the storage modulus (or viscoelastic quality of the material) of fibrillable collagen hydrogels increases from about 1 Pa (for collagen solutions) to about 400 Pa (for fibrillated collagen hydrogels).

[0185] As mentioned above and Figure 1 As shown in Figure 2, animal skin typically contains protofibrils arranged in an ordered, higher-order structure, including those present as bands (with regular, cavitary regions) and those that form multiple protofibrils into arrangements that can subsequently bundle into collagen bundles. In contrast, collagen hydrogels and therefore the biomanufactured leather described herein can have a predominantly disordered collagen fibrillary structure throughout the entire thickness of the material (and in some cases, the entire volume). Specifically, the collagen structure of biomanufactured leather formed from collagen hydrogels can be predominantly unbundled and unoriented along any particular axis. In some variations, the collagen fibrils can be unbundled (e.g., more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, etc. unbundled throughout the volume). Furthermore, the orientation of collagen fibrils within the volume (or throughout the entire volume) can be unoriented or randomly oriented, and this lack of orientation can be the same throughout the volume as in natural leather, rather than varying throughout the thickness of the volume, as seen in natural leather which can vary from vertically oriented collagen fibril bundles to horizontally oriented bundles throughout the thickness. Any properties that are the same at any horizontal thickness of the hydrogel, and therefore the resulting leather material, can be referred to herein as "uniform" throughout the thickness.

[0186] Furthermore, any bio-manufactured leather described herein exhibits a uniform fibril distribution throughout the gel thickness and thus the resulting leather material. This contrasts with natural leather, such as the material shown in Figure 2, which demonstrates an increase in the number of fiber bundles throughout the material thickness.

[0187] The lack of higher levels of tissue organization in fibrillated collagen hydrogels and the leather materials formed from them Figure 3A and Figure 3B The middle is obvious. Figure 3A A scanning electron micrograph of the fibrillated collagen hydrogel formed as described herein is shown. Similarly, Figure 4A transmission electron micrograph of a collagen hydrogel through fibrillation is shown. Both the transmission electron micrograph and the scanning electron micrograph show the fibrillated collagen hydrogel as collagen fibrils at disordered angles. As previously mentioned, the density of collagen fibril formation and, to a certain extent, the manner of collagen fibril formation can be controlled by adjusting the pH of the collagen solution during fibrillation induction and by adjusting the fibril concentration during dehydration. Figure 3 also shows a scanning electron micrograph of bovine dermis. Figure 3B Compared to natural bovine leather, the fibrillated collagen network shown is more random and lacks distinct striations. While the overall size of the fibrils may be similar, their arrangement is quite different. Such ultrastructural differences between fibrils in fibrillated collagen hydrogels and in natural tissues such as bovine leather (and leather derived from it) are not a problem in the final biomanufactured leather products, which can be as soft as or softer and more supple than natural leather and can have a similar appearance. To make the final biomanufactured leather products more durable, the fibrillated collagen may contain secondary materials (with collagen as the primary material). Suitable secondary materials include, but are not limited to, woven or braided fabrics, nonwovens including natural felts such as wool felt, synthetic felts such as polyester-polyurethane copolymers such as elastic fibers or LYCRA. Felt, poly(p-phenylene terephthalate) polymers such as KEVLAR Felt, nylon polymers (such as nylon 6, nylon 6,6 and similar felts), and polyester polymers (such as polyethylene terephthalate and similar felts), man-made short fibers (such as carbon fiber felt, silk fibers, etc.), cellulose microfibers, and combinations thereof. In one embodiment of the invention, the secondary material is surrounded by fibrillated collagen raw material to form a composite. One method of surrounding the secondary material with fibrillated collagen is to pour a collagen solution onto one side of the secondary material, then rapidly turn the secondary material over and pour the collagen solution onto the opposite side of the secondary material. This can be described as a sandwich-type structure.

[0188] In another embodiment of the invention, collagen can be converted into biomanufactured leather and a secondary material can be laminated to one side of the leather using an adhesive or the like. Suitable adhesives may include, but are not limited to, hot melt adhesives, emulsion polymeric adhesives, etc. The biomanufactured leather can be coated with an adhesive using known techniques such as channel casting, coincident coating, etc., and the secondary material can be applied to the leather and passed through rollers under heat to laminate the material.

[0189] In another embodiment, the secondary material may be distributed throughout the entire adhesive raw material to form a composite structure. The density of the secondary material may range from 1 μg / mL to 500 mg / mL. The ratio of fibrillated collagen to secondary material may range from 1:100 to 100:1. The ratio of dried collagen to secondary material in biomanufactured leather products may also range from 1:100 to 100:1.

[0190] Secondary materials can also be photoluminescent materials such as photoluminescent fabrics, nonwovens, felts, carbon fibers, or three-dimensional objects. As described above, the collagen solution can be poured onto one side of the secondary material, the secondary material can be quickly turned over, and the collagen solution can be poured onto the other side of the secondary material.

[0191] The fibrillated collagen (sometimes called a hydrogel) can then be dehydrated to remove most of its water content. Removing water from the fibrillated collagen hydrogel changes its physical properties from a hydrated gel to a flexible sheet. The material can be treated to prevent cracking / tearing. For example, care may be needed not to remove too much water from the fibrillated collagen. In some instances, it may be desirable to dehydrate the fibrillated collagen to have a water content of less than 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60%. The water content is determined by equilibration at 65% relative humidity, 25°C, and 1 atmosphere.

[0192] Dehydration can involve air drying, vacuum and pressure filtration, solvent exchange, etc. For example, fibrillated collagen hydrogels can also be dehydrated by replacing their water content with an organic solvent. Suitable organic solvents include, but are not limited to, acetone, ethanol, and ether. Subsequently, the organic solvent can be evaporated (e.g., air drying, vacuum drying, etc.). It is also possible to use one or more organic solvents for continuous dehydration steps to fine-tune the dehydration level in the final product.

[0193] After or during dehydration, fibrillated collagen raw materials can be treated with lubricants and / or oils to impart greater flexibility and bendability. Using a combination of oil and solvent allows the oil to penetrate the fibrillated collagen network more effectively than oil alone. In a reasonable amount of time, oil alone may only penetrate the exposed surface and may not easily penetrate the entire thickness of the fibrillated collagen raw material. Once the oil / solvent combination has penetrated the entire thickness of the material, the solvent can be removed. Suitable oils and lubricants include, but are not limited to, castor oil, pine oil, lanolin, mink oil, cow's foot oil, fish oil, shea butter, aloe vera, etc.

[0194] Lubricating dehydrated and cross-linked fibrillated collagen networks or hydrogels to form leather materials can produce materials with properties similar to or better than those of natural leather. Solutions containing a combination of oil and organic solvent increase the quality and softness of the dehydrated fibrillated collagen raw material (inversely proportional to the slope of the stress-strain curve). This is because the combination of oil and organic solvent penetrates the dehydrated fibrillated collagen raw material, and once penetrated, the oil remains distributed throughout the material, while the organic solvent can evaporate. Although not shown, using oil alone may be less effective in terms of overall penetration through the dehydrated fibrillated collagen raw material.

[0195] The raw material can then be processed similarly to fibrillated gum derived from natural leather made from animal hides or skin, and then retanned, dyed, and / or finished. Additional processing steps may include crosslinking, retanning, and surface coating. Crosslinking and retanning can include sub-processes such as rehydration (rehydrating semi-processed leather), homogenization (squeezing 45%-55% water out of the leather), separation (separating the leather into one or more layers), shaving (thinning the leather), neutralization (adjusting the pH of the leather to between 4.5 and 6.5), dyeing (coloring the leather), fatliquoring (fixing fats, oils, and waxes to the leather fibers), filling (intensive / heavy chemicals to make the leather harder and heavier), stuffing (adding fats, oils, and waxes between the leather fibers), fixing (bonding / capturing and removing unbound chemicals), shaping (smoothing the grain and removing excess water), drying (drying the leather to the desired moisture level, 10%-25%), conditioning (adding moisture to the leather to a level of 18%-28%), softening (physically softening the leather by separating the fibers), or polishing (brushing the leather surface to reduce fraying and grain defects). Surface coating may include any one or a combination of the following steps: oiling (applying crude oil or oil to the leather), polishing, spraying, roller coating, curtain coating, polishing, electroplating, embossing, ironing, or glazing.

[0196] Unlike animal hides, which must be trimmed to achieve the desired thickness or size, engineered leather materials can be manufactured with a wide range of thicknesses and desired dimensions for a specific end product.

[0197] The production of such engineered leather materials can also generate less waste by omitting the steps necessary for removing excess protein, fat, and hair from natural animal hides in leather production methods. This results in a smaller environmental impact from open methods and the products derived from them.

[0198] The biomanufacturing materials disclosed herein are advantageously combined, incorporated, or attached to other materials to form useful composites. For example, biomanufacturing coatings can be applied to secondary materials such as woven or nonwoven fabrics or plastic sieves by impregnation or spraying to form components of the biomanufacturing material. Biomanufacturing materials can be incorporated onto one or both sides of a flat secondary material or laminated to one or both sides of a flat secondary material. Specific embodiments of these composite materials are described below.

[0199] Implementation Plan

[0200] complex

[0201] This invention includes, but is not limited to, biomanufacturing material components having the features described below. The composites of this invention include those in which (i) one or more secondary components, such as particles, filaments, fabrics, or three-dimensional objects, are incorporated into or embedded in a collagen fibrillary network; (ii) the biomanufacturing material is coated or deposited, for example by filtration, on one side of one or more secondary components, such as woven or nonwoven fabrics (e.g., textiles, paper, or regenerated cellulose); (iii) the biomanufacturing component is coated or deposited on both sides of one or more secondary materials having an upper and lower side or an inner and outer side; or (iv) the biomanufacturing material component and one or more secondary components are adhered, attached, or laminated to each other, for example by direct lamination with or without an adhesive.

[0202] Immediately generated biomanufacturing materials can associate with one or more secondary components to form complexes. The complexes can be formed simultaneously with the biomanufacturing materials; for example, in the production described herein, the secondary components, such as particles or fibers, can be mixed with precursors of the biomanufacturing materials at any step. For example, the particulate or fibrous secondary material can be mixed with collagen, collagen fibrils, cross-linked collagen fibrils, lubricated collagen fibrils, dehydrated collagen fibrils (including in powder form), cross-linked, dehydrated, and lubricated collagen fibrils, and said substances are subsequently processed together with the secondary material into a complex containing biomanufacturing material components. The secondary components can be coated or embedded in the resulting biomanufacturing material. This example is a complex of cross-linked collagen fibrils deposited on filter paper and subsequently dehydrated and lubricated (the secondary component) and biomanufacturing material deposited by filtration through one side of the paper. Precursors to biomanufacturing material components may be coated or otherwise applied to the surface of secondary components and then processed into the final biomanufacturing material, for example, by at least one of fibrillated collagen, cross-linked collagen fibrils, dehydrated collagen fibrils or cross-linked collagen fibrils, and lubricated collagen fibrils or cross-linked collagen fibrils.

[0203] Alternatively, the biomanufacturing material component, once produced, can be coated or laminated onto at least one surface of a secondary component having an upper and lower surface or an inner and outer surface. In some embodiments, one or more layers of a planar secondary material will be sandwiched between two layers of the biomanufacturing component, the two layers forming an outer layer of a composite having the aesthetic qualities of the biomanufacturing component and the strength, thickness, or other properties imparted by the inner sandwiched secondary component.

[0204] The complex of the present invention may also contain a layered structure comprising an alternating or repeating series of one or more layers of biomanufacturing components and secondary components. These layers may appear in the complex in any order. Secondary component layers may be adjacent to each other or to the biomanufacturing layers. Biomanufacturing layers may be adjacent to each other or to layers of one or more secondary components. Such complexes may contain adjacent or multiple layers of biomanufacturing components, with or without non-collagenous secondary components. For example, multiple layers of biomanufacturing components may be deposited on one side of filter paper or a sieve to increase the thickness of the complex in terms of biomanufacturing material content.

[0205] The complexes of the present invention include, but are not limited to, (i) those relating to dispensing, encapsulating, incorporating, depositing, or otherwise introducing at least one biomanufacturing material into or onto at least one porous, permeable, or absorbable secondary component; (ii) those relating to layering, laminating, depositing, coating, or otherwise contacting at least one secondary component with at least one biomanufacturing material; or (iii) those relating to sandwiching, layering, laminating, coating, or otherwise covering the upper and lower surfaces or inner and outer surfaces of at least one secondary component with at least one biomanufacturing material.

[0206] These can involve incorporating or embedding one or more secondary materials into a collagen fibrillary network, for example, by mixing the secondary materials with a biomanufacturing material precursor or by adding them during the preparation of a biomanufacturing material comprising a cross-linked collagen fibrillary network. Examples of such secondary materials that can be incorporated into a biomanufacturing material to create a complex include particles, filaments, fabrics, or three-dimensional objects. Once the secondary component is incorporated into a precursor of the biomanufacturing material component, the mixture can be further processed into a biomanufacturing material incorporating, encapsulating, or incorporated secondary materials.

[0207] These methods include coating or depositing biomanufacturing materials or precursors of biomanufacturing materials, such as unfibrillated collagen, uncrosslinked collagen fibrils, undehydrated collagen fibrils, or unlubricated collagen fibrils, onto a secondary component substrate, such as woven or nonwoven fabric, paper, or regenerated cellulose. For example, deposition can be accomplished by filtering a solution or suspension of collagen fibrils or crosslinked fibrils through a secondary material (e.g., filter paper) that holds the collagen fibrils on one side. The deposited collagen fibrils can then be further processed into biomanufacturing materials incorporated into or on one side of the secondary material. In some embodiments, the material can be deposited on both sides of the substrate. In other embodiments, two substrates, each containing a layer of biomanufacturing material, can be laminated together, with the biomanufacturing material facing inwards or outwards. Preferably, for the purpose of providing a leather-like aesthetic, the layer of biomanufacturing material will face outwards.

[0208] Biomanufacturing materials can be deposited or coated on both sides of a secondary material substrate to provide a leather-like aesthetic to the outward-facing sides. Alternatively, biomanufacturing materials can form one or more inner layers of a composite, with the secondary material facing outwards.

[0209] Composite materials can be produced by attaching bio-manufacturing materials to one or more secondary components once they are generated, for example by coating or laminating the bio-manufacturing materials to at least one surface of a secondary component having an upper and lower surface or an inner and outer surface.

[0210] In some embodiments, the composite is produced by sandwiching one or more layers of flat secondary material between at least two outer layers of the biomanufacturing component, thus providing the aesthetic qualities of the biomanufacturing component and the strength, thickness, or other properties imparted by the secondary component sandwiched within.

[0211] The complexes of the present invention can be produced by alternating or repeating series of one or more layers of biomanufacturing components and secondary components. These layers can appear in the complex in any order. The method may include arranging the secondary component layers adjacent to each other or adjacent to the biomanufacturing layers. The biomanufacturing layers may be adjacent to each other or adjacent to layers of one or more secondary components. Such complexes may contain adjacent or multiple layers of biomanufacturing components with or without non-collagenous secondary components. For example, multiple layers of biomanufacturing components may be deposited on one side of filter paper or a sieve to increase the thickness of the complex in terms of biomanufacturing material content.

[0212] Specific embodiments of the composite material of the present invention include, but are not limited to, the following.

[0213] 1. A composite material comprising:

[0214] (i) at least one porous, permeable or absorbent secondary component, and

[0215] At least one biomanufactured material comprising a non-human collagen fibril network, wherein less than 10% by weight of the collagen fibrils in the material are in the form of collagen fibers having a diameter of 5 µm or greater, fibrils arranged in a manner with a length of 100 µm or greater, or both; wherein the material contains no more than 40% by weight of water; and wherein the material contains at least 1% of a lubricant; or

[0216] (i) at least one porous, permeable or absorbent secondary component, and

[0217] A biomanufactured material comprising at least one recombinant nonhuman collagen fibrillary network, wherein the collagen is substantially free of 3-hydroxyproline and optionally substantially free of hydroxylysine; wherein the material contains no more than 25% by weight of water; and wherein the material contains at least 1% of a lubricant; or

[0218] (ii) at least one layer of the secondary component, and

[0219] At least one layer of a biomanufacturing material comprising a network of non-human collagen fibrils, wherein less than 10% by weight of the collagen fibrils in the material are in the form of collagen fibers having a diameter of 5 µm or greater, fibrils arranged in a manner of 100 µm or greater in length, or both; wherein the material contains no more than 40% by weight of water; and wherein the material contains at least 1% of a lubricant.

[0220] (iii) at least one layer of the secondary component, and

[0221] A biomanufactured material comprising at least one recombinant nonhuman collagen fibrillary network, wherein the collagen is substantially free of 3-hydroxyproline and optionally substantially free of hydroxylysine; wherein the material contains no more than 25% by weight of water; and wherein the material contains at least 1% of a lubricant; or

[0222] (iv) at least one layer of the secondary components, and

[0223] A biomaterial comprising at least two outer layers having an upper and lower surface or an inner and outer surface, and containing a network of non-human collagen fibrils, wherein less than 10% by weight of the collagen fibrils in the material are in the form of collagen fibers having a diameter of 5 µm or greater, fibrils arranged in a manner of 100 µm or greater in length, or both; wherein the material contains no more than 40% by weight of water; and wherein the material contains at least 1% of a lubricant; or

[0224] (v) at least one layer of the secondary components, and

[0225] The material comprises at least two outer layers of at least one biomanufacturing material having an upper and lower surface or an inner and outer surface and containing a recombinant nonhuman collagen fibrillary network, wherein the collagen is substantially free of 3-hydroxyproline and optionally substantially free of hydroxylysine; wherein the material contains no more than 25% by weight of water; and wherein the material contains at least 1% of a lubricant.

[0226] 2. The complex as described in embodiment 1, i.e. (i) or (ii), wherein the secondary component has an upper surface and a lower surface or an inner surface and an outer surface.

[0227] 3. The complex according to embodiment 2, wherein the biomanufacturing material is located on or incorporated into only one of the upper, lower, inner, or outer surfaces.

[0228] 4. The complex according to embodiment 2, wherein the bio-manufacturing material is on or incorporated into both the upper and lower surfaces or both the inner and outer surfaces.

[0229] 5. The composite according to embodiment 2, wherein the secondary component is paper, regenerated cellulose, fabric or other nonwoven or woven fibrous material.

[0230] 6. The composite as described in embodiment 1, wherein the secondary component comprises at least one resin, polymer, or plastic.

[0231] 7. The complex as described in embodiment 1, wherein the secondary component comprises at least one fiber, bead, thread, particle, sieve, woven or nonwoven fabric.

[0232] 8. The complex as described in Embodiment 1, wherein the biomanufacturing material contains less than 1% by weight of actin, keratin, elastin, fibrin, albumin, globulin, mucin, mucinoids, non-collagen structural proteins and / or non-collagen non-structural proteins.

[0233] 9. The complex as described in embodiment 1, wherein the biomanufacturing material comprises at least 1% of at least one crosslinking agent.

[0234] 10. The complex according to embodiment 1, wherein the diameter of the protofibrils in the biomanufacturing material exhibits a substantially unimodal distribution, wherein at least 70% of the diameter of the protofibrils in the material is distributed around a single mode of diameter.

[0235] 11. The complex according to embodiment 1, wherein the bio-manufacturing material comprises at least one lubricant selected from the group consisting of at least one fat, bio-oil, mineral oil or synthetic oil, sulfonated oil, polymer, and organofunctional siloxane.

[0236] 12. The composite according to embodiment 1, wherein the biomanufacturing material has an elastic modulus between 100 kPa and 1,000 MPa, wherein the elastic modulus changes by no more than 20% when measured at the correct angle over the same length of the material, and the biomanufacturing material has a tensile strength in the range of 1 MPa to 100 MPa, wherein the tensile strength changes by no more than 20% when measured at the correct angle over the same length of the material.

[0237] 13. The complex according to embodiment 1, wherein the biomanufacturing material further comprises a surface coating or surface finishing; wherein the surface coating or surface finishing is uniformly distributed throughout the material such that its concentration variation by weight in or above the same unit volume of the material is no more than 20%.

[0238] 14. The complex according to embodiment 1, wherein the biomanufacturing material further comprises dyes, staining agents, resins, polymers, pigments or coatings, wherein the dyes, staining agents, resins, pigments or coatings are uniformly distributed throughout the material such that their concentration variation by weight in or above the same unit volume of the material is no more than 20%.

[0239] 15. The complex according to embodiment 1, wherein the biomanufacturing material further comprises at least one filler, wherein the filler is uniformly distributed throughout the material such that its concentration variation by weight in or above the same unit volume of the material is no more than 20%.

[0240] Methods for preparing complexes

[0241] Specific embodiments of the method for preparing the complex according to the present invention include, but are not limited to, the following:

[0242] 1. A method for preparing a composite material, the method comprising:

[0243] (i) Dispensing, encapsulating, incorporating, depositing, or otherwise introducing at least one biomanufacturing material into or onto at least one porous, permeable, or absorbable secondary component; wherein the at least one biomanufacturing material comprises a non-human collagen fibril network, wherein less than 10% by weight of the collagen fibrils in the material are in the form of collagen fibers having a diameter of 5 µm or greater, fibrils arranged in a manner of 100 µm or greater of their length, or both; wherein the material contains no more than 40% by weight of water; and wherein the material contains at least 1% of a lubricant; or

[0244] (ii) Dispensing, encapsulating, incorporating, depositing, or otherwise introducing at least one biomaterial into or onto at least one porous, permeable, or absorbable secondary component; wherein the at least one biomaterial comprises a recombinant nonhuman collagen fibrillary network, wherein the collagen is substantially free of 3-hydroxyproline and optionally substantially free of hydroxylysine; wherein the material contains no more than 25% by weight of water; and wherein the material contains at least 1% of a lubricant; or

[0245] (iii) Contacting at least one secondary component with at least one biomanufacturing material comprising a nonhuman collagen fibrillary network by layering, laminating, depositing, coating, or otherwise, wherein the secondary component has an upper and lower surface or an inner and outer surface, wherein less than 10% by weight of the collagen fibrils in the material are in the form of collagen fibers having a diameter of 5 µm or greater, fibrils arranged in a manner of 100 µm or greater of their length, or both; wherein the material contains no more than 40% by weight of water; and wherein the material contains at least 1% of a lubricant; or

[0246] (iv) Contacting at least one secondary component with at least one biomanufacturing material comprising a recombinant nonhuman collagen fibrillary network by layering, laminating, depositing, coating, or otherwise, wherein the secondary component has an upper and lower surface or an inner and outer surface, wherein the collagen is substantially free of 3-hydroxyproline and optionally substantially free of hydroxylysine; wherein the material contains no more than 25% by weight of water; and wherein the material contains at least 1% of a lubricant; or

[0247] (v) Sandwiching, layering, laminating, coating, or otherwise covering the upper and lower surfaces or inner and outer surfaces of at least one secondary component with at least one biomanufacturing material comprising a network of non-human collagen fibrils, wherein less than 10% by weight of the collagen fibrils in the material are in the form of collagen fibers having a diameter of 5 µm or greater, fibrils arranged in a manner of 100 µm or greater length, or both; wherein the material contains no more than 40% by weight of water; and wherein the material contains at least 1% of a lubricant; or

[0248] (vi) Sandwiching, layering, laminating, coating, or otherwise covering the upper and lower surfaces or inner and outer surfaces of at least one secondary component with at least one biomanufacturing material comprising a recombinant nonhuman collagen fibrillary network, wherein the collagen is substantially free of 3-hydroxyproline and optionally substantially free of hydroxylysine; wherein the material contains no more than 25% by weight of water; and wherein the material contains at least 1% of a lubricant.

[0249] 2. The method according to embodiment 1, wherein the method is (i), and wherein at least one biomaterial is produced by a method comprising the following steps in any order:

[0250] Non-human collagen molecules in aqueous solutions or suspensions are fibrillated into collagen fibrils.

[0251] The collagen fibrils are crosslinked by contacting them with at least one crosslinking agent.

[0252] Dehydrated cross-linked collagen fibrils so that they contain less than 40% by weight of water.

[0253] Lubrication is achieved by incorporating at least 1% by weight of at least one lubricant into the material.

[0254] 3. The method according to embodiment 2, wherein the bio-manufacturing material is generated by fibrillation recombinant gel.

[0255] 4. The method according to embodiment 1, wherein the method is (ii) and wherein at least one biomaterial is produced by a method comprising the following steps in any order:

[0256] The aqueous solution or suspension of recombinant non-human collagen molecules is fibrillated into collagen fibrils.

[0257] The collagen fibrils are crosslinked by contacting them with at least one crosslinking agent.

[0258] Dehydrated cross-linked collagen fibrils so that they contain less than 25% water by weight.

[0259] Lubrication is achieved by incorporating at least 1% by weight of at least one lubricant into the material.

[0260] 5. The method according to embodiment 4, wherein the fibrillation, crosslinking, dehydration and / or lubrication are performed under certain time and conditions to produce less than 10% by weight of collagen fibrils in the form of collagen fibers having a diameter of 5µm or greater, fibrils arranged in a length of 100µm or greater, or both, in a biomanufacturing material.

[0261] 6. The method according to embodiment 1, wherein the method is (i) or (ii), and wherein the bio-manufactured material is incorporated into or on at least one porous, permeable or absorbable secondary component.

[0262] 7. The method according to embodiment 1, wherein the secondary component comprises at least one resin, polymer or plastic.

[0263] 8. The method according to embodiment 1, wherein the secondary component includes at least one fiber, bead, thread, particle, sieve, woven or nonwoven fabric.

[0264] 9. The method according to embodiment 1, wherein the secondary component comprises at least one conductive material, magnetic material, fluorescent material, bioluminescent material, phosphorescent material, or a combination thereof.

[0265] 10. The method of embodiment 1, wherein the biomaterial is produced by fibrillating non-human collagen molecules to generate fibrils, which is achieved by adjusting at least one of the salt concentration of the aqueous solution containing the collagen molecules or adjusting the pH.

[0266] 11. The method of embodiment 1, wherein the bio-manufacturing material is produced by cross-linking collagen fibrils, which is achieved by contacting them with at least one compound selected from the group consisting of: amines, carboxylic acids, sulfates, sulfites, sulfonates, aldehydes, hydrazides, mercapto groups, diaziridines, aryl groups, azides, acrylates, epoxides, phenols, chromium compounds, vegetable tanning agents, and synthetic tanning agents.

[0267] 12. The method according to embodiment 1, wherein the biomanufacturing material is produced by a network of dehydrated collagen fibrils, which is achieved by contacting them with an agent that removes bound water from collagen.

[0268] 13. The method according to embodiment 1 includes lubricating collagen fibrils with at least one lubricant selected from the group consisting of: fats, bio-oils, mineral oils or synthetic oils, cod oils, sulfonated oils, polymers, and organofunctional siloxanes.

[0269] 14. The method according to embodiment 1, wherein the biomanufacturing material is produced by uniformly distributing a lubricant on or throughout the biomanufacturing material, such that the lubricant concentration variation in the same unit volume of the material is no more than 20%.

[0270] 15. The method according to embodiment 1, wherein the biomanufacturing material is produced by uniformly distributing dyes, colorants, pigments, resins, polymers or coatings in or on the biomanufacturing material, wherein the concentration variation of the dyes, colorants, pigments, resins, polymers or coatings in the same unit volume of the biomanufacturing material is not greater than 20%.

[0271] 16. The method according to embodiment 1, wherein the biomaterial is produced by incorporating at least one filler therein.

[0272] 17. The method of claim 1, further comprising introducing the biomanufacturing material into or on the upper or lower surface or inner and outer surface of the secondary component.

[0273] 18. The method of claim 17, further comprising crosslinking the bio-manufacturing material once introduced.

[0274] 19. The method of claim 17, further comprising dehydrating the biomaterial once introduced.

[0275] 20. The method of claim 17, further comprising lubricating the biomaterial once introduced.

[0276] 21. The method of claim 17, comprising introducing bio-manufacturing material into or onto a secondary component by means of filtration, impregnation, spraying, or coating.

[0277] 22. The method of claim 17, further comprising applying the bio-manufacturing material only to one side of the secondary component.

[0278] 23. The method of claim 17, further comprising applying bio-manufacturing material to both sides of a secondary component.

[0279] 24. The method of claim 17, wherein the biomanufacturing material is layered between two layers of the secondary component.

[0280] 25. The method of claim 17, wherein the secondary component comprises fibers, woven or unwoven fabrics or other fibrous materials.

[0281] 26. The method of claim 1, further comprising embedding or mixing the at least one secondary component in or together with the biomanufacturing material.

[0282] Biomanufacturing components of the complex

[0283] In one embodiment, the biomanufacturing material component includes a collagen fiber network, such as a biomanufacturing material or biomanufacturing leather:

[0284] (i) It contains a non-human collagen fibrillary network,

[0285] The material contains less than 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% collagen fibrils in the form of collagen fibers with a diameter of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 µm or greater and / or in the form of fibrils arranged with a length of 100 µm or greater; the material contains no more than 10%, 20%, 30%, 40%, 50%, or 60% water; the material contains at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, or 40% lubricant; and optionally the material comprises an upper surface and a lower surface or an inner surface and an outer surface; or

[0286] (ii) It comprises a recombinant nonhuman collagen fibrillary network, wherein the collagen is substantially free of 3-hydroxyproline and optionally substantially free of hydroxylysine; wherein the material contains no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% water; wherein the material contains at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, or 40% lubricant; and wherein optionally the material comprises an upper and lower surface or an inner and outer surface. The water content in this material is preferably no more than 25% to 40%. The lubricant content may be selected to match or not exceed the absorption capacity of the biomanufactured material for the lubricant. Such a material may comprise mammalian collagen, such as bovine type I or type III collagen. Preferably, it will not contain animal hair, hair follicles, or fat that naturally expresses the collagen molecules contained therein. For example, it may contain less than 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, or 10 wt% of actin, keratin, elastin, fibrin, albumin, globulin, mucin, mucinoids, non-collagen structural proteins, and / or non-collagen non-structural proteins present in conventional leather. It may substantially be free of other collagen proteins, carbohydrates, nucleic acids or lipids, or immunogens, antigens, or allergens present in conventional leather (such as animals that naturally express collagen molecules in biomanufacturing materials). Alternative embodiments may incorporate 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, or 10 wt% of one or more of actin, keratin, elastin, fibrin, albumin, globulin, mucin, mucinoids, non-collagen structural proteins, and / or non-collagen non-structural proteins present in conventional leather.

[0287] The collagen used to produce the fibrils in this material can be isolated from natural sources, preferably in purified form, or it can be recombinantly produced or chemically synthesized. Collagen typically contains 4-hydroxyproline. It may differ chemically from collagen obtained from natural sources, for example, if it contains lower amounts or substantially no hydroxylysine or 3-hydroxyproline, glycosylated or cross-linked amino acid residues, or other post-translational modifications to the collagen amino acid sequence. Alternatively, it may contain higher amounts of hydroxylated amino acid residues, glycosylated residues, cross-links, or other chemical modifications.

[0288] The biomanufacturing material components described above typically contain a network of collagen fibrils, which can exhibit fibril densities between 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 and 1,000 mg / cc, preferably between 100 and 500 mg / cc. These fibrils or fibril networks can impart grain textures to the biomanufacturing material or biomanufacturing leather, such as grain texture, feel, or aesthetics. However, biomanufacturing materials can exhibit more uniform porosity and other physical properties than their corresponding conventional leathers, which can be controlled or tuned by manipulating the composition, fibril size, crosslinking, and lubrication in the biomanufacturing product.

[0289] In many embodiments, the biomanufacturing material component of the complex described above will have an upper and lower surface, or an inner and outer surface, comprising collagen fibrils. One or more of these surfaces may be exposed. Individual layers of the biomanufacturing material can exhibit substantially the same grain and appearance on both sides, unlike conventional leather products in which the collagen fibrils or fiber diameter increases for the inner layers of the raw hide.

[0290] In other embodiments, the biomanufacturing material components of the complex may be cast, molded, or otherwise constructed into a specific shape, which may exhibit substantially uniform properties on its surface.

[0291] Collagen fibrils in the biomanufacturing material components of the complex can be tuned to have specific diameters. The distribution of fibril diameters can exhibit a substantially unimodal, bimodal, trimodal, or other multimodal distribution. A multimodal distribution can consist of two or more different fibril formulations produced using different fibrillation conditions. In a substantially unimodal distribution, >50%, 60%, 70%, 80%, 90%, 95%, or 99% of the fibril diameter is distributed around a single modality. In a bimodal distribution, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the fibrils will be distributed around a single modality. In trimodal and other multimodal distributions, typically at least about 5%, 10%, 15%, 20%, 25%, 30%, or greater (depending on the number of modalities) of the fibril diameter will be distributed around the modality.

[0292] The biomanufacturing material component may contain at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of collagen fibrils having a diameter between 1 nm and 1 µm. The fibril diameter can be determined by methods known in the art, including by visual microscopy or electron microscopy, such as scanning or transmission electron microscopy. For example, collagen fibrils may have a total average or individual fibril diameter in the range of 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 nm (1 µm).

[0293] The collagen fibrils in the biomanufacturing material components described above are typically cross-linked by contact with at least one agent that forms cross-links between the collagen fibrils. Such a cross-linking agent may be selected from one or more of the following: amines, carboxylic acids, sulfates, sulfites, sulfonates, aldehydes, hydrazides, mercapto groups, diaziridines, aryl groups, azides, acrylates, epoxides, phenols, chromium compounds, vegetable tanning agents, and synthetic tanning agents.

[0294] Crosslinking can be performed at crosslinking agent concentrations ranging from 1, 5, 10, 25, 50, 75 to 100 mM, and can be performed under conditions where collagen fibrils are uniformly exposed to the crosslinking agent, so that the average number of crosslinks formed is consistent and varies by no more than 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45% or 50% in the same unit volume of the material.

[0295] The biomanufacturing material component may contain at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of a crosslinking agent based on the weight of the material or based on the weight of collagen or collagen fibrils in the material. The crosslinking agent may be present in a covalent or non-covalent form; for example, it may be covalently bound to collagen fibrils. The crosslinking agent may be uniformly present in the biomanufacturing material, wherein its weight (or molar) concentration varies by no more than 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, or 50% per unit volume of the material.

[0296] The bio-manufactured materials or bio-manufactured leather components of the complexes described above contain lubricants. Non-lubricating materials containing collagen fibrillary networks can be generated for subsequent lubrication of such precursor substrates, but may lack the flexibility and other useful properties of lubricating products. Lubricants can be incorporated in any amount that facilitates fibrillary movement or imparts leather-like properties such as increased flexibility, reduced brittleness, durability, strength, shatter resistance, tear resistance, or water resistance. Lubricant content can range from approximately 0.1%, 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, and 60% by weight of the bio-manufactured leather.

[0297] Lubricants used in the biomanufacturing components of the complex include, but are not limited to, fats, bio-oils, mineral or synthetic oils, cod oil, sulfonated oils, polymers, resins, organofunctional siloxanes, and other agents used for fatliquoring conventional leather; mixtures thereof. Other lubricants include surfactants, anionic surfactants, cationic surfactants, cationic polymeric surfactants, anionic polymeric surfactants, amphoteric polymers, fatty acids, modified fatty acids, nonionic hydrophilic polymers, nonionic hydrophobic polymers, polyacrylic acid, polymethacrylic acid, acrylic acid, natural rubber, synthetic rubber, resins, amphoteric anionic polymers and copolymers, amphoteric cationic polymers and copolymers, mixtures thereof, and emulsions or suspensions of these in water, alcohols, ketones, and other solvents.

[0298] Solutions or emulsions containing lubricants can be used as lubricants; for example, resins and other hydrophobic lubricants can be applied as emulsions or in solvents suitable for dissolving them. Such solutions may contain any amount of lubricant suitable for application or incorporation into biomanufacturing leather. For example, they may contain 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, or 99% of lubricant or an equal or corresponding amount for other components, such as at least one aqueous solvent, such as water, alcohols (like C1-C6 alcohols, like ethanol), ketones (such as C1-C6 ketones), aldehydes (such as C1-C6 aldehydes), waxes, surfactants, dispersants, or other reagents. Lubricants can be in various forms, such as O / W or W / O emulsions in aqueous or hydrophobic solutions, in a sprayable form, or in other forms suitable for incorporation or application to biomanufacturing materials.

[0299] The lubricant can be uniformly distributed throughout the biomanufacturing material components, such that the concentration variation of the lubricant in the same unit volume of the material is no greater than 5%, 10%, 15%, 20%, 35%, 30%, 40%, or 50%, and can be mixed or blended into a form suitable for uniform application to or into the biomanufacturing material.

[0300] Some embodiments of biomanufacturing material components or complexes incorporating biomanufacturing material components along with secondary components will exhibit many of the advantageous properties similar to leather, or novel or superior properties compared to conventional leather.

[0301] The biomanufacturing material component or a complex containing said biomanufacturing material component may have an elastic modulus of at least 100 kPa. It may be in the range of 100 kPa to 1,000 MPa and any intermediate value within this range, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 MPa.

[0302] The biomanufacturing material component or a complex containing the biomanufacturing material component can exhibit uniform elasticity, wherein when measured at angles differing by 30, 60, or 90 degrees (or at other angles) over the same length or width (or volume or fixed cross-sectional area) of the material, the change in elastic modulus is no greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%.

[0303] The biomaterial component or complex containing the biomaterial component is stretchable and can be stretched in a relaxed state by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250% to 300% of its length. This range includes all intermediate values.

[0304] In some embodiments, the biomanufacturing material component or a complex containing said biomanufacturing material component may have a tensile strength of at least 1 kPa. It may be in the range of 1 kPa to 100 MPa and any intermediate value within this range, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 200, 300, 400, 500 kPa; 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 MPa. Some implementations will exhibit uniform tensile strength, wherein when measured over the same length or width (or volume or fixed cross-sectional area) of the material at angles differing by 30, 60 or 90 degrees (or at other angles), the tensile strength varies by no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%.

[0305] Certain biomanufacturing material components or complexes containing said biomanufacturing material components may exhibit tear strength or resistance that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 150%, or 200% greater than that of other leathers with the same grain surface or thickness containing the same type of collagen (e.g., bovine type I or III collagen), processed using the same crosslinking agent or lubricant. Some embodiments will exhibit uniform tear resistance, with the tear resistance varying by no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% when measured at angles differing by 30, 60, or 90 degrees (or other angles) over the same length or width (or volume or fixed cross-sectional area) of the material. Biomanufactured materials can have tear strengths in the range of about 1 to 500 N, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 or 500, and any intermediate tear strengths within this range.

[0306] The biomanufacturing material component or a complex containing said biomanufacturing material component may have a softness of 2, 3, 4, 5, 6, 7, 8, 10, 11, 12 mm or greater as defined by ISO 17235. Some embodiments will exhibit uniform softness, with variations in softness not exceeding 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 100% when measured in the same unit area or volume of the biomanufacturing material in another manner.

[0307] In other embodiments, the biomanufacturing material component or a complex containing said biomanufacturing material component exhibits a customized thickness to provide a grain-like product without a dermal backing. In some embodiments, the material or complex will have an upper and lower surface or an inner and outer surface having the same or substantially the same grain, grain texture, feel, and appearance. Other embodiments of the biomanufacturing material component or a complex incorporating said biomanufacturing material component are embossed, patterned, extruded or printed, dyed, or sprayed. Other embodiments of the biomanufacturing material component or a complex containing said biomanufacturing material component have a surface coating or surface finish that may be uniformly applied, either separately, over or throughout the material, such that the concentration variation by weight of said biomanufacturing material component in the same unit volume of the material or on a unit surface of the material is no greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. Some embodiments of biomanufacturing material components or complexes containing said biomanufacturing material components may contain dyes, colorants, resins, polymers, pigments, or coatings, optionally wherein the dyes, colorants, resins, pigments, or coatings are uniformly distributed throughout the material such that their concentration variation by weight in the same unit volume of the material or complex or on a unit area of ​​the material or complex is no greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%.

[0308] Some embodiments of the biomanufacturing material components described above may contain fillers and other substances or components incorporated into the collagen fibrillary network. For example, some embodiments will contain fillers as a second component, such as at least one of polymer microspheres, beads, fibers, filaments, or organic salts. These can be selected to control the organization of the dehydrated collagen fibrillary network by keeping the fibrils spaced apart during the drying process. The fillers may be soluble under certain conditions or in a form that allows them to be removed from the biomanufacturing material after drying or other processing.

[0309] Other embodiments include at least one woven or nonwoven material incorporated into a collagen fibrillary network, or a secondary component of a collagen fibrillary network incorporated into a nonwoven or woven material.

[0310] In some implementations, the biomanufacturing material component or a complex incorporated into the biomanufacturing material component will be incorporated into other products, such as footwear, clothing, sportswear, uniforms, wallets, watch straps, bracelets, luggage, home furnishings, or furniture.

[0311] Methods for preparing biomanufacturing components

[0312] The methods according to the present invention include, but are not limited to, the following embodiments of methods for preparing biomaterial components.

[0313] A method for preparing the following biomaterial components:

[0314] (i) Biomanufacturing material components comprising a non-human collagen fibril network, wherein less than 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, or 40 wt% of the collagen fibrils in the form of collagen fibers with a diameter of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 µm or greater and / or in the form of collagen fibers with a length of 25, 50, 100, 150, 200, 250, 300, 350, or 400 µm. The material is in the form of fibrils arranged in µm or larger; wherein the material contains no more than 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, 50 wt%, 55 wt%, or 60 wt% water; and wherein the material contains at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, or 40% lubricant, and the method comprises the following steps in any order: fibrillating an aqueous solution or suspension of non-human collagen molecules into collagen fibrils. The material comprises fibers, which are cross-linked by contacting collagen fibrils with at least one cross-linking agent, dehydrated cross-linked collagen fibrils such that they contain less than 40% by weight of water, and at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, or 40% by weight of at least one lubricant incorporated into the material, and optionally cast, molded, or otherwise formed the material comprising an upper and lower surface or an inner and outer surface; or

[0315] (ii) A biomanufacturing material component comprising a recombinant nonhuman collagen fibrillary network, wherein the collagen is substantially free of 3-hydroxyproline and optionally substantially free of hydroxylysine; wherein the material contains no more than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% water; and wherein the material contains at least 1% lubricant, the method comprising any sequence of the following steps: fibrillating an aqueous solution or suspension of nonhuman collagen molecules into collagen fibrillary fibers, by causing... Collagen fibrils are contacted with at least one crosslinking agent to crosslink the collagen fibrils, the crosslinked collagen fibrils are dehydrated such that they contain no more than 5 wt%, 10 wt%, 15 wt%, 20 wt%, or 25 wt% water, and at least 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 10 wt%, 15 wt%, 20 wt%, 30 wt%, 40 wt%, or 50 wt% of at least one lubricant is incorporated into the material, and optionally the material is cast, molded, or otherwise formed to include an upper and lower surface or an inner and outer surface.

[0316] The collagen or collagen raw material used in this method may include mammalian collagen, such as bovine type I, type III collagen, or other types and sources of collagen or collagen protein described herein. It may be obtained from mammals or other animals, or in some embodiments via Escherichia coli, Bacillus subtilis, or another bacterium; through Pichia pastoris, Saccharomyces cerevisiae Or another yeast or fungus; via plant cells; via insect cells or via mammalian cells for recombinant expression.

[0317] Collagen used in the methods disclosed herein can be isolated from cells such as those described above, cultured in vitro, such as cultured mammalian or animal cells. Alternatively, collagen or collagen protein can be obtained by other means, such as chemical synthesis. It may differ chemically from collagen obtained from natural sources, for example, if it may contain lower amounts or substantially no hydroxylysine or 3-hydroxyproline, glycosylated or cross-linked amino acid residues, or other post-translational modifications to the collagen amino acid sequence. Alternatively, it may contain higher amounts of hydroxylated amino acid residues, glycosylated residues, cross-links, or other chemical modifications.

[0318] Preferably, the collagen will be free of animal hair, hair follicles, or fat that naturally express the collagen molecules they contain, as these would impair its uniformity, strength, and aesthetic properties. For example, it may contain less than 1% by weight, 2% by weight, 3% by weight, 4% by weight, 5% by weight, 6% by weight, 7% by weight, 8% by weight, 9% by weight, or 10% by weight of actin, keratin, elastin, fibrin, albumin, globulin, mucin, mucoproteins, non-collagenous structural proteins, and / or non-collagenous non-structural proteins present in conventional leather. It may be substantially free of other collagen proteins, carbohydrates, nucleic acids or lipids, or immunogens, antigens, or allergens present in conventional leather (such as animals that naturally express collagen molecules in biomanufactured materials).

[0319] In some embodiments, collagen or collagen-like materials may be purified to be substantially homogeneous or may have a purity consistent with their ability to form fibrils, for example, it may contain 25 wt%, 30 wt%, 40 wt%, 50 wt%, 60 wt%, 70 wt%, 80 wt%, 90 wt%, 95 wt%, or 99 wt% collagen based on its total protein content or based on its total weight. Different types of collagen or mixtures of collagen from different biological sources may be used in some embodiments to balance the chemical and physical properties of collagen fibrils or to produce mixtures of fibrils with complementary properties. Such mixtures may contain 1 wt%, 5 wt%, 10 wt%, 25 wt%, 50 wt%, 75 wt%, 95 wt%, or 99 wt% of first collagen and 99 wt%, 95 wt%, 90 wt%, 75 wt%, 50 wt%, 25 wt%, 10 wt%, or 1 wt% of second, third, or subsequent collagen components. These ranges include all intermediate values ​​and collagen ratios, wherein the total collagen content of all collagen components is 100% by weight.

[0320] The methods disclosed herein can provide biomaterial components having substantially uniformly distributed fibrils, cross-linked fibrils, dehydrated fibrils, and / or lubricated fibrils. For example, fibrils can be distributed throughout the material such that the concentration variation of collagen fibrils by weight (or by number or average number) in the same unit volume of the material is no greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%.

[0321] In some implementations, bio-manufacturing material components are produced by scraping a softened material after cross-linking, dehydration, and / or lubrication.

[0322] In the embodiments described herein, collagen solutions or suspensions are fibrillated, for example, by adjusting the salt concentration of the solution or suspension, by adjusting its pH (e.g., increasing the pH of an acidic solution of collagen), or both. In some embodiments, fibrillation can be promoted by including a nucleating agent. Salts used for fibrillation include, but are not limited to, phosphate and chloride salts such as Na3PO4, K3PO4, KCl, and NaCl. The salt concentration during fibrillation can be adjusted to the range of 10 mM to 2 M, or the pH can be adjusted to pH 5.5, 6.0, 6.5, 7.0, 8.0, or greater using an acid, base, or buffer. Salt concentration and pH can be adjusted simultaneously to induce or promote fibrillation. In some embodiments of the methods described herein, aqueous solutions or suspensions of collagen molecules having a pH below 6.0 can be fibrillated by adjusting the pH to pH 6.0 to 8.0.

[0323] In some embodiments of the methods described herein, collagen fibrils are crosslinked during or after their formation process. Crosslinking can occur simultaneously with the incorporation of secondary components.

[0324] In other embodiments, collagen fibrils are crosslinked by contacting them with at least one of the following: amines, carboxylic acids, sulfates, sulfites, sulfonates, aldehydes, hydrazides, mercapto groups, diaziridines, aryl groups, azides, acrylates, epoxides, phenols, chromium compounds, vegetable tanning agents, and synthetic tanning agents.

[0325] One or more crosslinking agents may be added at concentrations ranging from 1 mM to 100 mM, for example at concentrations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 6, 70, 75, 80, 85, 90, 95 or 100 mM.

[0326] The crosslinking time, temperature, and other chemical and physical conditions can be selected to provide a specific degree of crosslinking within the collagen fibrils, such that the resulting crosslinked fibrils contain one or more different crosslinks to a specific degree. Based on the weight of the crosslinking agent and the weight of collagen, or based on the weight of the crosslinking network of the collagen fibrils (such as a hydrogel), the resulting crosslinked fibril formulation may contain at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or greater of the crosslinking agent. The crosslinking agent may be covalently or non-covalently bound to the collagen fibrils. After crosslinking, the number of crosslinks between or within collagen molecules, procollagen, or fibrils in the same unit volume of the material, or the average number of crosslinks between collagen molecules, procollagen, or collagen fibrils, may vary by no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%.

[0327] The methods described herein require a dehydration or removal step, which may occur during fibrillation or crosslinking, or both, or after fibrillation and crosslinking are substantially complete. These steps may be performed concurrently with the incorporation of secondary components.

[0328] In some embodiments, dehydration involves contacting the collagen fibrillary network with acetone, a synthetic tanning agent, or other agents that remove water bound to the collagen. In other embodiments, some water may be removed from the fibrillary formulation or cross-linked fibrillary formulation by filtration or evaporation, and residual water associated with the collagen fibrillary network may then be removed using solvents such as acetone or other water-removing chemical agents.

[0329] The methods described herein typically require lubrication of the resulting collagen fibrillation network. Lubrication can occur during fibrillation, crosslinking, dehydration, or any of these steps, or substantially after one or more of these steps are completed. Lubrication can be performed concurrently with the incorporation of secondary components.

[0330] In some implementations, lubrication will involve contacting a cross-linked collagen fibrillary network with one or more lubricants, such as fats, bio-oils, mineral or synthetic oils, cod oil, sulfonated oils, polymers, organofunctional siloxanes, and other agents used for fatliquoring conventional leather; or mixtures thereof.

[0331] In other embodiments, the lubricant is applied using a method that facilitates uniform lubrication and dehydration of the collagen fibrillary cross-linked network, such that the variation in lubricant concentration by weight in the same unit volume of the material is no greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. This application can be achieved by dip coating, spraying, vapor deposition, spin coating, scalpel coating, brush coating, and other known coating or deposition methods.

[0332] In another embodiment of the methods described herein, a surface coating or surface finishing is applied to a biomanufacturing material. While these can be applied to the surface of a material containing a collagen fibrillary network during various steps in the preparation of a biomanufacturing material, they are typically applied to crosslinked, dehydrated, and lubricated products. Uniform lubrication is made possible by the methods described herein that facilitate the successful and uniform application and adhesion of such coatings or finishing.

[0333] In other embodiments, the methods described herein may include incorporation or contact with the biomanufacturing material during its various preparation steps or after crosslinking, dehydration, and lubrication with other functional ingredients, including but not limited to dyes, stains, pigments, resins, polymers, or coatings. In further embodiments, these functional ingredients may be applied or incorporated under conditions where these agents are uniformly distributed on or throughout the material, such that the variation in their concentration by weight in the same unit volume of the material is no greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%.

[0334] In other embodiments, the methods described herein involve incorporating fillers of secondary components into biomanufacturing materials during various steps of their preparation or after crosslinking, dehydration, and lubrication. Typically, these fillers are incorporated prior to dehydration, such as during fibrillation or crosslinking. Such fillers include, but are not limited to, polymer microspheres, beads, fibers, filaments, or organic salts.

[0335] Some embodiments of the methods described above involve incorporating biomaterials into or on top of the preparation of at least one woven or nonwoven material. For example, this can be achieved by filtering cross-linked fibrils using woven or nonwoven paper or fabric materials. Other embodiments involve incorporating biomaterials into the preparation of at least one woven or nonwoven material.

[0336] Commercial implementations of the method involve incorporating bio-manufactured materials into products such as footwear, apparel, sportswear, uniforms, wallets, watch straps, bracelets, luggage, home furnishings, furniture, or other industrial, commercial, or consumer products.

[0337] The following non-limiting embodiments illustrate the present invention. The scope of the invention is not limited to the details described in these embodiments.

[0338] Example 1

[0339] Controlling the thickness of bio-manufactured leather

[0340] The thickness of the biomaterial used for the complex can be controlled by adjusting the collagen content. Hydrogels of extracted bovine type I collagen were formed at different collagen concentrations and volumes to produce dried collagen raw materials of varying thicknesses. Collagen was dissolved in 0.01 N HCl at 5 g / L or 9 g / L, and then 1 part of 10x PBS was added to 9 parts of the dissolved collagen to induce collagen fibrillation and gel formation.

[0341] Solutions of 0.8 L or 1.6 L of fibrillated collagen were then cast into molds and incubated at 25°C to allow hydrogel formation. The 0.8 L solution produced a gel 1.5 cm thick, while the 1.7 L solution produced a gel 3.0 cm thick. These gels were dehydrated and lubricated in acetone, then dried and mechanically scraped to soften a leather-like material. The final thickness of the dried material was related to the total amount of collagen in the initial hydrogel.

[0342] The thickness of bio-manufactured leather is controlled by altering its total collagen content. At 525 cm... 2 Samples A, B, and C were generated using 4, 7.2, or 14.4 grams of collagen per 1000 cubic meters of hydrated gel area, respectively. Bio-manufactured leather was produced from each sample through cross-linking, lubrication, and dehydration. As shown in Table 1, increasing the collagen content in the gel resulted in an increase in the thickness of the obtained bio-manufactured leather.

[0343] Table 1

[0344]

[0345] Example 2

[0346] Leather produced from type I collagen

[0347] The biomanufacturing components of the complex described in this article can be produced from type I collagen.

[0348] Type I collagen was purchased from Wuxi Biot Biotechnology Co., Ltd. (medical collagen sponge). Collagen was isolated from bovine tendon by acid treatment followed by pepsin digestion, and then purified by size exclusion chromatography, frozen, and lyophilized.

[0349] Using an overhead mixer, the lyophilized protein (4.1 g) was dissolved in 733 ml of 0.01 N HCl. After adequate collagen dissolution, as confirmed by the absence of solid collagen sponges in the solution (mixing at 1600 rpm for at least 1 hour), 82 μL of the tanning agent Relugan GTW was added to the solution, followed by the addition of 81 mL of 10x PBS (pH 11.2) to raise the pH of the solution to 7.2.

[0350] The solution was then mixed for 3 minutes before being poured into the silicone mold. The collagen solution was incubated in the silicone mold at 25°C for 2 hours to allow the collagen fibrillation into a viscoelastic hydrogel.

[0351] The stationary period of rheological properties, along with solution opacity (as measured by absorbing 425 nm light), indicates that fibrillation is complete at this point, and the presence of collagen fibrils was determined using scanning electron microscopy (Figure 3) and transmission electron microscopy (TEM). Figure 4(to confirm)

[0352] The fibrillated collagen hydrogel was removed from the mold and placed in 700 mL of acetone in a plastic container, then agitated on a track-mounted shaker at 25°C and 40 rpm. The hydrogel was dehydrated by replacing the acetone after an overnight incubation, followed by 5 x 1-hour washes and another overnight incubation. The acetone was replaced after each wash to remove water from the gel.

[0353] After dehydration with acetone, the collagen gel was incubated overnight in a fattening solution containing 20% ​​(v / v) cod liver oil or castor oil in 80% acetone or ethanol, while shaking at 40 rpm.

[0354] After incubation in a fatliquoring solution, the collagen gel is dried at 37°C. Upon drying, the material becomes soft and leather-like or bio-manufactured leather. Excess oil can be removed to improve the leather-like appearance of the material.

[0355] Sample weight and mechanical analysis confirmed that the oil permeated into the fibrous gel. The oil was able to penetrate the fibrous collagen network by dissolving it in a suitable solvent, as evidenced by the increased dry weight and decreased elastic modulus of the material compared to hydrogels that were not dehydrated or fatliquored in a solvent.

[0356] Bio-manufactured leather has a grainy texture on both the upper and lower surfaces and absorbs dyes consistently on both surfaces.

[0357] Example 3

[0358] Leather produced from type III collagen

[0359] The bio-manufacturing components of the complex described in this article can be generated using type III collagen.

[0360] The pH was increased to 7 by adding 1 part of 200 mM sodium phosphate solution (22 mL) (pH 11.2) to 9 parts of collagen solution (200 mL) to a solution of recombinant type III collagen at 2.5 mg / mL in 0.01 N HCl (FibroGen) and stirring at room temperature for 2 hours.

[0361] fibrillation was confirmed by measuring the absorbance of the solution at 400 nm over time.

[0362] After fibrillation, the fibrils are tanned by adding Relugan GTW (provided at 2% w / w based on collagen) to the fibril suspension and mixing for 30 min.

[0363] The tanned collagen fibrils were then centrifuged at 3,500 RPM for 30 minutes to concentrate the fibrils to a concentration of 10 mg / ml. The fibril precipitate was further centrifuged at 21,000 RPM for 30 minutes to obtain a fibril gel with a concentration of approximately 40-50 mg / ml.

[0364] Rheometers were used to evaluate the physical properties of fibril gels.

[0365] Storage modulus and complex viscosity confirm the properties of materials that are typically elastic.

[0366] The fibrillary gel was then dried in a food dehydrator set to 37°C for 18 hours.

[0367] After drying, the material is dyed and retanned by incubation in a solution of Lowepel Acid Black dye (provided at 2% w / w based on collagen) and Lubritan WP (provided at 20% w / w based on collagen).

[0368] The material is blown and squeezed in this solution to ensure that the dyes and synthetic tanning agents penetrate into the material. Finally, the material is dried and scraped to soften it and produce a leather-like material.

[0369] Example 4

[0370] Leather produced from type III collagen

[0371] The bio-manufacturing components of the complex described in this article can be generated using type II collagen.

[0372] Recombinant type III collagen was purchased from Fibrogen. Collagen was supplied at a concentration of 2.5 mg / mL in 0.01 N HCl.

[0373] To initiate collagen fibril assembly, 1 part of 200 mM Na2HPO4 (pH 11.2) (100 mL) was added to 9 parts of stock type III collagen solution at room temperature to bring the solution to pH 7.2. The solution was mixed for 1 hour at 1600 rpm using a top-mounted mixer.

[0374] After stirring for 1 hour, the collagen fibrils were reacted with Relugan GTW, which was added to the solution at 2% (w / w) based on the collagen mass. The solution was mixed for 1 hour at 1600 rpm using an overhead mixer.

[0375] Lipoderm A1 and Tanigan FT were then added to the solution, which was provided at 80% (w / w) of each collagen mass. The solution was mixed for 30 min at 1600 rpm using a top-mounted mixer. The pH of the solution was then lowered to 4 using a 10% (v / v) formic acid solution. The solution was mixed for 30 min at 1600 rpm using a top-mounted mixer.

[0376] Then, using a Buchner funnel attached to a vacuum pump (-27 in Hg pressure) and a rubber barrier on top of the Buchner funnel, 144 mL of the solution was filtered through a 47 mm Waterman 1 membrane. Vacuuming was maintained for 18 hours.

[0377] The concentrated fibrillary tissue was then allowed to dry under environmental conditions, and a leather-like material was produced by manually softening it for 30 minutes through rolling, bending, and stretching.

[0378] Example 5

[0379] Expancell

[0380] Type I bovine collagen isolated from bovine tendons by acid treatment followed by pepsin digestion, purification by size exclusion chromatography, freezing, and lyophilization was purchased from Wuxi Biot Biotechnology Co., Ltd. (medical collagen sponge).

[0381] Using an overhead mixer, dissolve 10 g of lyophilized collagen protein in 1 L of 0.01 N HCl (pH 2) at 1,600 rpm for at least one hour until no solid collagen sponge remains.

[0382] Then add 111.1 ml of 200 mM sodium phosphate (adjust the pH to 11.2 using sodium hydroxide) to raise the pH of the collagen solution to 7.2.

[0383] The pH 7.2 collagen solution was then stirred for 10 minutes, and 0.1 ml of 20% Relugan GTW (BASF) as a crosslinking agent, based on 2% of the collagen weight, was added to produce crosslinked collagen fibrils.

[0384] The cross-linked collagen fibrils were then mixed with 5 ml of 20% Tanigan FT (Lanxess) and stirred for one hour.

[0385] Then, add 1 gram of Expansion microspheres 461 WE 20 d36 (AkzoNobel) (10% of the collagen weight) and 40 ml of Truposol Ben (Trumpler) (80% of the collagen weight), and stir for another hour using a top mixer.

[0386] The pH of the solution was lowered to pH 4.0 by adding 10% formic acid and then stirred for one hour.

[0387] After the pH was lowered, 150 ml of the solution was filtered through a 90 mm Waterman 1 membrane using a Buchner funnel attached to a vacuum pump at -27 mmHg pressure.

[0388] The concentrated fibrillary tissue was then allowed to dry under ambient conditions, and a leather-like material was produced by manually softening it for 30 minutes through rolling, bending, and stretching. This material can be incorporated into the composite described herein.

[0389] Example 6

[0390] Titanium dioxide (white pigment)

[0391] Type I bovine collagen was purchased from Wuxi Biot Biotechnology Co., Ltd. (medical collagen sponge). This collagen was derived from type I collagen isolated from bovine tendon by acid treatment followed by pepsin digestion, purification by size exclusion chromatography, freeze-drying, and rehydration. The rehydration-dried protein (10 g) was dissolved in 1 L of 0.01 N HCl (pH 2) using an overhead mixer. After proper collagen dissolution, as confirmed by the absence of solid collagen sponge in the solution (mixing at 1,600 rpm for at least 1 hour), 111.1 ml of 200 mmol sodium phosphate (adjusted to pH 11.2 using sodium hydroxide) was added to raise the pH of the solution to 7.2. The resulting collagen solution was stirred for 10 minutes, and 0.1 ml of 20% Relugan GTW (BASF) cross-linking agent solution (2% by weight of collagen) was added.

[0392] Add 5 mL of 20% Tanigan FT (Lanxess) to the cross-linked collagen fibrils solution and stir for one hour.

[0393] After adding the Tanigan-FT, add 1 gram of Expansion microspheres (10% of collagen weight) 461 WE 20d36 (AkzoNobel), 40 ml of Truposol Ben (80% of collagen weight) (Trumpler), and 2 ml of PPE White HS a pa (10% of collagen weight), and mix for another hour using a top mixer.

[0394] The pH of the solution was lowered to pH 4.0 using 10% formic acid and stirred for one hour.

[0395] After the pH change, 150 ml of the solution was filtered through a 90 mM Waterman 1 membrane using a Buchner funnel attached to a vacuum pump at -27 mmHg pressure.

[0396] The concentrated fibrillary tissue was then allowed to dry under ambient conditions, and a leather-like material was produced by manually softening it for 30 minutes through rolling, bending, and stretching. This material can be incorporated into the composite described herein.

[0397] Example 7

[0398] Hycar resin (26552)

[0399] Bovine collagen was purchased from Wuxi Biot Biotechnology Co., Ltd. (medical collagen sponge). This collagen is type I collagen isolated from bovine tendons through acid treatment followed by pepsin digestion, purification by size exclusion chromatography, freezing, and lyophilization.

[0400] Dissolve 10 g of lyophilized protein in 1 L of 0.01 N HCl (pH 2) using an overhead mixer. After adequately dissolving the collagen, as confirmed by the absence of solid collagen sponges in the solution (mixing at 1600 rpm for at least 1 hour), add 111.1 ml of 200 mM sodium phosphate (adjusting the pH to 11.2 using sodium hydroxide) to raise the pH of the solution to 7.2.

[0401] Stir the obtained collagen solution for 10 minutes, and add 0.1 ml of 20% Relugan GTW (BASF) crosslinking agent solution and tanning agent solution, which is 2% of the collagen weight.

[0402] Add 5 mL of 20% Tanigan FT (Lanxess) to the cross-linked collagen fibrils solution and stir for one hour. After the addition of Tanigan-FT, add 1 g of Expancel microspheres (10% of collagen weight) 461 WE 20d36 (AkzoNobel), 40 mL of Truposol Ben (Trumpler) (80% of collagen weight), and 2 mL of PPE White HS a pa (Stahl) (10% of collagen weight), and stir again for one hour using a top stirrer.

[0403] The pH of the solution was lowered to 4.0 using 10% formic acid, and Hycar Resin 26552 (Lubrizol) was added and stirred for another hour. After pH adjustment and resin addition, 150 ml of the solution was filtered through a 90 mm Waterman 1 membrane using a Buchner funnel attached to a vacuum pump at -27 mmHg. To promote activation, Hycar Resin 26552 was mixed with the fibril solution and heated at 50°C for 2 hours.

[0404] The concentrated fibrillary tissue was then allowed to dry under ambient conditions, and a leather-like material was produced by manually softening it for 30 minutes through rolling, bending, and stretching. This material can be incorporated into the composite described herein.

[0405] The addition of resin causes the following Figure 1 The mechanical properties are improved as shown.

[0406] Following the pH change, 150 ml of the solution was filtered through a 90 mm Waterman 1 membrane using a Buchner funnel attached to a vacuum pump at -27 mmHg pressure. The solution immediately formed a green precipitate and could not be filtered further.

[0407]

[0408] Relugan is followed by retanning agents based on polymers, resins, or aldehydes. Tanigan is a sulfone-based synthetic tanning agent. Truposol Ben is a fatliquoring agent for chrome-free leather. Lipoderm Liquor A1 is a water-based fatliquoring agent based on long-chain alcohols, paraffin, and anionic surfactants. Hycar Resin 26552: a formaldehyde-free acrylic emulsion.

[0409] Example 8

[0410] Encapsulated carbon fiber

[0411] Bovine collagen was purchased from Wuxi Biot Biotechnology Co., Ltd. (medical collagen sponge). This collagen was derived from type I collagen isolated from bovine tendon by acid treatment followed by pepsin digestion, purification by size exclusion chromatography, freeze-drying, and rehydration. The rehydration-dried protein (4.1 g) was dissolved in 733 mL of 0.01 N HCl (pH 2) using an overhead mixer. After proper collagen dissolution, as confirmed by the absence of solid collagen sponge in the solution (mixing at 1,600 rpm for at least 1 hour), 82 μL of the tanning agent Relugan GTW was added to the solution, followed by 81 mL of 10x PBS (pH 11.2) to raise the pH to 7.2. The solution was mixed for 3 min and then poured into a mold containing 0.25-inch shredded carbon fibers. The carbon fibers were purchased from Fibre Glast Developments. The carbon fibers were mixed into the collagen solution to distribute the fibers throughout the collagen matrix. Collagen solution was incubated in a silicon mold at 25°C for 2 hours to allow collagen fibrillation into a viscoelastic hydrogel, thereby encapsulating carbon fibers.

[0412] The fibrillated collagen hydrogel with encapsulated carbon fibers was removed from the mold and dehydrated in a series of acetone solutions (5 x 1 h, 40 rpm at 25°C). After acetone dehydration, the collagen gel was incubated overnight in a fatliquoring solution containing 20% ​​(v / v) cod liver oil in 80% acetone with shaking at 40 rpm. After incubation in the cod liver oil solution, the collagen gel was dried at 37°C. The fibrillated collagen hydrogel was then removed from the mold and placed in 700 mL of acetone in a plastic container and... 25℃ The gel was oscillated at 40 rpm on a fixed-track oscillator. The hydrogel was dehydrated by replacing the acetone after an overnight incubation, followed by 5 x 1-hour washes and another overnight incubation. The acetone was replaced after each wash to remove water from the gel. After acetone dehydration, the collagen gel was incubated overnight in a fatliquoring solution containing 20% ​​(v / v) cod liver oil or castor oil in 80% acetone or ethanol, oscillated at 40 rpm. After incubation in the fatliquoring solution, the collagen gel was dried at 37°C. After drying, the material became soft and leather-like. Furthermore, the carbon fibers were encapsulated within a tanned and fatliquoring collagen network and could be processed without delamination or pulling out bio-manufactured leather.

[0413] Example 9

[0414] Layered nonwoven fabrics

[0415] Bovine collagen was dissolved as described in Example 8. Once the collagen was dissolved, as confirmed by the absence of solid collagen sponges in the solution (mixing at 1600 rpm for at least 1 hour), 0.2 g of Lowepel Acid Black dye dissolved in 5 mL of deionized water was added dropwise to the stirred collagen solution. The dye was mixed at 1600 rpm for 1 hour to allow the dye to be fixed to the collagen. Then 82 μL of the tanning agent Relugan GTW was added to the solution, followed by 81 mL of 10x PBS (pH 11.2) to increase the pH of the solution to 7.2. The solution was mixed for 3 min and integrating with a secondary material of nonwoven wool felt using a vacuum technique. The wool felt was purchased from US Felts and treated overnight at 50°C with 1 M hydroxylamine and 1 g / L Triton n-57 surfactant (pH 8) to remove surface lipids and increase the wettability and reactivity of the wool fibers. 60 mL of the collagen precursor solution was drawn into the wool felt under a vacuum. A dye gradient is visible from the top to the bottom surface of the felt. After integration with the collagen solution, the wool felt is placed top-down on a freshly cast collagen precursor solution. The collagen and wool felt are incubated at 25°C for 2 hours to allow fibrillation. After fibrillation, the material is dried in a dehydrator at 37°C. This dried material is then scraped to create a soft, leather-like material with a wool backing.

[0416] Example 10

[0417] Fabric with embedded photoluminescent patterns

[0418] Quantum dots functionalized with primary amines and PEG spacers were purchased from Sigma. The quantum dots were diluted 1:10 in an ice-quenched collagen precursor solution (5 wt% type I collagen, 1xPBS, 0.02 μL GTW / mg collagen). The quantum dot / collagen solution was then screen-printed in an “M” shape onto a secondary material of a silk-woven fabric. The quantum dot / collagen screen-printed fabric was incubated at room temperature for 1 hour before encapsulating the fabric in the collagen gel. As in Example 2, the collagen precursor solution (5 mg / mL type I collagen, 1xPBS, 0.02 μL GTW / mg collagen) was cast into a silicone mold 3 minutes after the addition of PBS, and the fabric was placed in the middle of the collagen solution. The solution was incubated at 25°C for 1 hour to allow fibrillation, followed by dehydration of the gel with the encapsulated fabric in a series of acetones, fatliquoring in cod oil / acetone, and drying. After drying and softening, the material is exposed to a UV light source to illuminate the embedded quantum dots “M”.

[0419] Example 11

[0420] Embedded 3D objects

[0421] Quantum dots functionalized with primary amines and PEG spacers were purchased from Sigma. The quantum dots were diluted 1:10 in a Slygard 184 polydimethylsiloxane (PDMS) substrate, followed by a 10:1 mixture of quantum dots / substrate and curing agent. After mixing, the quantum dot / substrate / curing agent solution was cast into a mold in the shape of an “M”. The PDMS “M” was cured overnight at 40°C and then removed from the mold to produce an elastomer and photoluminescent “M”. As in Example 2, a collagen precursor solution (5 mg / mL type I collagen, 1 x PBS, 0.02 μL GTW / mg collagen) was cast into a silicone mold 3 minutes after the addition of PBS, with the PDMS “M” placed in the center of the collagen solution. The solution was incubated at 25°C for 1 hour to allow fibrillation, followed by dehydration of the gel with the encapsulated fabric in a series of acetones, then fatliquoring in cod oil / acetone and drying (see Example 2 for details). After drying and softening, the encapsulated three-dimensional “M” creates an “M”-shaped tactile pattern on the surface of the bio-manufactured leather. Additionally, the material is exposed to a UV light source to illuminate the quantum dots embedded within the PDMS “M”.

[0422] Example 12

[0423] wool felt compound

[0424] The method of Example 9 was repeated using wool felt and the collagen precursor solution of Example 6 to form composite leather.

[0425] Example 13

[0426] Lycra® Complex

[0427] Use 3” x 3” polyester-polyurethane copolymer felt (Lycra) A 3” x 3” sample of the leather from Example 2 was laminated using a hot melt adhesive at 50°C. This formed a leather-secondary material backing composite.

[0428] Examples 14-20

[0429] As shown in Examples 14-20 below, the biomanufacturing material of the present invention can be successfully applied or integrated into secondary components to produce a robust leather-like complex.

[0430] Example 14

[0431] Spacer fabric

[0432] Bovine collagen was purchased from Wuxi Biot Biotechnology Co., Ltd. (medical collagen sponge). This collagen was derived from type I collagen isolated from bovine tendons through acid treatment followed by pepsin digestion, purification by size exclusion chromatography, freezing, and lyophilization. The lyophilized protein (10 g) was dissolved in 1 L of 0.01 N HCl (pH 2) using an overhead mixer.

[0433] After the collagen has been properly dissolved, as confirmed by the absence of solid collagen sponges in the solution (mixing at 1,600 rpm for at least 1 hour), add 111.1 mL of 200 mmol sodium phosphate (adjusting the pH to 11.2 using sodium hydroxide) to raise the pH of the solution to 7.2. Stir the resulting collagen solution for 10 minutes and add 0.1 mL of a 20% Relugan GTW (BASF) tanning agent solution (2% based on collagen weight).

[0434] Add 5 mL of 20% Tanigan FT (Lanxess) to the cross-linked collagen fibrils solution and stir for one hour.

[0435] Following the addition of Tanigan-FT, 1 gram of Expancel microspheres (based on 10% of collagen weight) 461 WE 20d36 (AkzoNobel) and 40 ml of Truposol Ben (based on 80% of collagen weight) were added, and the mixture was stirred for another hour using a top stirrer. The pH of the solution was adjusted to 4.0 using 10% formic acid and stirred for another hour.

[0436] After the pH change, a 75 mm disc of 100% polyester 3D spacer fabric was cut off and placed on top of a 90 mm Waterman No. 1 membrane. A thin layer of high vacuum grease (Dow Corning) was applied around the edge of the membrane to retain the material while filtering.

[0437] Then, using a Buchner funnel attached to a vacuum pump (-27 inHg pressure), 150 mL of the solution was filtered through the textile and Waterman 1 membrane. Vacuum was maintained for 40 minutes.

[0438] The concentrated fibrillary tissue was then allowed to dry in a chamber at 20°C and 65% humidity. When the concentrated fibrillary tissue had reached 20% moisture content, it was pressed into a carving press and subjected to 1 metric ton of pressure at 50°C for 10 minutes. The material was then manually softened for 30 minutes by rolling, bending, and stretching to produce a leather-like material.

[0439] The spacer fabric remains integrated into the original fiber structure, resulting in a leather-like material with an exposed fabric backing on one side and an embossed pattern formed on its surface by embedded textiles. The material is finished with a highly efficient coating conventionally used in the footwear industry.

[0440] Example 15

[0441] The procedure of Example 2 was repeated by replacing the smaller 75 mm discs that were regionally integrated into the final material.

[0442] Example 16

[0443] Polyester screen

[0444] Bovine collagen was purchased from Wuxi Biot Biotechnology Co., Ltd. (medical collagen sponge). This collagen was derived from type I collagen isolated from bovine tendons through acid treatment followed by pepsin digestion, purification by size exclusion chromatography, freezing, and lyophilization. The lyophilized protein (10 g) was dissolved in 1 L of 0.01 N HCl (pH 2) using an overhead mixer.

[0445] After the collagen has been properly dissolved, as confirmed by the absence of solid collagen sponges in the solution (mixing at 1600 rpm for at least 1 hour), add 111.1 mL of 200 mmol sodium phosphate (adjust the pH to 11.2 using sodium hydroxide) to raise the pH of the solution to 7.2.

[0446] Stir the resulting collagen solution for 10 minutes and add 0.1 mL of 20% Relugan GTW (BASF) (2% based on collagen weight) tanning agent solution.

[0447] Add 5 mL of 20% Tanigan FT (Lanxess) to the crosslinked collagen fibrils solution and stir for one hour. After the addition of Tanigan-FT, add 1 g of Expancel microspheres (10% of collagen weight) 461 WE 20d36 (AkzoNobel) and 40 mL of Truposol Ben (Trumpler) (80% of collagen weight) and stir again for one hour using a top stirrer. Adjust the pH of the solution to 4.0 using 10% formic acid and stir for one hour.

[0448] After the pH change, a 75 mm disc of polyester screen was cut off and placed on top of a 90 mm Waterman 1 membrane. A thin layer of high-vacuum grease (Dow Corning) was applied around the edges of the membrane to retain the material during filtration. Then, using a Buchner funnel attached to a vacuum pump (-27 inHg pressure), 150 mL of the solution was filtered through the textile and the Waterman 1 membrane. Vacuum was maintained for 40 minutes.

[0449] The concentrated fibrillary tissue was then allowed to dry in a chamber at 20°C and 65% humidity.

[0450] When the concentrated fibrillated tissue has reached 20% moisture content, it is pressed into a carving press and subjected to 50°C and 1 metric ton of pressure for 10 minutes. The material is then manually softened for 30 minutes by rolling, bending, and stretching to produce a leather-like material.

[0451] The fabric is removed after 15 minutes of scraping, resulting in a double-sided grained surface with different textures and aesthetics on each side of the material. The material is then finished with a high-efficiency coating commonly used in the footwear industry.

[0452] Example 17

[0453] Polyester textiles

[0454] Repeat the procedure of Example 3 by further steps of laminating 100% polyester technical textiles to one side of the material.

[0455] Example 18

[0456] coating

[0457] Bovine collagen was purchased from Wuxi Biot Biotechnology Co., Ltd. (medical collagen sponge). This collagen was derived from type I collagen isolated from bovine tendons through acid treatment followed by pepsin digestion, purification by size exclusion chromatography, freezing, and lyophilization. The lyophilized protein (10 g) was dissolved in 1 L of 0.01 N HCl (pH 2) using an overhead mixer.

[0458] After the collagen has been properly dissolved, as confirmed by the absence of solid collagen sponges in the solution (mixing at 1600 rpm for at least 1 hour), add 111.1 mL of 200 mmol sodium phosphate (adjust the pH to 11.2 using sodium hydroxide) to raise the pH of the solution to 7.2.

[0459] Stir the resulting collagen solution for 10 minutes and add 0.1 mL of 20% Relugan GTW (BASF) (2% based on collagen weight) tanning agent solution.

[0460] Add 5 mL of 20% Tanigan FT (Lanxess) to the crosslinked collagen fibrils solution and stir for one hour. After the addition of Tanigan-FT, add 1 g of Expancel microspheres (10% of collagen weight) 461 WE 20d36 (AkzoNobel) and 40 mL of Truposol Ben (Trumpler) (80% of collagen weight) and stir again for one hour using a top stirrer. Adjust the pH of the solution to 4.0 using 10% formic acid and stir for one hour.

[0461] Following the pH change, 150 mL of the solution was filtered through a 90 mm Waterman 1 membrane using a Buchner funnel attached to a vacuum pump (-27 inHg pressure). Vacuum was maintained for 40 minutes. The concentrated fibrillary tissue was then allowed to dry in a chamber at 20°C and 65% humidity.

[0462] When the concentrated fibrillated tissue has reached 20% moisture content, it is pressed into a carving press and subjected to 50°C and 1 metric ton of pressure for 10 minutes. The material is then manually softened for 30 minutes by rolling, bending, and stretching to produce a leather-like material.

[0463] The material is finished using a high-efficiency coating commonly used in the footwear industry. The finished material is then glued together on three leather plates to create a three-dimensional surface texture and aesthetic appeal.

[0464] Example 19

[0465] Polyester screen

[0466] Bovine collagen was purchased from Wuxi Biot Biotechnology Co., Ltd. (medical collagen sponge). This collagen was derived from type I collagen isolated from bovine tendon through acid treatment followed by pepsin digestion, purification by size exclusion chromatography, freezing, and lyophilization. The lyophilized protein (10 g) was dissolved in 1 L of 0.01 N HCl (pH 2) using an overhead mixer. After proper collagen dissolution, as confirmed by the absence of solid collagen sponge in the solution (mixing at 1600 rpm for at least 1 hour), 111.1 mL of 200 mmol sodium phosphate (adjusted to pH 11.2 using sodium hydroxide) was added to raise the pH of the solution to 7.2.

[0467] Stir the resulting collagen solution for 10 minutes and add 0.1 mL of 20% Relugan GTW (BASF) (2% based on collagen weight) tanning agent solution.

[0468] Add 5 mL of 20% Tanigan FT (Lanxess) to the crosslinked collagen fibrils solution and stir for one hour. After the addition of Tanigan-FT, add 1 g of Expancel microspheres (10% of collagen weight) 461 WE 20d36 (AkzoNobel) and 40 mL of Truposol Ben (Trumpler) (80% of collagen weight) and stir again for one hour using a top stirrer. Adjust the pH of the solution to 4.0 using 10% formic acid and stir for one hour.

[0469] After the pH change, three strips of 100% polyester screen fabric (each 10 mm wide) were cut off and placed horizontally on top of the 90 mm Waterman No. 1 membrane (with a 5 mm gap between each strip). A thin layer of high vacuum grease (Dow Corning) was applied around the edge of the membrane to retain the material while filtering.

[0470] Then, using a Buchner funnel attached to a vacuum pump (-27 inHg pressure), 150 mL of the solution was filtered through the textile and Waterman 1 membrane. Vacuum was maintained for 40 minutes.

[0471] The concentrated fibrillary structure is then allowed to dry at 20°C in a 65% humidity chamber, and when it reaches 20% moisture content, it is placed in an oven at 50°C for 2 hours. The screen fabric remains integrated into the fibrillary structure, thereby producing a fabric backing material having a fabric embossed pattern formed on its surface by embedded textiles.

[0472] The concentrated fibrillary tissue is compressed around the screen to form a self-assembled three-dimensional end material; this method can be controlled to form the desired end shape.

[0473] Example 20

[0474] Polyester screen

[0475] Bovine collagen was purchased from Wuxi Biot Biotechnology Co., Ltd. (medical collagen sponge). This collagen was derived from type I collagen isolated from bovine tendon by acid treatment followed by pepsin digestion, purification by size exclusion chromatography, freezing, and lyophilization. The lyophilized protein (4.1 g) was dissolved in 733 mL of 0.01 N HCl (pH 2) using an overhead mixer.

[0476] After the collagen has been properly dissolved, as confirmed by the absence of solid collagen sponges in the solution (mixing at 1600 rpm for at least 1 hour), 82 μL of the tanning agent Relugan GTW is added to the solution, followed by the addition of 81 mL of 10x PBS (pH 11.2) to raise the pH of the solution to 7.2.

[0477] Mix the solution for 3 minutes, then pour it into a mold containing a 100% polyester screen (measured as 75 mm x 200 mm), which is fixed in place 5 mm above the bottom of the mold.

[0478] The collagen solution was incubated in a silicone mold at 25°C for 2 hours to allow the collagen fibrillation into a viscoelastic hydrogel, thereby encapsulating the polyester fabric in the middle of the gel. The fibrillated collagen hydrogel was removed from the mold and placed in 700 mL of acetone in a plastic container and oscillated on a fixed-track oscillator at 25°C and 40 rpm.

[0479] The hydrogel was dehydrated by replacing the acetone after overnight incubation, followed by 5 x 1-hour washes and another overnight incubation. The acetone was replaced after each wash to remove water from the gel. After acetone dehydration, the collagen gel was incubated overnight in a lubricating solution containing 20% ​​(v / v) cod liver oil or castor oil in 80% acetone or ethanol, with shaking at 40 rpm. After incubation in the fatliquoring solution, the collagen gel was dried at 37°C.

[0480] After drying, the material becomes soft and leathery. Furthermore, the encapsulated mesh creates a double-sided grained surface, which can be modified almost infinitely depending on the type and structure of the embedded fabric.

[0481] Explanation

[0482] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. For example, as used herein, the singular forms “an” and “described” are intended to also include the plural forms unless the context clearly indicates otherwise. It will be further understood that, when used in this specification, the terms “comprises” and / or “comprising” indicate the presence of the specified features, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed entries and may be abbreviated to “ / ”.

[0483] Spatial relative terms such as “down,” “below,” “lower,” “up,” and “above” are used herein for ease of description to describe the relationship between one element or feature and another, as shown in the accompanying drawings. It will be understood that spatial relative terms are intended to cover different orientations of the device in use or operation other than those depicted in the drawings. For example, if the device in the drawings is inverted, an element described as “down” or “below” other elements or features will be oriented “up” to those other elements or features. Thus, the exemplary term “down” can cover both up and down orientations. Devices may be oriented in other ways (rotated 90 degrees or in other orientations), and the spatial relative descriptive terms used herein will be interpreted accordingly. Similarly, terms such as “up,” “down,” “vertical,” and “horizontal” are used herein for explanatory purposes only, unless otherwise explicitly stated.

[0484] While the terms “first” and “second” may be used herein to describe various features / elements (including steps), these features / elements should not be limited by these terms unless the context otherwise indicates. These terms may be used to distinguish one feature / element from another. Thus, the first feature / element discussed below may be referred to as the second feature / element, and similarly, the second feature / element discussed below may be referred to as the first feature / element without departing from the doctrine of the invention.

[0485] Throughout this specification and the following claims, unless the context otherwise requires, the word “comprise” and variations such as “comprises” and “comprising” mean that various components may be used together in a method and article (e.g., a composition and an instrument comprising a device and a method). For example, the term “comprising” will be understood to imply the inclusion of any specified element or step, but does not exclude any other element or step.

[0486] As used herein, in the specification and claims, including as used in the embodiments and unless otherwise expressly stated, all figures may be read as prefixed with the words “substantially,” “approximately,” or “approximately,” even if the term is not explicitly stated. The phrase “approximately” or “approximately” may be used when describing magnitude and / or location to indicate that the described numerical value and / or location is within a reasonably expected range of numerical value and / or location. For example, numerical values ​​may have values ​​of + / - 0.1% (or a range of values) of a specified value, + / - 1% (or a range of values) of a specified value, + / - 2% (or a range of values) of a specified value, + / - 5% (or a range of values) of a specified value, + / - 10% (or a range of values) of a specified value, etc. Any range of figures listed herein is intended to include all subranges included therein.

[0487] When a feature or element is referred to herein as being “on top of” another feature or element, it may be directly situated on top of the other feature or element, or there may be inserted features and / or elements present. In contrast, when a feature or element is referred to as being “directly on” another feature or element, there is no inserted feature or element. It will also be understood that when a feature or element is referred to as being “connected,” “attached,” or “joined” to another feature or element, it may be directly connected, attached, or joined to the other feature or element, or there may be inserted features or elements present. In contrast, when a feature or element is referred to as being “directly connected,” “directly attached,” or “directly joined” to another feature or element, there is no inserted feature or element. Although described or illustrated with respect to one embodiment, the features and elements thus described or illustrated may be applied to other embodiments. Those skilled in the art will also appreciate that a structure or feature referring to being “adjacent” to another feature may have portions overlapping the adjacent feature or located below the adjacent feature.

[0488] While various illustrative embodiments have been described above, any number of variations may be made to the various embodiments without departing from the scope of the invention as defined in the claims. For example, the order of the method steps described may generally vary in alternative embodiments, and in other alternative embodiments, one or more method steps may be skipped. Optional features of the various apparatus and system embodiments may be included in some embodiments but not in others. Therefore, the foregoing description is provided primarily for illustrative purposes and should not be construed as limiting the scope of the invention as set forth in the claims.

[0489] The embodiments and descriptions included herein illustrate specific implementations of the subject matter by way of illustration and not limitation. As mentioned, other embodiments may be used and derived therefrom, allowing for structural and logical substitutions and changes without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein, individually or collectively, solely for convenience and are not intended to automatically limit the scope of this application to any single invention or inventive concept, if in fact more than one is disclosed. Therefore, while specific embodiments have been illustrated and described herein, any arrangement that achieves the same purpose may replace the specific embodiments shown. This invention is intended to cover any and all changes or variations of the various embodiments. Combinations of the above embodiments and other embodiments not specifically described herein will be apparent to those skilled in the art upon review of the foregoing description.

[0490] Incorporate by reference

[0491] All publications and patent applications mentioned in this specification are incorporated herein by reference to the same extent as each individual publication or patent application is explicitly and individually indicated to be incorporated by reference, especially the disclosure appearing in the same sentences, paragraphs, page numbers or portions of the specification in which it appears by reference. sequence list <110> Modern Meadow, Inc. <120> Methods for biomanufacturing composite materials <130> 500567WO <150> US 62 / 295,435 <151> 2016-02-15 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 1463 <212> PRT <213> domestic cattle <220> <221> MISC_FEATURE <222> (1)..(1463) <223> Collagen α-1(I) chain precursor <400> 1 Met Phe Ser Phe Val Asp Leu Arg Leu Leu Leu Leu Leu Ala Ala Thr 1 5 10 15 Ala Leu Leu Thr His Gly Gln Glu Glu Gly Gln Glu Glu Gly Gln Glu 20 25 30 Glu Asp Ile Pro Pro Val Thr Cys Val Gln Asn Gly Leu Arg Tyr His 35 40 45 Asp Arg Asp Val Trp Lys Pro Val Pro Cys Gln Ile Cys Val Cys Asp 50 55 60 Asn Gly Asn Val Leu Cys Asp Asp Val Ile Cys Asp Glu Leu Lys Asp 65 70 75 80 Cys Pro Asn Ala Lys Val Pro Thr Asp Glu Cys Cys Pro Val Cys Pro 85 90 95 Glu Gly Gln Glu Ser Pro Thr Asp Gln Glu Thr Thr Gly Val Glu Gly 100 105 110 Pro Lys Gly Asp Thr Gly Pro Arg Gly Pro Arg Gly Pro Ala Gly Pro 115 120 125 Pro Gly Arg Asp Gly Ile Pro Gly Gln Pro Gly Leu Pro Gly Pro Pro 130 135 140 Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Leu Gly Gly Asn Phe Ala 145 150 155 160 Pro Gln Leu Ser Tyr Gly Tyr Asp Glu Lys Ser Thr Gly Ile Ser Val 165 170 175 Pro Gly Pro Met Gly Pro Ser Gly Pro Arg Gly Leu Pro Gly Pro Pro 180 185 190 Gly Ala Pro Gly Pro Gln Gly Phe Gln Gly Pro Pro Gly Glu Pro Gly 195 200 205 Glu Pro Gly Ala Ser Gly Pro Met Gly Pro Arg Gly Pro Pro Gly Pro 210 215 220 Pro Gly Lys Asn Gly Asp Asp Gly Glu Ala Gly Lys Pro Gly Arg Pro 225 230 235 240 Gly Glu Arg Gly Pro Pro Gly Pro Gln Gly Ala Arg Gly Leu Pro Gly 245 250 255 Thr Ala Gly Leu Pro Gly Met Lys Gly His Arg Gly Phe Ser Gly Leu 260 265 270 Asp Gly Ala Lys Gly Asp Ala Gly Pro Ala Gly Pro Lys Gly Glu Pro 275 280 285 Gly Ser Pro Gly Glu Asn Gly Ala Pro Gly Gln Met Gly Pro Arg Gly 290 295 300 Leu Pro Gly Glu Arg Gly Arg Pro Gly Ala Pro Gly Pro Ala Gly Ala 305 310 315 320 Arg Gly Asn Asp Gly Ala Thr Gly Ala Ala Gly Pro Pro Gly Pro Thr 325 330 335 Gly Pro Ala Gly Pro Pro Gly Phe Pro Gly Ala Val Gly Ala Lys Gly 340 345 350 Glu Gly Gly Pro Gln Gly Pro Arg Gly Ser Glu Gly Pro Gln Gly Val 355 360 365 Arg Gly Glu Pro Gly Pro Pro Gly Pro Ala Gly Ala Ala Gly Pro Ala 370 375 380 Gly Asn Pro Gly Ala Asp Gly Gln Pro Gly Ala Lys Gly Ala Asn Gly 385 390 395 400 Ala Pro Gly Ile Ala Gly Ala Pro Gly Phe Pro Gly Ala Arg Gly Pro 405 410 415 Ser Gly Pro Gln Gly Pro Ser Gly Pro Pro Gly Pro Lys Gly Asn Ser 420 425 430 Gly Glu Pro Gly Ala Pro Gly Ser Lys Gly Asp Thr Gly Ala Lys Gly 435 440 445 Glu Pro Gly Pro Thr Gly Ile Gln Gly Pro Pro Gly Pro Ala Gly Glu 450 455 460 Glu Gly Lys Arg Gly Ala Arg Gly Glu Pro Gly Pro Ala Gly Leu Pro 465 470 475 480 Gly Pro Pro Gly Glu Arg Gly Gly Pro Gly Ser Arg Gly Phe Pro Gly 485 490 495 Ala Asp Gly Val Ala Gly Pro Lys Gly Pro Ala Gly Glu Arg Gly Ala 500 505 510 Pro Gly Pro Ala Gly Pro Lys Gly Ser Pro Gly Glu Ala Gly Arg Pro 515 520 525 Gly Glu Ala Gly Leu Pro Gly Ala Lys Gly Leu Thr Gly Ser Pro Gly 530 535 540 Ser Pro Gly Pro Asp Gly Lys Thr Gly Pro Pro Gly Pro Ala Gly Gln 545 550 555 560 Asp Gly Arg Pro Gly Pro Pro Gly Pro Pro Gly Ala Arg Gly Gln Ala 565 570 575 Gly Val Met Gly Phe Pro Gly Pro Lys Gly Ala Ala Gly Glu Pro Gly 580 585 590 Lys Ala Gly Glu Arg Gly Val Pro Gly Pro Pro Gly Ala Val Gly Pro 595 600 605 Ala Gly Lys Asp Gly Glu Ala Gly Ala Gln Gly Pro Pro Gly Pro Ala 610 615 620 Gly Pro Ala Gly Glu Arg Gly Glu Gln Gly Pro Ala Gly Ser Pro Gly 625 630 635 640 Phe Gln Gly Leu Pro Gly Pro Ala Gly Pro Pro Gly Glu Ala Gly Lys 645 650 655 Pro Gly Glu Gln Gly Val Pro Gly Asp Leu Gly Ala Pro Gly Pro Ser 660 665 670 Gly Ala Arg Gly Glu Arg Gly Phe Pro Gly Glu Arg Gly Val Gln Gly 675 680 685 Pro Pro Gly Pro Ala Gly Pro Arg Gly Ala Asn Gly Ala Pro Gly Asn 690 695 700 Asp Gly Ala Lys Gly Asp Ala Gly Ala Pro Gly Ala Pro Gly Ser Gln 705 710 715 720 Gly Ala Pro Gly Leu Gln Gly Met Pro Gly Glu Arg Gly Ala Ala Gly 725 730 735 Leu Pro Gly Pro Lys Gly Asp Arg Gly Asp Ala Gly Pro Lys Gly Ala 740 745 750 Asp Gly Ala Pro Gly Lys Asp Gly Val Arg Gly Leu Thr Gly Pro Ile 755 760 765 Gly Pro Pro Gly Pro Ala Gly Ala Pro Gly Asp Lys Gly Glu Ala Gly 770 775 780 Pro Ser Gly Pro Ala Gly Pro Thr Gly Ala Arg Gly Ala Pro Gly Asp 785 790 795 800 Arg Gly Glu Pro Gly Pro Pro Gly Pro Ala Gly Phe Ala Gly Pro Pro 805 810 815 Gly Ala Asp Gly Gln Pro Gly Ala Lys Gly Glu Pro Gly Asp Ala Gly 820 825 830 Ala Lys Gly Asp Ala Gly Pro Pro Gly Pro Ala Gly Pro Ala Gly Pro 835 840 845 Pro Gly Pro Ile Gly Asn Val Gly Ala Pro Gly Pro Lys Gly Ala Arg 850 855 860 Gly Ser Ala Gly Pro Pro Gly Ala Thr Gly Phe Pro Gly Ala Ala Gly 865 870 875 880 Arg Val Gly Pro Pro Gly Pro Ser Gly Asn Ala Gly Pro Pro Gly Pro 885 890 895 Pro Gly Pro Ala Gly Lys Glu Gly Ser Lys Gly Pro Arg Gly Glu Thr 900 905 910 Gly Pro Ala Gly Arg Pro Gly Glu Val Gly Pro Pro Gly Pro Pro Gly 915 920 925 Pro Ala Gly Glu Lys Gly Ala Pro Gly Ala Asp Gly Pro Ala Gly Ala 930 935 940 Pro Gly Thr Pro Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly Val Val 945 950 955 960 Gly Leu Pro Gly Gln Arg Gly Glu Arg Gly Phe Pro Gly Leu Pro Gly 965 970 975 Pro Ser Gly Glu Pro Gly Lys Gln Gly Pro Ser Gly Ala Ser Gly Glu 980 985 990 Arg Gly Pro Pro Gly Pro Met Gly Pro Pro Gly Leu Ala Gly Pro Pro 995 1000 1005 Gly Glu Ser Gly Arg Glu Gly Ala Pro Gly Ala Glu Gly Ser Pro 1010 1015 1020 Gly Arg Asp Gly Ser Pro Gly Ala Lys Gly Asp Arg Gly Glu Thr 1025 1030 1035 Gly Pro Ala Gly Pro Pro Gly Ala Pro Gly Ala Pro Gly Ala Pro 1040 1045 1050 Gly Pro Val Gly Pro Ala Gly Lys Ser Gly Asp Arg Gly Glu Thr 1055 1060 1065 Gly Pro Ala Gly Pro Ala Gly Pro Ile Gly Pro Val Gly Ala Arg 1070 1075 1080 Gly Pro Ala Gly Pro Gln Gly Pro Arg Gly Asp Lys Gly Glu Thr 1085 1090 1095 Gly Glu Gln Gly Asp Arg Gly Ile Lys Gly His Arg Gly Phe Ser 1100 1105 1110 Gly Leu Gln Gly Pro Pro Gly Pro Pro Gly Ser Pro Gly Glu Gln 1115 1120 1125 Gly Pro Ser Gly Ala Ser Gly Pro Ala Gly Pro Arg Gly Pro Pro 1130 1135 1140 Gly Ser Ala Gly Ser Pro Gly Lys Asp Gly Leu Asn Gly Leu Pro 1145 1150 1155 Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly Arg Thr Gly Asp Ala 1160 1165 1170 Gly Pro Ala Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro 1175 1180 1185 Gly Pro Pro Ser Gly Gly Tyr Asp Leu Ser Phe Leu Pro Gln Pro 1190 1195 1200 Pro Gln Glu Lys Ala His Asp Gly Gly Arg Tyr Tyr Arg Ala Asp 1205 1210 1215 Asp Ala Asn Val Val Arg Asp Arg Asp Leu Glu Val Asp Thr Thr 1220 1225 1230 Leu Lys Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro Glu 1235 1240 1245 Gly Ser Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met 1250 1255 1260 Cys His Ser Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn 1265 1270 1275 Gln Gly Cys Asn Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu 1280 1285 1290 Thr Gly Glu Thr Cys Val Tyr Pro Thr Gln Pro Ser Val Ala Gln 1295 1300 1305 Lys Asn Trp Tyr Ile Ser Lys Asn Pro Lys Glu Lys Arg His Val 1310 1315 1320 Trp Tyr Gly Glu Ser Met Thr Gly Gly Phe Gln Phe Glu Tyr Gly 1325 1330 1335 Gly Gln Gly Ser Asp Pro Ala Asp Val Ala Ile Gln Leu Thr Phe 1340 1345 1350 Leu Arg Leu Met Ser Thr Glu Ala Ser Gln Asn Ile Thr Tyr His 1355 1360 1365 Cys Lys Asn Ser Val Ala Tyr Met Asp Gln Gln Thr Gly Asn Leu 1370 1375 1380 Lys Lys Ala Leu Leu Leu Gln Gly Ser Asn Glu Ile Glu Ile Arg 1385 1390 1395 Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser Val Thr Tyr Asp Gly 1400 1405 1410 Cys Thr Ser His Thr Gly Ala Trp Gly Lys Thr Val Ile Glu Tyr 1415 1420 1425 Lys Thr Thr Lys Thr Ser Arg Leu Pro Ile Ile Asp Val Ala Pro 1430 1435 1440 Leu Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe Asp Val Gly 1445 1450 1455 Pro Ala Cys Phe Leu 1460 <210> 2 <211> 1364 <212> PRT <213> Bos taurus <220> <221> MISC_FEATURE <222> (1)..(1364) <223> collagen alpha-2(I) chain precursor <400> 2 Met Leu Ser Phe Val Asp Thr Arg Thr Leu Leu Leu Leu Ala Val Thr 1 5 10 15 Ser Cys Leu Ala Thr Cys Gln Ser Leu Gln Glu Ala Thr Ala Arg Lys 20 25 30 Gly Pro Ser Gly Asp Arg Gly Pro Arg Gly Glu Arg Gly Pro Pro Gly 35 40 45 Pro Pro Gly Arg Asp Gly Asp Asp Gly Ile Pro Gly Pro Pro Gly Pro 50 55 60 Pro Gly Pro Pro Gly Pro Pro Gly Leu Gly Gly Asn Phe Ala Ala Gln 65 70 75 80 Phe Asp Ala Lys Gly Gly Gly Pro Gly Pro Met Gly Leu Met Gly Pro 85 90 95 Arg Gly Pro Pro Gly Ala Ser Gly Ala Pro Gly Pro Gln Gly Phe Gln 100 105 110 Gly Pro Pro Gly Glu Pro Gly Glu Pro Gly Gln Thr Gly Pro Ala Gly 115 120 125 Ala Arg Gly Pro Pro Gly Pro Pro Gly Lys Ala Gly Glu Asp Gly His 130 135 140 Pro Gly Lys Pro Gly Arg Pro Gly Glu Arg Gly Val Val Gly Pro Gln 145 150 155 160 Gly Ala Arg Gly Phe Pro Gly Thr Pro Gly Leu Pro Gly Phe Lys Gly 165 170 175 Ile Arg Gly His Asn Gly Leu Asp Gly Leu Lys Gly Gln Pro Gly Ala 180 185 190 Pro Gly Val Lys Gly Glu Pro Gly Ala Pro Gly Glu Asn Gly Thr Pro 195 200 205 Gly Gln Thr Gly Ala Arg Gly Leu Pro Gly Glu Arg Gly Arg Val Gly 210 215 220 Ala Pro Gly Pro Ala Gly Ala Arg Gly Ser Asp Gly Ser Val Gly Pro 225 230 235 240 Val Gly Pro Ala Gly Pro Ile Gly Ser Ala Gly Pro Pro Gly Phe Pro 245 250 255 Gly Ala Pro Gly Pro Lys Gly Glu Leu Gly Pro Val Gly Asn Pro Gly 260 265 270 Pro Ala Gly Pro Ala Gly Pro Arg Gly Glu Val Gly Leu Pro Gly Leu 275 280 285 Ser Gly Pro Val Gly Pro Pro Gly Asn Pro Gly Ala Asn Gly Leu Pro 290 295 300 Gly Ala Lys Gly Ala Ala Gly Leu Pro Gly Val Ala Gly Ala Pro Gly 305 310 315 320 Leu Pro Gly Pro Arg Gly Ile Pro Gly Pro Val Gly Ala Ala Gly Ala 325 330 335 Thr Gly Ala Arg Gly Leu Val Gly Glu Pro Gly Pro Ala Gly Ser Lys 340 345 350 Gly Glu Ser Gly Asn Lys Gly Glu Pro Gly Ala Val Gly Gln Pro Gly 355 360 365 Pro Pro Gly Pro Ser Gly Glu Glu Gly Lys Arg Gly Ser Thr Gly Glu 370 375 380 Ile Gly Pro Ala Gly Pro Pro Gly Pro Pro Gly Leu Arg Gly Asn Pro 385 390 395 400 Gly Ser Arg Gly Leu Pro Gly Ala Asp Gly Arg Ala Gly Val Met Gly 405 410 415 Pro Ala Gly Ser Arg Gly Ala Thr Gly Pro Ala Gly Val Arg Gly Pro 420 425 430 Asn Gly Asp Ser Gly Arg Pro Gly Glu Pro Gly Leu Met Gly Pro Arg 435 440 445 Gly Phe Pro Gly Ser Pro Gly Asn Ile Gly Pro Ala Gly Lys Glu Gly 450 455 460 Pro Val Gly Leu Pro Gly Ile Asp Gly Arg Pro Gly Pro Ile Gly Pro 465 470 475 480 Ala Gly Ala Arg Gly Glu Pro Gly Asn Ile Gly Phe Pro Gly Pro Lys 485 490 495 Gly Pro Ser Gly Asp Pro Gly Lys Ala Gly Glu Lys Gly His Ala Gly 500 505 510 Leu Ala Gly Ala Arg Gly Ala Pro Gly Pro Asp Gly Asn Asn Gly Ala 515 520 525 Gln Gly Pro Pro Gly Leu Gln Gly Val Gln Gly Gly Lys Gly Glu Gln 530 535 540 Gly Pro Ala Gly Pro Pro Gly Phe Gln Gly Leu Pro Gly Pro Ala Gly 545 550 555 560 Thr Ala Gly Glu Ala Gly Lys Pro Gly Glu Arg Gly Ile Pro Gly Glu 565 570 575 Phe Gly Leu Pro Gly Pro Ala Gly Ala Arg Gly Glu Arg Gly Pro Pro 580 585 590 Gly Glu Ser Gly Ala Ala Gly Pro Thr Gly Pro Ile Gly Ser Arg Gly 595 600 605 Pro Ser Gly Pro Pro Gly Pro Asp Gly Asn Lys Gly Glu Pro Gly Val 610 615 620 Val Gly Ala Pro Gly Thr Ala Gly Pro Ser Gly Pro Ser Gly Leu Pro 625 630 635 640 Gly Glu Arg Gly Ala Ala Gly Ile Pro Gly Gly Lys Gly Glu Lys Gly 645 650 655 Glu Thr Gly Leu Arg Gly Asp Ile Gly Ser Pro Gly Arg Asp Gly Ala 660 665 670 Arg Gly Ala Pro Gly Ala Ile Gly Ala Pro Gly Pro Ala Gly Ala Asn 675 680 685 Gly Asp Arg Gly Glu Ala Gly Pro Ala Gly Pro Ala Gly Pro Ala Gly 690 695 700 Pro Arg Gly Ser Pro Gly Glu Arg Gly Glu Val Gly Pro Ala Gly Pro 705 710 715 720 Asn Gly Phe Ala Gly Pro Ala Gly Ala Ala Gly Gln Pro Gly Ala Lys 725 730 735 Gly Glu Arg Gly Thr Lys Gly Pro Lys Gly Glu Asn Gly Pro Val Gly 740 745 750 Pro Thr Gly Pro Val Gly Ala Ala Gly Pro Ser Gly Pro Asn Gly Pro 755 760 765 Pro Gly Pro Ala Gly Ser Arg Gly Asp Gly Gly Pro Pro Gly Ala Thr 770 775 780 Gly Phe Pro Gly Ala Ala Gly Arg Thr Gly Pro Pro Gly Pro Ser Gly 785 790 795 800 Ile Ser Gly Pro Pro Gly Pro Pro Gly Pro Ala Gly Lys Glu Gly Leu 805 810 815 Arg Gly Pro Arg Gly Asp Gln Gly Pro Val Gly Arg Ser Gly Glu Thr 820 825 830 Gly Ala Ser Gly Pro Pro Gly Phe Val Gly Glu Lys Gly Pro Ser Gly 835 840 845 Glu Pro Gly Thr Ala Gly Pro Pro Gly Thr Pro Gly Pro Gln Gly Leu 850 855 860 Leu Gly Ala Pro Gly Phe Leu Gly Leu Pro Gly Ser Arg Gly Glu Arg 865 870 875 880 Gly Leu Pro Gly Val Ala Gly Ser Val Gly Glu Pro Gly Pro Leu Gly 885 890 895 Ile Ala Gly Pro Pro Gly Ala Arg Gly Pro Pro Gly Asn Val Gly Asn 900 905 910 Pro Gly Val Asn Gly Ala Pro Gly Glu Ala Gly Arg Asp Gly Asn Pro 915 920 925 Gly Asn Asp Gly Pro Pro Gly Arg Asp Gly Gln Pro Gly His Lys Gly 930 935 940 Glu Arg Gly Tyr Pro Gly Asn Ala Gly Pro Val Gly Ala Ala Gly Ala 945 950 955 960 Pro Gly Pro Gln Gly Pro Val Gly Pro Val Gly Lys His Gly Asn Arg 965 970 975 Gly Glu Pro Gly Pro Ala Gly Ala Val Gly Pro Ala Gly Ala Val Gly 980 985 990 Pro Arg Gly Pro Ser Gly Pro Gln Gly Ile Arg Gly Asp Lys Gly Glu 995 1000 1005 Pro Gly Asp Lys Gly Pro Arg Gly Leu Pro Gly Leu Lys Gly His 1010 1015 1020 Asn Gly Leu Gln Gly Leu Pro Gly Leu Ala Gly His His Gly Asp 1025 1030 1035 Gln Gly Ala Pro Gly Ala Val Gly Pro Ala Gly Pro Arg Gly Pro 1040 1045 1050 Ala Gly Pro Ser Gly Pro Ala Gly Lys Asp Gly Arg Ile Gly Gln 1055 1060 1065 Pro Gly Ala Val Gly Pro Ala Gly Ile Arg Gly Ser Gln Gly Ser 1070 1075 1080 Gln Gly Pro Ala Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro 1085 1090 1095 Pro Gly Pro Ser Gly Gly Gly Tyr Glu Phe Gly Phe Asp Gly Asp 1100 1105 1110 Phe Tyr Arg Ala Asp Gln Pro Arg Ser Pro Thr Ser Leu Arg Pro 1115 1120 1125 Lys Asp Tyr Glu Val Asp Ala Thr Leu Lys Ser Leu Asn Asn Gln 1130 1135 1140 Ile Glu Thr Leu Leu Thr Pro Glu Gly Ser Arg Lys Asn Pro Ala 1145 1150 1155 Arg Thr Cys Arg Asp Leu Arg Leu Ser His Pro Glu Trp Ser Ser 1160 1165 1170 Gly Tyr Tyr Trp Ile Asp Pro Asn Gln Gly Cys Thr Met Asp Ala 1175 1180 1185 Ile Lys Val Tyr Cys Asp Phe Ser Thr Gly Glu Thr Cys Ile Arg 1190 1195 1200 Ala Gln Pro Glu Asp Ile Pro Val Lys Asn Trp Tyr Arg Asn Ser 1205 1210 1215 Lys Ala Lys Lys His Val Trp Val Gly Glu Thr Ile Asn Gly Gly 1220 1225 1230 Thr Gln Phe Glu Tyr Asn Val Glu Gly Val Thr Thr Lys Glu Met 1235 1240 1245 Ala Thr Gln Leu Ala Phe Met Arg Leu Leu Ala Asn His Ala Ser 1250 1255 1260 Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Ile Ala Tyr Met Asp 1265 1270 1275 Glu Glu Thr Gly Asn Leu Lys Lys Ala Val Ile Leu Gln Gly Ser 1280 1285 1290 Asn Asp Val Glu Leu Val Ala Glu Gly Asn Ser Arg Phe Thr Tyr 1295 1300 1305 Thr Val Leu Val Asp Gly Cys Ser Lys Lys Thr Asn Glu Trp Gln 1310 1315 1320 Lys Thr Ile Ile Glu Tyr Lys Thr Asn Lys Pro Ser Arg Leu Pro 1325 1330 1335 Ile Leu Asp Ile Ala Pro Leu Asp Ile Gly Gly Ala Asp Gln Glu 1340 1345 1350 Ile Arg Leu Asn Ile Gly Pro Val Cys Phe Lys 1355 1360 <210> 3 <211> 1466 <212> PRT <213> Bos taurus <220> <221> MISC_FEATURE <222> (1)..(1466) <223> collagen alpha-1(III) chain precursor <400> 3 Met Met Ser Phe Val Gln Lys Gly Thr Trp Leu Leu Phe Ala Leu Leu 1 5 10 15 His Pro Thr Val Ile Leu Ala Gln Gln Glu Ala Val Asp Gly Gly Cys 20 25 30 Ser His Leu Gly Gln Ser Tyr Ala Asp Arg Asp Val Trp Lys Pro Glu 35 40 45 Pro Cys Gln Ile Cys Val Cys Asp Ser Gly Ser Val Leu Cys Asp Asp 50 55 60 Ile Ile Cys Asp Asp Gln Glu Leu Asp Cys Pro Asn Pro Glu Ile Pro 65 70 75 80 Phe Gly Glu Cys Cys Ala Val Cys Pro Gln Pro Pro Thr Ala Pro Thr 85 90 95 Arg Pro Pro Asn Gly Gln Gly Pro Gln Gly Pro Lys Gly Asp Pro Gly 100 105 110 Pro Pro Gly Ile Pro Gly Arg Asn Gly Asp Pro Gly Pro Pro Gly Ser 115 120 125 Pro Gly Ser Pro Gly Ser Pro Gly Pro Pro Gly Ile Cys Glu Ser Cys 130 135 140 Pro Thr Gly Gly Gln Asn Tyr Ser Pro Gln Tyr Glu Ala Tyr Asp Val 145 150 155 160 Lys Ser Gly Val Ala Gly Gly Gly Ile Ala Gly Tyr Pro Gly Pro Ala 165 170 175 Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Thr Ser Gly His Pro Gly 180 185 190 Ala Pro Gly Ala Pro Gly Tyr Gln Gly Pro Pro Gly Glu Pro Gly Gln 195 200 205 Ala Gly Pro Ala Gly Pro Pro Gly Pro Pro Gly Ala Ile Gly Pro Ser 210 215 220 Gly Pro Ala Gly Lys Asp Gly Glu Ser Gly Arg Pro Gly Arg Pro Gly 225 230 235 240 Glu Arg Gly Phe Pro Gly Pro Pro Gly Met Lys Gly Pro Ala Gly Met 245 250 255 Pro Gly Phe Pro Gly Met Lys Gly His Arg Gly Phe Asp Gly Arg Asn 260 265 270 Gly Glu Lys Gly Glu Thr Gly Ala Pro Gly Leu Lys Gly Glu Asn Gly 275 280 285 Val Pro Gly Glu Asn Gly Ala Pro Gly Pro Met Gly Pro Arg Gly Ala 290 295 300 Pro Gly Glu Arg Gly Arg Pro Gly Leu Pro Gly Ala Ala Gly Ala Arg 305 310 315 320 Gly Asn Asp Gly Ala Arg Gly Ser Asp Gly Gln Pro Gly Pro Pro Gly 325 330 335 Pro Pro Gly Thr Ala Gly Phe Pro Gly Ser Pro Gly Ala Lys Gly Glu 340 345 350 Val Gly Pro Ala Gly Ser Pro Gly Ser Ser Gly Ala Pro Gly Gln Arg 355 360 365 Gly Glu Pro Gly Pro Gln Gly His Ala Gly Ala Pro Gly Pro Pro Gly 370 375 380 Pro Pro Gly Ser Asn Gly Ser Pro Gly Gly Lys Gly Glu Met Gly Pro 385 390 395 400 Ala Gly Ile Pro Gly Ala Pro Gly Leu Ile Gly Ala Arg Gly Pro Pro 405 410 415 Gly Pro Pro Gly Thr Asn Gly Val Pro Gly Gln Arg Gly Ala Ala Gly 420 425 430 Glu Pro Gly Lys Asn Gly Ala Lys Gly Asp Pro Gly Pro Arg Gly Glu 435 440 445 Arg Gly Glu Ala Gly Ser Pro Gly Ile Ala Gly Pro Lys Gly Glu Asp 450 455 460 Gly Lys Asp Gly Ser Pro Gly Glu Pro Gly Ala Asn Gly Leu Pro Gly 465 470 475 480 Ala Ala Gly Glu Arg Gly Val Pro Gly Phe Arg Gly Pro Ala Gly Ala 485 490 495 Asn Gly Leu Pro Gly Glu Lys Gly Pro Pro Gly Asp Arg Gly Gly Pro 500 505 510 Gly Pro Ala Gly Pro Arg Gly Val Ala Gly Glu Pro Gly Arg Asp Gly 515 520 525 Leu Pro Gly Gly Pro Gly Leu Arg Gly Ile Pro Gly Ser Pro Gly Gly 530 535 540 Pro Gly Ser Asp Gly Lys Pro Gly Pro Pro Gly Ser Gln Gly Glu Thr 545 550 555 560 Gly Arg Pro Gly Pro Pro Gly Ser Pro Gly Pro Arg Gly Gln Pro Gly 565 570 575 Val Met Gly Phe Pro Gly Pro Lys Gly Asn Asp Gly Ala Pro Gly Lys 580 585 590 Asn Gly Glu Arg Gly Gly Pro Gly Gly Pro Gly Pro Gln Gly Pro Ala 595 600 605 Gly Lys Asn Gly Glu Thr Gly Pro Gln Gly Pro Pro Gly Pro Thr Gly 610 615 620 Pro Ser Gly Asp Lys Gly Asp Thr Gly Pro Pro Gly Pro Gln Gly Leu 625 630 635 640 Gln Gly Leu Pro Gly Thr Ser Gly Pro Pro Gly Glu Asn Gly Lys Pro 645 650 655 Gly Glu Pro Gly Pro Lys Gly Glu Ala Gly Ala Pro Gly Ile Pro Gly 660 665 670 Gly Lys Gly Asp Ser Gly Ala Pro Gly Glu Arg Gly Pro Pro Gly Ala 675 680 685 Gly Gly Pro Pro Gly Pro Arg Gly Gly Ala Gly Pro Pro Gly Pro Glu 690 695 700 Gly Gly Lys Gly Ala Ala Gly Pro Pro Gly Pro Pro Gly Ser Ala Gly 705 710 715 720 Thr Pro Gly Leu Gln Gly Met Pro Gly Glu Arg Gly Gly Pro Gly Gly 725 730 735 Pro Gly Pro Lys Gly Asp Lys Gly Glu Pro Gly Ser Ser Gly Val Asp 740 745 750 Gly Ala Pro Gly Lys Asp Gly Pro Arg Gly Pro Thr Gly Pro Ile Gly 755 760 765 Pro Pro Gly Pro Ala Gly Gln Pro Gly Asp Lys Gly Glu Ser Gly Ala 770 775 780 Pro Gly Val Pro Gly Ile Ala Gly Pro Arg Gly Gly Pro Gly Glu Arg 785 790 795 800 Gly Glu Gln Gly Pro Pro Gly Pro Ala Gly Phe Pro Gly Ala Pro Gly 805 810 815 Gln Asn Gly Glu Pro Gly Ala Lys Gly Glu Arg Gly Ala Pro Gly Glu 820 825 830 Lys Gly Glu Gly Gly Pro Pro Gly Ala Ala Gly Pro Ala Gly Gly Ser 835 840 845 Gly Pro Ala Gly Pro Pro Gly Pro Gln Gly Val Lys Gly Glu Arg Gly 850 855 860 Ser Pro Gly Gly Pro Gly Ala Ala Gly Phe Pro Gly Gly Arg Gly Pro 865 870 875 880 Pro Gly Pro Pro Gly Ser Asn Gly Asn Pro Gly Pro Pro Gly Ser Ser 885 890 895 Gly Ala Pro Gly Lys Asp Gly Pro Pro Gly Pro Pro Gly Ser Asn Gly 900 905 910 Ala Pro Gly Ser Pro Gly Ile Ser Gly Pro Lys Gly Asp Ser Gly Pro 915 920 925 Pro Gly Glu Arg Gly Ala Pro Gly Pro Gln Gly Pro Pro Gly Ala Pro 930 935 940 Gly Pro Leu Gly Ile Ala Gly Leu Thr Gly Ala Arg Gly Leu Ala Gly 945 950 955 960 Pro Pro Gly Met Pro Gly Ala Arg Gly Ser Pro Gly Pro Gln Gly Ile 965 970 975 Lys Gly Glu Asn Gly Lys Pro Gly Pro Ser Gly Gln Asn Gly Glu Arg 980 985 990 Gly Pro Pro Gly Pro Gln Gly Leu Pro Gly Leu Ala Gly Thr Ala Gly 995 1000 1005 Glu Pro Gly Arg Asp Gly Asn Pro Gly Ser Asp Gly Leu Pro Gly 1010 1015 1020 Arg Asp Gly Ala Pro Gly Ala Lys Gly Asp Arg Gly Glu Asn Gly 1025 1030 1035 Ser Pro Gly Ala Pro Gly Ala Pro Gly His Pro Gly Pro Pro Gly 1040 1045 1050 Pro Val Gly Pro Ala Gly Lys Ser Gly Asp Arg Gly Glu Thr Gly 1055 1060 1065 Pro Ala Gly Pro Ser Gly Ala Pro Gly Pro Ala Gly Ser Arg Gly 1070 1075 1080 Pro Pro Gly Pro Gln Gly Pro Arg Gly Asp Lys Gly Glu Thr Gly 1085 1090 1095 Glu Arg Gly Ala Met Gly Ile Lys Gly His Arg Gly Phe Pro Gly 1100 1105 1110 Asn Pro Gly Ala Pro Gly Ser Pro Gly Pro Ala Gly His Gln Gly 1115 1120 1125 Ala Val Gly Ser Pro Gly Pro Ala Gly Pro Arg Gly Pro Val Gly 1130 1135 1140 Pro Ser Gly Pro Pro Gly Lys Asp Gly Ala Ser Gly His Pro Gly 1145 1150 1155 Pro Ile Gly Pro Pro Gly Pro Arg Gly Asn Arg Gly Glu Arg Gly 1160 1165 1170 Ser Glu Gly Ser Pro Gly His Pro Gly Gln Pro Gly Pro Pro Gly 1175 1180 1185 Pro Pro Gly Ala Pro Gly Pro Cys Cys Gly Ala Gly Gly Val Ala 1190 1195 1200 Ala Ile Ala Gly Val Gly Ala Glu Lys Ala Gly Gly Phe Ala Pro 1205 1210 1215 Tyr Tyr Gly Asp Glu Pro Ile Asp Phe Lys Ile Asn Thr Asp Glu 1220 1225 1230 Ile Met Thr Ser Leu Lys Ser Val Asn Gly Gln Ile Glu Ser Leu 1235 1240 1245 Ile Ser Pro Asp Gly Ser Arg Lys Asn Pro Ala Arg Asn Cys Arg 1250 1255 1260 Asp Leu Lys Phe Cys His Pro Glu Leu Gln Ser Gly Glu Tyr Trp 1265 1270 1275 Val Asp Pro Asn Gln Gly Cys Lys Leu Asp Ala Ile Lys Val Tyr 1280 1285 1290 Cys Asn Met Glu Thr Gly Glu Thr Cys Ile Ser Ala Ser Pro Leu 1295 1300 1305 Thr Ile Pro Gln Lys Asn Trp Trp Thr Asp Ser Gly Ala Glu Lys 1310 1315 1320 Lys His Val Trp Phe Gly Glu Ser Met Glu Gly Gly Phe Gln Phe 1325 1330 1335 Ser Tyr Gly Asn Pro Glu Leu Pro Glu Asp Val Leu Asp Val Gln 1340 1345 1350 Leu Ala Phe Leu Arg Leu Leu Ser Ser Arg Ala Ser Gln Asn Ile 1355 1360 1365 Thr Tyr His Cys Lys Asn Ser Ile Ala Tyr Met Asp His Ala Ser 1370 1375 1380 Gly Asn Val Lys Lys Ala Leu Lys Leu Met Gly Ser Asn Glu Gly 1385 1390 1395 Glu Phe Lys Ala Glu Gly Asn Ser Lys Phe Thr Tyr Thr Val Leu 1400 1405 1410 Glu Asp Gly Cys Thr Lys His Thr Gly Glu Trp Gly Lys Thr Val 1415 1420 1425 Phe Gln Tyr Gln Thr Arg Lys Ala Val Arg Leu Pro Ile Val Asp 1430 1435 1440 Ile Ala Pro Tyr Asp Ile Gly Gly Pro Asp Gln Glu Phe Gly Ala 1445 1450 1455 Asp Ile Gly Pro Val Cys Phe Leu 1460 1465

Claims

1. A method for preparing a composite material comprising a biomanufacturing material and a secondary component, wherein the biomanufacturing material is dispensed, encapsulated, incorporated, deposited, or otherwise introduced into or onto at least one porous, permeable, or absorbent secondary component to form the composite material, wherein the at least one porous, permeable, or absorbent secondary component comprises at least one of an upper surface, a lower surface, an inner surface, and an outer surface. The biomaterial is produced by a method comprising: The aqueous solution or suspension of non-human collagen molecules is fibrillated into multiple non-human collagen fibrils, wherein the fibrillation includes increasing the pH of the solution or suspension and / or adjusting the salt concentration of the solution or suspension. Crosslinking the plurality of non-human collagen fibrils to form a non-human collagen fibril network comprising the plurality of crosslinked non-human collagen fibrils, wherein crosslinking includes contacting the plurality of non-human collagen fibrils with at least one crosslinking agent; Dehydrate the plurality of cross-linked non-human collagen fibrils such that the plurality of cross-linked collagen fibrils contain water ranging from 10% by weight to 40% by weight of the biomaterial; The plurality of cross-linked non-human collagen fibrils are lubricated such that the plurality of cross-linked collagen fibrils contain a lubricant ranging from 1% to 40% by weight of the biomanufacturing material; and Less than 10% by weight of the collagen fibrils are in the form of collagen fibrils with a diameter of at least 5 µm and / or in the form of fibrils arranged with a length of 100 μm or more.

2. The method according to claim 1, wherein the non-human collagen molecule comprises recombinant non-human collagen molecule.

3. The method according to claim 1, further comprising: The plurality of cross-linked collagen fibrils are dehydrated such that the composite material contains water ranging from 10% to 25% by weight of the biomanufacturing material.

4. The method of claim 1, wherein dispensing, encapsulating, depositing, or otherwise introducing the biomanufacturing material into or onto the at least one porous, permeable, or absorbent secondary component comprises at least one of the following: layering, stacking, coating, or sandwiching the at least one porous, permeable, or absorbent secondary component with the plurality of cross-linked non-human collagen fibrils.

5. The method according to claim 2, wherein the recombinant nonhuman collagen molecule does not contain 3-hydroxyproline.

6. The method of claim 1, wherein dispensing, encapsulating, depositing, or otherwise introducing the biomanufacturing material into or onto the at least one porous, permeable, or absorbent secondary component comprises at least one of the following: filtration, impregnation, spraying, or coating.

7. The method of claim 1, wherein the bio-manufacturing material is incorporated only into or on one of the upper, lower, inner, or outer surfaces of the at least one porous, permeable, or absorbent secondary component.

8. The method of claim 1, wherein the biomanufacturing material is incorporated into or on the upper and lower surfaces of the at least one porous, permeable or absorbent secondary component, or wherein the plurality of cross-linked non-human collagen fibrils are incorporated into or on the inner and outer surfaces of the at least one porous, permeable or absorbent secondary component.

9. The method of claim 1, wherein the at least one porous, permeable or absorbent secondary component comprises a plurality of layers, and wherein the biomanufacturing material is disposed between at least two layers of the at least one porous, permeable or absorbent secondary component.

10. The method of claim 1, wherein the secondary component comprises fibers, woven fabrics, or unwoven fabrics.

11. The method of claim 1, wherein dispensing, encapsulating, depositing, or otherwise incorporating the biomanufacturing material into or onto the at least one porous, permeable, or absorbent secondary component comprises embedding or mixing the at least one porous, permeable, or absorbent secondary component into or together with the biomanufacturing material.

12. A composite material comprising: A biomanufactured material comprising a non-human collagen fibrillary network and a crosslinking agent comprising at least 1% by weight of the material or by weight of collagen or collagen fibrillary in the material, wherein the collagen fibrillary is formed by fibrillating collagen molecules, the fibrillation comprising increasing the pH of a collagen solution or suspension and / or adjusting the salt concentration of the collagen solution or suspension. The biomanufacturing material is dispensed, encapsulated, incorporated, deposited, or otherwise incorporated into or onto at least one porous, permeable, or absorbable secondary component. The at least one porous, permeable, or absorbent secondary component comprises at least one of an upper surface, a lower surface, an inner surface, and an outer surface. The biomaterial contains no more than 40% by weight of water and at least 1% by weight of lubricant, and Less than 10% by weight of the collagen fibrils are in the form of collagen fibrils with a diameter of at least 5 µm and / or in the form of fibrils arranged with a length of 100 μm or greater.

13. The composite material according to claim 12, wherein the lubricant is a grease, bio-oil, mineral oil, synthetic oil, polymer, or organofunctional siloxane or any combination thereof.

14. The composite material according to claim 13, wherein the bio-oil is cod oil or sulfonated oil, or the polymer is a resin.

15. The composite material of claim 12, wherein the crosslinked non-human collagen fibrils comprise crosslinked recombinant non-human collagen fibrils.

16. The composite material of claim 15, further comprising water ranging from 10% to 25% by weight of the biomaterial.

17. The composite material according to claim 15, wherein the recombinant non-human collagen fibrils do not contain 3-hydroxyproline.

18. The composite material of claim 12, wherein the biomanufacturing material is distributed within the at least one porous, permeable or absorbable secondary component.

19. The composite material of claim 12, wherein the biomanufacturing material is incorporated into or on at least one of the upper, lower, inner, and outer surfaces of the at least one porous, permeable, or absorbent secondary component.

20. The composite material of claim 12, wherein the biomanufacturing material is incorporated into or on the upper and lower surfaces of the at least one porous, permeable or absorbent secondary component, or the inner and outer surfaces of the at least one porous, permeable or absorbent secondary component.

21. The composite material according to claim 12, wherein the at least one porous, permeable or absorbent secondary component is paper or fabric.

22. The composite material according to claim 12, wherein the at least one porous, permeable or absorbent secondary component is an unwoven fiber material or a woven fiber material.

23. The composite material of claim 12, wherein the at least one porous, permeable or absorbent secondary component comprises at least one of particles, fibers, filaments, sieves, woven materials or unwoven materials.

24. The composite material of claim 23, wherein the particles are beads.

25. The composite material of claim 12, wherein the at least one porous, permeable or absorbable secondary component is encapsulated in the biomanufacturing material.

26. The composite material of claim 12, wherein the biomanufacturing material is incorporated into or onto the at least one porous, permeable or absorbent secondary component using an adhesive.

27. The composite material of claim 12, wherein the biomanufacturing material is laminated onto the at least one porous, permeable or absorbable secondary component.

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