Composite biofabricated material

Abstract

The invention is directed to a composite material comprising a biofabricated material and a secondary component. The secondary component may 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 crosslinked collagen fibrilsand provides strength, elasticity and an aesthetic appearance to the composite material.

Claims

1. A composite material, comprising: a secondary component substrate; a collagen-based material coated on the secondary component substrate, the collagen-based material comprising: a network of recombinant non-human collagen fibrils, 5% to 40% water by weight of the collagen-based material, and 1% to 60% of a lubricant by weight of the collagen-based material; and a leather dye, wherein 1% to 40% by weight of the recombinant non-human collagen fibrils within the network are assembled in the form of recombinant collagen fibers comprising a plurality of recombinant non-human collagen fibrils, wherein each of the recombinant collagen fibers has a diameter in a range of 1 μm to 10 μm.

2. The composite material of claim 1, wherein the secondary component substrate has a top surface and a bottom surface, or an inner surface and an outer surface.

3. The composite material of claim 2, wherein the collagen-based material is adhered to, laminated to, or attached to one of the top surface, the bottom surface, the inner surface, or the outer surface.

4. The composite material of claim 2, wherein the collagen-based material is incorporated into both the top surface and the bottom surface, or wherein the collagen-based material is incorporated into both the inner surface and the outer surface.

5. The composite material of claim 1, wherein the collagen-based material-comprises an amount up to 1% of a protein by weight of the collagen-based material, wherein the protein is at least one of actin, keratin, elastin, fibrin, albumin, globulin, mucin, mucinoids, a noncollagen structural protein, or a noncollagen nonstructural protein.

6. The composite material of claim 1, wherein the non-human collagen fibrils have a substantially unimodal distribution of diameters, further wherein 70% to 99% of the diameters are distributed around a single mode.

7. The composite material of claim 1, wherein the lubricant is selected from the group consisting of a fat, a biological lubricant, a mineral oil, a synthetic oil, a sulfonated oil, a polymer, and an organofunctional siloxane.

8. The composite material of claim 1, wherein the composite material further comprises a surface coating or a surface finish, and wherein the surface coating or the surface finish is distributed uniformly on a surface of the composite material.

9. The composite material of claim 1, wherein the collagen-based material further comprises a stain, a resin, a polymer, a pigment, or a paint, and wherein the stain, the resin, the pigment, or the paint is distributed uniformly throughout the collagen-based material.

10. The composite material of claim 1, wherein the composite material further comprises at least one filler distributed uniformly throughout the composite material.

11. The composite material of claim 1, wherein the recombinant non-human collagen fibrils comprise substantially no 3-hydroxyproline.

12. The composite material of claim 1, wherein the secondary component substrate comprises at least one of a nonwoven material or a woven material.

13. The composite material of claim 1, wherein the composite material has a collagen fibril density in a range of 70 mg/cc to 1,000 mg/cc.

14. A composite material, comprising: a secondary component substrate, the secondary component substrate comprising a first surface and a second surface; a collagen-based material coated on at least one of the first surface of the secondary component substrate or the second surface of the secondary component substrate, the collagen-based material comprising: a network of recombinant non-human collagen fibrils, 5% to 40% water by weight of the collagen-based material, and 1% to 60% of a lubricant by weight of the collagen-based material; and a leather dye, wherein 1% to 40% by weight of the recombinant non-human collagen fibrils within the network are assembled in the form of recombinant collagen fibers comprising a plurality of recombinant non-human collagen fibrils, wherein each of the recombinant collagen fibers has a diameter in a range of 1 μm to 10 μm.

15. The composite material of claim 14, wherein the secondary component substrate comprises at least one of a nonwoven material or a woven material.

16. The composite material of claim 14, wherein the at least one collagen-based material is incorporated into both the first surface of the secondary component substrate and the second surface of the secondary component.

17. The composite material of claim 14, wherein the composite material has an elastic modulus in a range of 100 kPa to 1,000 MPa.

18. The composite material of claim 14, wherein the composite material has a tensile strength in a range of 1 kPa to 100 MPa.

19. The composite material of claim 14, wherein the composite material has a tear strength in a range of 1 N to 500 N.

20. The composite material of claim 14, wherein the composite material has a softness in a range of 2 mm to 12 mm.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a drawing showing the composition of collagen in a hierarchical fashion. Reference character (1) shows each triple helical collagen monomer and how they are assembled with respect to neighboring collagen monomers; (2) shows assembled collagen that makes up banded collagen fibrils; (3) shows the collagen fibrils at larger scale; (4) shows collagen fibrils aligned into fibers; and (5) shows bundles of collagen fibers.

(2) FIG. 2A is a picture showing the composition of buffalo hide. The top grain layer and the corium layer underneath are shown and the relative degrees of higher order organization from collagen fibrils to collagen fiber bundles are indicated. The top grain layer is mostly composed of fine collagen fibrils while the corium layer is mostly composed of coarser collagen fibers and fiber bundles.

(3) FIGS. 2B and 2C compare the textures and grains of the outer and inner surfaces of leather depicting fine grain on one side and coarser corium on the other.

(4) FIG. 3A is a scanning electron micrograph of the fibrillated collagen hydrogel showing a network of fine collagen fibrils.

(5) FIG. 3B is a scanning electron micrograph of bovine corium showing coarser fiber bundles.

(6) FIG. 4 is a transmission electron micrograph of a fibrillated collagen network or hydrogel showing fibril banding.

DETAILED DESCRIPTION OF THE INVENTION

(7) “Biofabricated material” or “biofabricated leather” as used herein is a material produced from collagen or a collagen-like protein. It can be produced from non-human collagens such as bovine, buffalo, ox, dear, sheep, goat, or pig collagen, which may be isolated from a natural source like animal hide, by in vitro culture of mammalian or animal cells, recombinantly produced or chemically synthesized. It is not a conventional material or leather which is produced from animal skins. Methods for producing this biofabricated material or biofabricated leather are disclosed herein and usually involve fibrillating an isolated or purified solution or suspension of collagen molecules to produce collagen fibrils, crosslinking the fibrils, dehydrating the fibrils and lubricating the fibrils.

(8) In contrast to natural leathers which exhibit heterogeneous internal collagen structures, a biofabricated material or biofabricated leather can exhibit a substantially uniform internal structure characterized by unbundled and randomly-oriented collagen fibrils throughout its volume.

(9) The resulting biofabricated material may be used in any way that natural leather is used and may be grossly similar in appearance and feel to real leather, while having compositional, functional or aesthetic features that differentiate it from ordinary leather. For example, unlike natural leather, a biofabricated leather need not contain potentially allergenic non-collagen proteins or components found in a natural leather, a biofabricated leather may exhibit a similar flexibility and strength in all directions (non-anisotropy) due to substantial non-alignment of its collagen fibrils, and aesthetically may have a smooth grain texture on both sides. A biofabricated leather can exhibit uniformity of properties including uniform thickness and consistency, uniform distribution of lubricants, crosslinkers and dyes, uniform non-anisotropic strength, stretch, flexibility and resistance to piping (or the tendency for natural leather to separate or split parallel to a plane of a sheet). By selecting the content of collagen and processing conditions, biofabricated leather can be “tuned” to a particular thickness, consistency, flexibility, softness, drape. surface texture or other functionality. Laminated, layered or composite products may comprise a biofabricated leather.

(10) A “composite” is a combination of a biofabricated material or biofabricated leather component and a secondary material. The secondary component may be incorporated into the biofabricated material; the biofabricated material may be at least partially incorporated into a secondary material, or coated on, layered on, or laminated to a secondary material. Examples of composites include a biofabricated material encapsulating a secondary material, a secondary material coated on one side with a biofabricated material, a secondary material coated on both external sides with a biofabricated material, and one or more layers of a secondary material laminated to one or more layers of a biofabricated material. This term encompasses all forms and combinations of a biofabricated material and one or more secondary materials.

(11) The term “collagen” refers to any one of the known collagen types, including collagen types I through XX, as well as to any other collagens, whether natural, synthetic, semi-synthetic, or recombinant. It includes all of the collagens, modified collagens and collagen-like proteins described herein. The two also encompasses procollagens and collagen-like proteins or collagenous proteins comprising the motif (Gly-X-Y)n where n is an integer. It encompasses molecules of collagen and collagen-like proteins, trimers of collagen molecules, fibrils of collagen, and fibers of collagen fibrils. It also refers to chemically, enzymatically or recombinantly-modified collagens or collagen-like molecules that can be fibrillated as well as fragments of collagen, collagen-like molecules and collagenous molecules capable of assembling into a nanofiber.

(12) In some embodiments, amino acid residues, such as lysine and proline, in a collagen or collagen-like protein may lack hydroxylation or may have a lesser or greater degree of hydroxylation than a corresponding natural or unmodified collagen or collagen-like protein. In other embodiments, amino acid residues in a collagen or collagen-like protein may lack glycosylation or may have a lesser or greater degree of glycosylation than a corresponding natural or unmodified collagen or collagen-like protein.

(13) The collagen in a collagen composition may homogenously contain a single type of collagen molecule, such as 100% bovine Type I collagen or 100% Type III bovine collagen, or may contain a mixture of different kinds of collagen molecules or collagen-like molecules, such as a mixture of bovine Type I and Type III molecules. Such mixtures may include >0%, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99 or <100% of the 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 may contain 33.3% of Type I collagen, 33.3% of Type II collagen, and 33.3% of Type III collagen, where the percentage of collagen is based on the total mass of collagen in the composition or on the molecular percentages of collagen molecules.

(14) “Collagen fibrils” are nanofibers composed of tropocollagen (triple helices of collagen molecules). Tropocollagens also include tropocollagen-like structures exhibiting triple helical structures. The collagen fibrils of the invention may have diameters ranging from 1 nm and 1 μm. For example, the collagen fibrils of the invention may have an average or individual fibril diameter ranging from 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 subranges. In some of the embodiments of the invention collagen fibrils will form networks, for example, as depicted by FIGS. 3 and 4. Collagen fibrils can associate into fibrils exhibiting a banded pattern as shown in FIG. 1 and these fibrils can associate into larger aggregates of fibrils. In some embodiments the collagen or collagen-like fibrils will have diameters and orientations similar to those in the top grain or surface layer of a bovine or other conventional leather. In other embodiments, the collagen fibrils may have diameters comprising the top grain and those of a corium layer of a conventional leather.

(15) A “collagen fiber” is composed of collagen fibrils that are tightly packed and exhibit a high degree of alignment in the direction of the fiber as shown in FIG. 1. It can vary in diameter from more than 1 μm to more than 10 μm, for example >1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 μm or more. Some embodiments of the network of collage fibrils of the invention do not contain substantial content of collagen fibers having diameters greater than 5 μm As shown in FIG. 2, the composition of the grain surface of a leather can differ from its more internal portions, such as the corium which contains coarser fiber bundles.

(16) “Fibrillation” refers to a process of producing collagen fibrils. It may be performed by raising the pH or by adjusting the salt concentration of a collagen solution or suspension. In forming the fibrillated collagen, the collagen may be incubated to form the fibrils for any appropriate length of time, including between 1 min and 24 hrs and all intermediate values.

(17) The fibrillated collagen described herein may generally be formed in any appropriate shape and/or thickness, including flat sheets, curved shapes/sheets, cylinders, threads, and complex shapes. These sheets and other forms may have virtually any linear dimensions including a thickness, width or height greater of 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 μm or more.

(18) The fibrillated collagen in a biofabricated leather may lack any or any substantial amount of higher order structure. In a preferred embodiment, the collagen fibrils in a biofabricated leather will be unbundled and not form the large collagen fibers found in animal skin and provide a strong and uniform non-anisotropic structure to the biofabricated leather.

(19) In other embodiments, some collagen fibrils can be bundled or aligned into higher order structures. Collagen fibrils in a biofabricated 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, wherein an orientation index of 0 describes collagen fibrils that lack alignment with other fibrils and an orientation index of 1.0 describes collagen fibrils that are completely aligned. This range includes all intermediate values and subranges. Those of skill in the art are familiar with the orientation index which 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).

(20) The methods disclosed herein make it possible to produce a biofabricated leather comprising collagen fibrils differing in diameter from those produced by an animal expressing the same type of collagen. The characteristics of natural collagens, such as fibril diameter and degree of crosslinking between fibrils are affected by genetic and environmental factors such as the species or breed of the animal and by the condition of the animal, for example the amount of fat, type of feed (e.g. grain, grass), and level of exercise.

(21) A biofabricated leather may be fibrillated and processed to contain collagen fibrils that resemble or mimic the properties of collagen fibrils produced by particular species or breeds of animals or by animals raised under particular conditions.

(22) Alternatively, fibrillation and processing conditions can be selected to provide collagen fibrils distinct from those found in nature, such as by decreasing or increasing the fibril diameter, degree of alignment, or degree of crosslinking compared to fibrils in natural leather.

(23) A crosslinked network of collagen, sometimes called a hydrogel, may be formed as the collagen is fibrillated, or it may form a network after fibrillation; in some variations, the process of fibrillating the collagen also forms gel-like network. Once formed, the fibrillated collagen network may be further stabilized by incorporating molecules with di-, tri-, or multifunctional reactive groups that include chromium, amines, carboxylic acids, sulfates, sulfites, sulfonates, aldehydes, hydrazides, sulfhydryls, diazarines, aryl-, azides, acrylates, epoxides, or phenols.

(24) The fibrillated collagen network may also be polymerized with other agents (e.g. polymers that are capable of polymerizing or other suitable fibers), which could be used to further stabilize the matrix and provide the desired end structure. Hydrogels based upon acrylamides, acrylic acids, and their salts may be prepared using inverse suspension polymerization. Hydrogels described herein may be prepared from polar monomers. The hydrogels used may be natural polymer hydrogels, synthetic polymer hydrogels, or a combination of the two. The hydrogels used may be obtained using graft polymerization, crosslinking polymerization, networks formed of water soluble polymers, radiation crosslinking, and so on. A small amount of crosslinking agent may be added to the hydrogel composition to enhance polymerization.

(25) Average or individual collagen fibril length may range from 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 (1 μm); 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 μm (1 mm) throughout the entire thickness of a biofabricated leather. These ranges include all intermediate values and subranges.

(26) Fibrils may align with other fibrils over 50, 100, 200, 300, 400, 500 μm or more of their lengths or may exhibit little or no alignment. In other embodiments, some collagen fibrils can be bundled or aligned into higher order structures.

(27) Collagen fibrils in a biofabricated 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, wherein an orientation index of 0 describes collagen fibrils that lack alignment with other fibrils and an orientation index of 1.0 describes collagen fibrils that are completely aligned. This range includes all intermediate values and subranges. Those of skill in the art are familiar with the orientation index which 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).

(28) Collagen fibril density of a biofabricated leather may 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.

(29) The collagen fibrils in a biofabricated leather may exhibit a unimodal, bimodal, trimiodal, or multimodal distribution, for example, a biofabricated leather may be composed of two different fibril preparations each having a different range of fibril diameters arranged around one of two different modes. Such mixtures may be selected to impart additive, synergistic or a balance of physical properties on a biofabricated leather conferred by fibrils having different diameters.

(30) Natural leather products may contain 150-300 mg/cc collagen based on the weight of the leather product. A biofabricated leather may contain a similar content of collagen or collagen fibrils as conventional leather based on the weight of the biofabricated leather, such as a collagen concentration of 100, 150, 200, 250, 300 or 350 mg/cc.

(31) The fibrillated collagen, sometimes called a hydrogel, may have a thickness selected based on its ultimate use. Thicker or more concentrated preparations of the fibrillated collagen generally produce thicker biofabricated leathers. The final thickness of a biofabricated leather may be only 10, 20, 30, 40, 50, 60, 70, 80 or 90% that of the fibril preparation prior to shrinkage caused by crosslinking, dehydration and lubrication.

(32) “Crosslinking” refers to formation (or reformation) of chemical bonds within between collagen molecules. A crosslinking reaction stabilizes the collagen structure and in some cases forms a network between collagen molecules. Any suitable crosslinking agent known in the art can be used including, without limitation, mineral salts such as those based on chromium, formaldehyde, hexamethylene diisocyanate, glutaraldehyde, polyepoxy compounds, gamma irradiation, and ultraviolet irradiation with riboflavin. The crosslinking can be performed by any known method; see, e.g., 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. U.S.A. 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 by reference.

(33) Crosslinkers include isocyantes, carbodiimide, poly(aldehyde), poly(azyridine), mineral salts, poly(epoxies), enzymes, thiirane, phenolics, novolac, resole as well as other compounds that have chemistries that react with amino acid side chains such as lysine, arginine, aspartic acid, glutamic acid, hydroxylproline, or hydroxylysine.

(34) A collagen or collagen-like protein may be chemically modified to promote chemical and/or physical crosslinking between the collagen fibrils. Chemical crosslinking may be possible because reactive groups such as lysine, glutamic acid, and hydroxyl groups on the collagen molecule project from collagen's rod-like fibril structure. Crosslinking that involve these groups prevent the collagen molecules from sliding past each other under stress and thus increases the mechanical strength of the collagen fibers. Examples of chemical crosslinking reactions include but are not limited to reactions with the ε-amino group of lysine, or reaction with carboxyl groups of the collagen molecule. Enzymes such as transglutaminase may also be used to generate crosslinks between glutamic acid and lysine to form a stable γ-glutamyl-lysine crosslink. Inducing crosslinking between functional groups of neighboring collagen molecules is known in the art. Crosslinking is another step that can be implemented here to adjust the physical properties obtained from the fibrillated collagen hydrogel-derived materials.

(35) Still fibrillating or fibrillated collagen may be crosslinked or lubricated. Collagen fibrils can be treated with compounds containing chromium or at least one aldehyde group, or vegetable tannins prior to network formation, during network formation, or network gel formation. Crosslinking further stabilizes the fibrillated collagen leather. For example, collagen fibrils pre-treated with acrylic polymer followed by treatment with a vegetable tannin, such as Acacia Mollissima, can exhibit increased hydrothermal stability. In other embodiments, glyceraldehyde may be used as a cross-linking agent to increase the thermal stability, proteolytic resistance, and mechanical characteristics, such as Young's modulus and tensile stress, of the fibrillated collagen.

(36) A biofabricated material containing a network of collagen fibrils may contain 0, >0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20% or more of a crosslinking agent including tanning agents used for conventional leather. The crosslinking agents may be covalently bound to the collagen fibrils or other components of a biofabricated material or non-covalently associated with them. Preferably, a biofabricated leather will contain no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10% of a crosslinking agent.

(37) “Lubricating” describes a process of applying a lubricant, such as a fat or other hydrophobic compound or any material that modulates or controls fibril-fibril bonding during dehydration to leather or to biofabricated products comprising collagen. A desirable feature of the leather aesthetic is the stiffness or hand of the material. In order to achieve this property, water-mediated hydrogen bonding between fibrils and/or fibers is limited in leather through the use of lubricants. Examples of lubricants include fats, biological, mineral or synthetic oils, cod oil, sulfonated oil, polymers, organofunctional siloxanes, and other hydrophobic compounds or agents used for fatliquoring conventional leather as well as mixtures thereof. While lubricating is in some ways analogous to fatliquoring a natural leather, a biofabricated product can be more uniformly treated with a lubricant due to its method of manufacture, more homogenous composition and less complex composition.

(38) Other lubricants include surfactants, anionic surfactants, cationic surfactants, cationic polymeric surfactants, anionic polymeric surfactants, amphiphilic polymers, fatty acids, modified fatty acids, nonionic hydrophilic polymers, nonionic hydrophobic polymers, poly acrylic acids, poly methacrylic, acrylics, natural rubbers, synthetic rubbers, resins, amphiphilic anionic polymer and copolymers, amphiphilic cationic polymer and copolymers and mixtures thereof as well as emulsions or suspensions of these in water, alcohol, ketones, and other solvents.

(39) Lubricants may be added to a biofabricated material containing collagen fibrils. Lubricants may be incorporated in any amount that facilitates fibril movement or that confers leather-like properties such as flexibility, decrease in brittleness, durability, or water resistance. A lubricant content can range from about 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 biofabricated leather.

(40) “Dehydrating” or “dewatering” describes a process of removing water from a mixture containing collagen fibrils and water, such as an aqueous solution, suspension, gel, or hydrogel containing fibrillated collagen. Water may be removed by filtration, evaporation, freeze-drying, solvent exchange, vacuum-drying, convection-drying, heating, irradiating or microwaving, or by other known methods for removing water. In addition, chemical crosslinking of collagen is known to remove bound water from collagen by consuming hydrophilic amino acid residues such as lysine, arginine, and hydroxylysine among others. The inventors have found that acetone quickly dehydrates collagen fibrils and may also remove water bound to hydrated collagen molecules. Water content of a biofabricated material or leather after dehydration 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 biofabricated leather. This range includes all intermediate values. Water content is measured by equilibration at 65% relative humidity at 25° C. and 1 atm.

(41) “Grain texture” describes a leather-like texture which is aesthetically or texturally the similar to the texture of a full grain leather, top grain leather, corrected grain leather (where an artificial grain has been applied), or coarser split grain leather texture. Advantageously, the biofabricated material of the invention can be tuned to provide a fine grain, resembling the surface grain of a leather such as that depicted by FIGS. 2A, 2B and 2C.

(42) A “biofabricated leather product” includes products comprising at least one component of a biofabricated leather such as foot ware, garments, gloves, furniture or vehicle upholstery and other leather goods and products. It includes but is not limited to clothing, such as overcoats, coats, jackets, shirts, trousers, pants, shorts, swimwear, undergarments, unifoinis, emblems or letters, costumes, ties, skirts, dresses, blouses, leggings, gloves, mittens, foot ware, shoes, shoe components such as sole, quarter, tongue, cuff, welt, and counter, dress shoes, athletic shoes, running shoes, casual shoes, athletic, running or casual shoe components such as toe cap, toe box, outsole, midsole, upper, laces, eyelets, collar, lining, Achilles notch, heel, and counter, fashion or women's shoes and their shoe components such as upper, outer sole, toe spring, toe box, decoration, vamp, lining, sock, insole, platform, counter, and heel or high heel, boots, sandals, buttons, sandals, hats, masks, headgear, headbands, head wraps, and belts; jewelry such as bracelets, watch bands, and necklaces; gloves, umbrellas, walking sticks, wallets, mobile phone or wearable computer coverings, purses, backpacks, suitcases, handbags, folios, folders, boxes, and other personal objects; athletic, sports, hunting or recreational gear such as harnesses, bridles, reins, bits, leashes, mitts, tennis rackets, golf clubs, polo, hockey, or lacrosse gear, chessboards and game boards, medicine balls, kick balls, baseballs, and other kinds of balls, and toys; book bindings, book covers, picture frames or artwork; furniture and home, office or other interior or exterior furnishings including chairs, sofas, doors, seats, ottomans, room dividers, coasters, mouse pads, desk blotters, or other pads, tables, beds, floor, wall or ceiling coverings, flooring; automobile, boat, aircraft and other vehicular products including seats, headrests, upholstery, paneling, steering wheel, joystick or control coverings and other wraps or coverings.

(43) Many uses of leather products require a durable product that doesn't rip or tear, even when the leather has been stitched together. Typical products that include stitched leather and require durable leather include automobile steering wheel covers, automobile seats, furniture, sporting goods, sport shoes, sneakers, watch straps and the like. There is a need to increase the durability of biofabricated leather to improve performance in these products. A biofabricated leather according to the invention can be used to make any of these products.

(44) Physical Properties of a biofabricated network of collagen fibrils or a biofabricated leather may be selected or tuned by selecting the type of collagen, the amount of concentration of collagen fibrillated, the degree of fibrillation, crosslinking, dehydration and lubrication. Many advantageous properties are associated with the network structure of the collagen fibrils which can provide strong, flexible and substantially uniform properties to the resulting biofabricated material or leather. Preferable physical properties of the biofabricated leather according to the invention include a tensile strength ranging from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more MPa, a flexibility determined by elongation at break ranging from 1, 5, 10, 15, 20, 25, 30% or more, softness as determined by ISO 17235 of 4, 5, 6, 7, 8 mm or more, a thickness ranging from 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 mm or more, and a collagen density (collagen fibril density) of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 mg/cc or more, preferably 100-500 mg/cc. The above ranges include all subranges and intermediate values.

(45) Thickness. Depending on its ultimate application a biofabricated material or leather may have any thickness. Its thickness preferably ranges from about 0.05 mm to 20 mm as well as any intermediate value 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 more. The thickness of a biofabricated leather can be controlled by adjusting collagen content.

(46) Elastic modulus. The elastic modulus (also known as Young's modulus) is a number that measures an object or substance's resistance to being defouned elastically (i.e., non-permanently) 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 elastic deformation region. A stiffer material will have a higher elastic modulus. The elastic modulus can be measured using a texture analyzer.

(47) A biofabricated leather can have an elastic modulus of at least 100 kPa. It can range from 100 kPa to 1,000 MPa as well as any intermediate value in 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. A biofabricated leather may be able to elongate up to 300% from its relaxed state length, for example, by >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 state length.

(48) Tensile strength (also known as ultimate tensile strength) is the capacity of a material or structure to withstand loads tending to elongate, as opposed to compressive strength, which withstands loads tending to reduce size. Tensile strength resists tension or being pulled apart, whereas compressive strength resists compression or being pushed together.

(49) A sample of a biofabricated material may be tested for tensile strength using an Instron machine. Clamps are attached to the ends of the sample and the sample is pulled in opposite directions until failure. Good strength is demonstrated when the sample has a tensile strength of at least 1 MPa. A biofabricated leather can have a tensile strength of at least 1 kPA. It can range from 1 kPa to 100 MPa as well as any intermediate value in 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.

(50) Tear strength (also known as tear resistance) is a measure of how well a material can withstand the effects of tearing. More specifically however it is how well a material (normally rubber) resists the growth of any cuts when under tension, it is usually measured in kN/m. Tear resistance can be measured by the ASTM D 412 method (the same used to measure tensile strength, modulus and elongation). ASTM D 624 can be used to measure the resistance to the formation of a tear (tear initiation) and the resistance to the expansion of a tear (tear propagation). Regardless of which of these two is being measured, the sample is held between two holders and a uniform pulling force applied until the aforementioned deformation occurs. Tear resistance is then calculated by dividing the force applied by the thickness of the material. A biofabricated leather may exhibit tear resistance of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150 or 200% more than that of a conventional top grain or other leather of the same thickness comprising the same type of collagen, e.g., bovine Type I or Type III collagen, processed using the same crosslinker(s) or lubricants. A biofabricated material may have a tear strength ranging from about 1 to 500 N, for example, 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 as well as any intermediate tear strength within this range.

(51) Softness. ISO 17235:2015 specifies a non-destructive method for determining the softness of leather. It is applicable to all non-rigid leathers, e.g. shoe upper leather, upholstery leather, leather goods leather, and apparel leather. A biofabricated leather may have a softness as determined by ISO 17235 of 2, 3, 4, 5, 6, 7, 8, 10, 11, 12 mm or more.

(52) Grain, The top grain surface of leather is often regarded as 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 is often a limitation for practical applications of the top grain alone and conventional leather products are often backed with corium having a much coarser grain. FIGS. 2A, 2B and 2C compare top grain and corium leather surfaces. A biofabricated material as disclosed herein which can be produced with strong and uniform physical properties or increased thickness can be used to provide top grain like products without the requirement for corium backing.

(53) Content of other components. In some embodiments, the collagen is free of 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, noncollagen structural proteins, and/or noncollagen nonstructural proteins in a biofabricated leather may range from 0, 1, 2, 3,4, 5, 6, 7, 8, 9 to 10% by weight of the biofabricated leather. In other embodiments, a content of actin, keratin, elastin, fibrin, albumin, globulin, mucin, mucinoids, noncollagen structural proteins, and/or noncollagen nonstructural proteins may be incorporated into a biofabricated 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 more by weight of a biofabricated leather. Such components may be introduced during or after fibrillation, cross-linking, dehydration or lubrication.

(54) A “leather dye” refers to dyes which can be used to color leather or biofabricated leather. These include acidic dyes, direct dyes, lakes, sulfur dyes, basic dyes and reactive dyes. Dyes and pigments can also be incorporated into a precursor of a biofabricated leather, such as into a suspension or network gel comprising collagen fibrils during production of the biofabricated leather.

(55) “Fillers”. In some embodiments a biofabricated leather may comprise fillers, other than components of leather, such as microspheres. One way to control the organization of the dehydrated fibril network is to include filling materials that keep the fibrils spaced apart during dehydration. These filler materials include nanoparticles, microparticles, or various polymers such as syntans commonly used in the tanning industry. These filling materials could be part of the final dehydrated leather material, or the filling materials could be sacrificial, that is they are degraded or dissolved away leaving open space for a more porous fibril network. The shape and dimension of these fillers may also be used to control the orientation of the dehydrated fibril network.

(56) In some embodiments a filler or secondary component may comprise polymeric microsphere(s), bead(s), fiber(s), wire(s), or organic salt(s). Other materials may also be embedded or otherwise incorporated into a biofabricated leather or into a network of collagen fibrils according to the invention. These include, but are not limited to one fibers, including both woven and nonwoven fibers as well as cotton, wool, cashmere, angora, linen, bamboo, bast, hemp, soya, seacell, fibers produced from milk or milk proteins, silk, spider silk, other peptides or polypeptides including recombinantly produced peptides or polypeptides, chitosan, mycelium, cellulose including bacterial cellulose, wood including wood fibers, rayon, lyocell, vicose, antimicrobial yarn (A.M.Y.), Sorbtek, nylon, polyester, elastomers such as lycra®, spandex or elastane and other .sup.1polyester-polyurethane copolymers, aramids, 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 comprising iron, steel, lead, gold, silver, platinum, copper, zinc and titanium, which may be in the form of particles, fibers, wires or other forms suitable for incorporating into biofabricated leather. Such fillers may include an electrically conductive material, magnetic material, fluorescent material, bioluminescent material, phosphorescent material or other photoluminescent material, or combinations thereof. Mixtures or blends of these components may also be embedded or incorporated into a biofabricated leather, for example, to modify the chemical and physical properties disclosed herein.

(57) Method of Making the Biofabricated Material Component of a Composite.

(58) A method of forming biofabricated material component from collagen for use in a composite material includes the steps of fibrillating, crosslinking, dehydrating/dewatering and lubricating in any order. For example, a collagen solution may be fibrillated, the fibrils may be crosslinked with an agent such as glutaraldehyde, then coated with a lubricant such as a sulfited oil, and then dehydrated through filtration to form a fibrillated collagen leather. However, the method of making is not limited to this particular order of steps.

(59) Alternatively, following fibril crosslinking, the fibrils can be dehydrated through a solvent exchange with acetone, followed by fat liquoring with a sulfited oil before evaporating away the solvent to form a fibrillated collagen leather. In addition, the incorporation of chemical or physical crosslinks between fibrils (to impart material strength) can be accomplished at any point during the process. For example, a solid fibrillated collagen, sometimes called a hydrogel, can be formed, then this fibril network can be dehydrated through a solvent exchange with acetone, followed by fat liquoring with a sulfited oil. Further, the collagen fibrils can be crosslinked into a network through the incorporation of other polymers such as those typically used in resin formulations.

(60) Materials such as lubricants, humectants, dyes and other treating agents can be uniformly distributed through a biofabricated leather product during the biofabrication process This is an advantage compared to conventional leather tanning and fat liquoring which due to its structural heterogeneity often makes uniform treatment impossible. Further, as chemical agents can be incorporated before network formation, smaller amounts of treatment chemicals would be necessary as there is reduced chemical loss by not having to penetrate a collagen network from a float containing the treatment chemicals. Unlike high temperatures often used to treat natural leather, a biofabricated can be heated at ambient temperature or at a temperature no greater than 37° C. during processing before evaporating away the solvent to form a fibrillated collagen leather. Alternatively, collagen fibrils can be crosslinked and lubricated in suspension before forming a network between fibrils during dehydration or through the addition of a binding agent to the suspension or to the dehydrated material.

(61) A method of forming a biofabricated leather material may include inducing fibrillation of collagen in a solution; crosslinking (e.g., tanning) and dehydrating the fibrillated collagen, which may appear in the form of a hydrogel, to obtain a fibrillated collagen sheet or other product, and incorporating at least one humectant or lubricant, such as a fat or oil into the fibrillated collagen sheet or product to obtain a flexible biofabricated leather.

(62) A method of biofabricating a leather from fibrils may include inducing fibrillation of collagen or collagen-like proteins in a solution to obtain a fibrillated collagen hydrogel; crosslinking the fibrillated collagen hydrogel to obtain a fibrillated collagen hydrogel leather; and incorporating at least one lubricating oil into the fibrillated collagen hydrogel leather.

(63) In the processes described herein for producing a biofabricated leather, the order of the steps for forming biofabricated leather may be varied or two or more steps may be performed simultaneously. For example, fibrillating and crosslinking may be performed together or by addition of one or more agents, or crosslinker and lubricant may be incorporated in the solution prior to fibrillating the collagen, etc.

(64) The collagen or collagen-like proteins may be obtained through extraction of collagen from an animal source, such as, but not limited to bovine hide or tendon collagen extraction. Alternatively, the collagen or collagen-like proteins may be obtained from a non-animal source, for example through recombinant DNA techniques, cell culture techniques, or chemical peptide synthesis.

(65) Any of these methods may include polymerizing the collagen or collagen-like proteins into dimers, trimers, and higher order oligomers prior to fibrillation, and/or chemically modifying the collagen or collagen-like proteins to promote crosslinking between the collagen or collagen-like proteins.

(66) Any of these methods may include functionalizing the collagen or collagen-like proteins with one or a combination of chromium, amine, carboxylic acid, sulfate, sulfite, sulfonate, aldehyde, hydrazide, sulfhydryl, diazirine, aryl, azide, acrylate, epoxide, or phenol group.

(67) Inducing fibrillation may include adding a salt or a combination of salts, for example, the salt or combination of salts may include: Na.sub.3PO.sub.4, K.sub.3PO.sub.4, KCl, and NaCl, the salt concentration of each salt may be between 10 mM to 5M, etc.

(68) In general, inducing fibrillation may comprise adjusting the pH with an acid or a base, adding a nucleating agent, such as a branched collagen microgel, wherein the nucleating agent has a concentration between 1 mM to 100 mM.

(69) The fibrillated collagen may be stabilized with a chromium compound, an aldehyde compound, or vegetable tannins, or any other crosslinking agent. For example, the fibrillated collagen may be stabilized with a chromium compound, an aldehyde compound, or vegetable tannins, wherein the chromium, aldehyde, or vegetable tannin compounds having a concentration of between 1 mM to 100 mM.

(70) Any of these methods may include adjusting the water content of the fibrillated collagen to 5, 10, 20, 25, 30, 40, 50 or 60% or less by weight to obtain the fibrillated collagen hydrogel leather. For example, the fibrillated collagen material may be dehydrated. Any of these methods may also include dyeing and/or applying a surface finish to the fibrillated collagen leather.

(71) The selection of collagen starting materials for biofabricating the engineered leather materials described herein can be controlled, the resulting product may differential formed with physical and aesthetic properties for distinct end uses, such as with features useful in footware and different features useful in apparel. In general, the biofabricated fibrillated collagen hydrogel-derived leathers described herein are formed from solutions of collagen that are induced to self-assemble into collagen fibrils.

(72) The collagen fibrils, unlike endogenous collagen fibrils, are not assembled into any high-order structures (e.g., bundles of fibers), but remain somewhat disordered, more particularly unbundled fibrils. When assembled in vivo, collagen fibrils are typically aligned laterally to form bundles having a higher order of structure and make up tough, micron-sized collagen fibers found, e.g., in skin. A characteristic feature of native collagen fibrils is their banded structure. The diameter of the native fibril changes slightly along the length, with a highly reproducible D-band repeat of approximately 67 nm. In some of the methods described herein, collagen fibrils may be unbanded and unbundled or may be banded and unbundled or may have a D-band of different spacing ranging from 1 to 100 nm and all intermediate values in this range). The collagen fibrils may be randomly oriented (e.g., un-oriented or not oriented in any particular direction or axis).

(73) The starting material used to form the biofabricated leather material as described herein may include any appropriate non-human collagen source or modified or engineered collagens that can be fibrillated.

(74) Various forms of collagen are found throughout the animal kingdom. The collagen used herein may be obtained from animal sources, including both vertebrates and invertebrates, or from synthetic sources. Collagen may also be sourced from byproducts of existing animal processing. Collagen obtained from animal sources may be isolated using standard laboratory techniques known in the art, for example, Silva et. Al., Marine Origin Collagens and its Potential Applications, Mar. Drugs, 2014 Dec. 12(12); 5881-5901).

(75) One major benefit of the biofabricated leather materials and methods for forming them described herein is that collagen may be obtained from sources that do not require killing of an animal.

(76) The collagen described herein also may be obtained by cell culture techniques including from cells grown in a bioreactor.

(77) Collagen may also be obtained via recombinant DNA techniques. Constructs encoding non-human collagen may be introduced into host organisms to produce non-human collagen. For instance, collagen may also be produced with yeast, such as Hansenula polymorpha, Saccharomyces cerevisiae, Pichia pastoris and the like as the host. Further, in recent years, bacterial genomes have been identified that provide the signature (Gly-Xaa-Yaa)n repeating amino acid sequence that is characteristic of triple helix collagen. For example, gram positive bacterium Streptococcus pyogenes contains two collagen-like proteins, Scl1 and Scl2 that now have well characterized structure and functional properties. Thus, it would be possible to obtain constructs in recombinant E. coli systems with various sequence modifications of either Scl1 or Scl2 for establishing large scale production methods. Collagen may also be obtained through standard peptide synthesis techniques. Collagen obtained from any of the techniques mentioned may be further polymerized. Collagen dimers and trimers are formed from self-association of collagen monomers in solution.

(78) As an initial step in the formation of the collagen materials described herein, the starting collagen material may be placed in solution and fibrillated. Collagen fibrillation may be induced through the introduction of salts to the collagen solution. The addition of a salt or a combination of salts such as sodium phosphate, potassium phosphate, potassium chloride, and sodium chloride to the collagen solution may change the ionic strength of the collagen solution. Collagen fibrillation may occur as a result of increasing electrostatic interactions, through greater hydrogen bonding, Van der Waals interactions, and covalent bonding. Suitable salt concentrations may range, for example, from approximately 10 mM, 50 mM, 100 mM, 500 mM, 1M, 2M, 3M, 4M to 5M as well as any intermediate value within this range.

(79) Collagen fibrillation may also be induced or enhanced with a nucleating agent other than salts. Nucleating agents provide a surface on which collagen monomers can come into close contact with each other to initiate fibrillation or can act as a branch point in which multiple fibrils are connected through the nucleating agent. Examples of suitable nucleating agents include but are not limited to: microgels containing collagen, collagen micro or nanoparticles, metallic particles or naturally or synthetically derived fibers. Suitable nucleating agent concentrations may range from approximately 1 mM to 100 mM.

(80) A collagen network may also be highly sensitive to pH. During the fibrillation step, the pH may be adjusted to control fibril dimensions such as diameter and length. The overall dimensions and organization of the collagen fibrils will affect the toughness, stretch-ability, and breathability of the resulting fibrillated collagen derived materials. This may be of use for fabricating fibrillated collagen derived leather for various uses that may require different toughness, flexibility, and breathability. Adjustment of pH, with or without a change in salt concentration may be used for fibrillation.

(81) One way to control the organization of the dehydrated fibril network is to include filling materials that keep the fibrils spaced apart during drying. These filler materials could include nanoparticles, microparticles, or various polymers such as syntans commonly used in the tanning industry. These filling materials could be part of the final dehydrated leather material, or the filling materials could be sacrificial, that is they are degraded or dissolved away leaving open space for a more porous fibril network.

(82) The collagen or collagen-like proteins may be chemically modified to promote chemical and physical crosslinking between the collagen fibrils. Chemical crosslinking may be possible because reactive groups such as lysine, glutamic acid, and hydroxyl groups on the collagen molecule project from collagen's rod-like fibril structure. Crosslinking that involve these groups prevent the collagen molecules from sliding past each other under stress and thus increases the mechanical strength of the collagen fibers. Examples of chemical crosslinking reactions include but are not limited to reactions with the ε-amino group of lysine, or reaction with carboxyl groups of the collagen molecule. Enzymes such as transglutaminase may also be used to generate crosslinks between glutamic acid and lysine to form a stable γ-glutamyl-lysine crosslink. Inducing crosslinking between functional groups of neighboring collagen molecules is known in the art. Crosslinking is another step that can be implemented here to adjust the physical properties obtained from the fibrillated collagen hydrogel-derived materials.

(83) Once formed, the fibrillated collagen network may be further stabilized by incorporating molecules with di-, tri-, or multifunctional reactive groups that include chromium, amines, carboxylic acids, sulfates, sulfites, sulfonates, aldehydes, hydrazides, sulfhydryls, diazarines, aryl-, azides, acrylates, epoxides, or phenols.

(84) The fibrillated collagen network may also be polymerized with other agents (e.g. polymers that are capable of polymerizing or other suitable fibers) that form a hydrogel or have fibrous qualities, which could be used to further stabilize the matrix and provide the desired end structure. Hydrogels based upon acrylamides, acrylic acids, and their salts may be prepared using inverse suspension polymerization. Hydrogels described herein may be prepared from polar monomers. The hydrogels used may be natural polymer hydrogels, synthetic polymer hydrogels, or a combination of the two. The hydrogels used may be obtained using graft polymerization, crosslinking polymerization, networks formed of water soluble polymers, radiation crosslinking, and so on. A small amount of crosslinking agent may be added to the hydrogel composition to enhance polymerization.

(85) Any appropriate thickness of the fibrillated collagen hydrogel may be made as described herein. Because the final thickness will be much less (e.g., between 10-90% thinner) than the hydrogel thickness, the initial hydrogel thickness may depend on the thickness of the final product desired, presuming the changes to the thickness (or overall volume) including shrinkage during crosslinking, dehydration and/or adding one or more oils or other lubricants as described herein.

(86) A hydrogel thickness may be between 0.1 mm and 50 cm or any intermediate value within this range. In forming the fibrillated hydrogel, the hydrogel may be incubated to form the thickness for any appropriate length of time, including between 1 min and 24 hrs.

(87) The fibrillated collagen hydrogels described herein may generally be formed in any appropriate shape and/or thickness, including flat sheets, curved shapes/sheets, cylinders, threads, and complex shapes. Further, virtually any linear size of these shapes. For example, any of these hydrogels may be formed into a sheet having a thickness as described and a length of greater than 10 mm (e.g., greater than, in cm, 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, etc.) and width that is greater than 10 mm, such as greater than, in cm, 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, etc.

(88) Once the collagen fibrils, often characterized as a hydrogel, have formed or during formation, they may be crosslinked. For example, the fibrillated collagen hydrogel be treated with compounds containing chromium or at least one aldehyde group, or vegetable tannins prior to gel formation, during gel formation, or after gel formation, to further stabilize the fibrillated collagen hydrogel. For example, collagen fibrils may be pre-treated with acrylic polymer followed by treatment with a vegetable tannin (e.g., Acacia Mollissima) may exhibit increased hydrothermal stability. In other examples, glyceraldehyde may be used as a cross-linking agent that may increase the thermal stability, proteolytic resistance, and mechanical characteristics (e.g. Young's modulus, tensile stress) of the fibrillated collagen hydrogel.

(89) Depending on the temperature and volume of starting material, the fibrillation and hydrogel formation may occur somewhat quickly after induction and be largely complete after an hour and a half, as shown by the absorbance values leveling off after 70 minute's time passing. An increase in storage modulus (or viscoelastic qualities of the material) of the fibrillated collagen hydrogel after induction from around 1 Pa (for the solution of collagen) to approximately 400 Pa for the fibrillated collagen hydrogel may occur.

(90) As mentioned above and illustrated in FIGS. 1 and 2, animal skin typically includes fibrils that are ordered into higher-order structures, including the presence of banding (having regular lacunar regions) and formation of multiple fibrils into aligned fibers which may then bundled into collagen bundles. In contrast, the collagen hydrogels and therefore the biofabricated leathers described herein may have a primary disorganized collagen fibril structure throughout the entire thickness (in some cases entire volume) of the material. Specifically, the collagen structure of the biofabricated leathers formed from collagen hydrogels may be primarily unbundled and un-oriented along any particular axis. In some variations the collagen fibrils may be unbanded (e.g., greater than 10% unbanded, greater than 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, etc. unbanded throughout the volume). Furthermore, the orientation of the collagen fibrils within the volume (or throughout the volume) may be un-oriented, or randomly oriented, and this lack of orientation may be the same throughout the volume, rather than changing through the volume thickness as in native leather, which may have change from bundles of collagen fibrils that are vertically oriented to bundles that are horizontally oriented in the thickness. Any of the properties which are the same at any level thickness of the hydrogel and therefore resulting leather material may be referred to herein as “uniformly” the same throughout the thickness.

(91) In addition, any of the biofabricated leathers described herein may have a uniform distribution of fibrils throughout the thickness of the gel and therefore resulting leather material. This is in contrast with native leathers, such as the material shown in FIG. 2, showing an increase in the number of fiber bundles through the thickness of the material.

(92) The lack of higher-level organization of the fibrillated collagen hydrogels and leather material formed from them is apparent in FIGS. 3A and 3B. FIG. 3A shows a scanning electron micrograph of a fibrillated collagen hydrogel formed as described herein. Similarly, FIG. 4 shows a transmission electron micrograph through a fibrillated collagen hydrogel. The transmission electron micrograph and the scanning electron micrograph both show the fibrillated collagen hydrogel as being a disordered tangle of collagen fibrils. As previously mentioned, the density and to some extent, the pattern of collagen fibril formation may be controlled by adjusting the pH of the collagen solution during fibrillation induction along with the concentration of fibrils during dehydration. FIG. 3 also shows a scanning electron micrograph of bovine corium. In comparison with a natural bovine corium shown in FIG. 3B, the fibrillated collagen network is much more random and lacks the apparent striations. Although the overall size of the fibrils may be similar, the arrangement of these fibrils is quite different. Such ultrastructural differences between the collagen fibrils within the fibrillated collagen hydrogel and natural tissue such as bovine corium (and resulting leather made therefrom) may not be an issue in the final biofabricated leather product may be as soft or softer, and more pliable than natural leather, and may have a similar appearance. In order to make the final biofabricated leather product more durable, the fibrillated collagen may include a secondary material (collagen being the primary material). Suitable secondary materials include, but are not limited to, woven or knitted fabrics, nonwovens including natural felts such as wool felts and the like, synthetic felts such as polyester-polyurethane copolymers such as elastane or LYCRA® felts, polyparaphenylene terephthalamide polymers such as KEVLAR® felts, nylon polymers such as nylon 6, nylon 6,6 and the like felts, and polyester polymers such as polyethylene terephthalatepolyethylene and the like felts, staple fibers such as carbon fibers felts, silk fibers and the like, cellulosic microfibers and combinations thereof. In one embodiment of the present invention, the secondary material is surrounded by the fibrillated collagen material to create a composite. One method of surrounding a secondary material with fibrillated collagen is to pour a collagen solution over one side of the secondary material, then the secondary material may be flipped and collagen solution poured onto the opposite side of the secondary material. This may be described as a sandwich type structure.

(93) In another embodiment of the present invention, the collagen may be converted into a biofabricated leather and the secondary material may be laminated to one side of the leather using adhesives and the like. Suitable adhesives may include but are not limited to hot melt adhesives, emulsion polymer adhesives and the like. The biofabricated leather may be coated with adhesive by known techniques such as slot die casting, kiss coating and the like and the secondary material may be applied to the leather and passed through rollers under heat to laminate the materials.

(94) In another embodiment, the secondary material may be dispersed throughout the collagen material to create the 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 the biofabricated leather product may range from 1:100 to 100:1.

(95) The secondary material may also be a photoluminescent material such as a photoluminescent fabric, nonwoven, felt, carbon fiber or 3 dimensional object. As described above, the collagen solution may be poured over one side of the secondary material, the secondary material may be flipped over and collagen solution may be poured over the other side of the secondary material.

(96) The fibrillated collagen, sometimes called a hydrogel, may then be dehydrated to rid the fibrillated collagen hydrogel of the majority of its water content. Removing the water from the fibrillated collagen hydrogel may change its physical quality from a hydrated gel to a pliable sheet. The material may be treated to prevent breakage/tearing. For example, care may be taken not to remove too much water from the fibrillated collagen. In some examples, 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%. Water content is determined by equilibration at 25° C. at 1 atm pressure at a relative humidity of 65%.

(97) Dehydration may involve air drying, vacuum and pressure filtration, solvent exchange or the like. For example, fibrillated collagen hydrogel may also undergo dehydration through replacement of its water content with organic solvents. Suitable organic solvents may include, but are not limited to acetone, ethanol, diethyl ether, and so forth. Subsequently, the organic solvents may be evaporated (e.g. air drying, vacuum drying, etc.). It is also possible to perform successive steps of dehydration using one or more than one organic solvent to fine tune the level of dehydration in the final product.

(98) After or during dehydration, the fibrillated collagen material may be treated with lubricants and/or oils to impart greater flexibility and suppleness to the fibrillated collagen material. Using a combination of oil and solvent may allow the oil to better penetrate the fibrillated collagen network compared to using oil by itself. Oil by itself will only likely penetrate the exposed surfaces but may not readily infiltrate the entire thickness of the fibrillated collagen material in a reasonable amount of time. Once the oil/solvent composition has penetrated the entire thickness of the material, the solvent may then be removed. Suitable oils and lubricants may include but are not limited to castor oil, pine oil, lanolin, mink oil, neatsfoot oil, fish oil, shea butter, aloe, and so forth.

(99) Lubricating the dehydrated and crosslinked fibrillated collagen network or hydrogel to form a leather material may result in a material having properties that are similar, or better, than the properties of natural leather. The solutions that included a combination of oils and organic solvent increased the mass and the softness (inversely proportional to the slope of the stress-strain curve) of the dehydrated fibrillated collagen material. This is due to the combination of oils and organic solvents penetrating the dehydrated fibrillated collagen material and once penetrated through, the oils remained distributed throughout the material, while the organic solvents are able to evaporate away. While not shown, the use of oils alone may not be as effective in penetrating entirely through the dehydrated fibrillated collagen material.

(100) The resulting fibrillated collagen materials then may be treated similarly to natural leather derived from animal hide or skin, and be re-tanned, dyed, and/or finished. Additional processing steps may include: crosslinking, re-tanning, and surface coating. Crosslinking and re-tanning may include sub-processes such as wetting back (re-hydrating semi-processed leather), sammying (45-55% water is squeezed from the leather), splitting (leather is split into one or more layers), shaving (leather is thinned), neutralization (pH of leather is adjusted to between 4.5 and 6.5), dyeing (leather is colored), fat liquoring (fats, oils, waxes are fixed to the leather fibers), filling (dense/heavy chemicals to make leather harder and heavier), stuffing (fats, oils, waxes added between leather fibers), fixation (unbound chemicals are bonded/trapped and removed), setting (grain flatness are imparted and excess water removed), drying (leather is dried to desired moisture levels, 10-25%), conditioning (moisture is added to leather to a 18-28% level), softening (physical softening of leather by separating the fibers), or buffing (abrading surface of leather to reduce nap and grain defects). Surface coating may include any one or combination of the following steps: oiling (leather coated with raw oil or oils), buffing, spraying, roller coating, curtain coating, polishing, plating, embossing, ironing, or glazing.

(101) Unlike animal hides, where the hide has to be trimmed to obtain the desired thickness or dimensions, the engineered leather material may be fabricated with a wide range of thicknesses as well as the desired dimensions with a particular end product in mind.

(102) The production of such engineered leather materials may also generate less waste by bypassing the step of removing excess proteins, fats, and hair necessary for treating natural animal hide in the leather production process, which results in less environmental impact from the disclosed process and the products derived from these methods.

(103) The biofabricated materials disclosed herein are advantageously combined, incorporated or attached to other materials to form useful composites. For example, a biofabricated coating may be applied to a secondary material such as a woven or nonwoven fabric or a plastic mesh by dipping or spraying components forming the biofabricated material. A biofabricated material may be incorporated on or laminated to one or both sides of a flat secondary material. Specific embodiments of these composite materials are described below.

EMBODIMENTS

Composites

(104) The invention includes, but is not limited to biofabricated materials components having the features described below. The composites of the invention include those where (i) one or more secondary components, such as a particle, wire, fabric, or three dimensional object is incorporated or embedded in a network of collagen fibrils, (ii) where a biofabricated material is coated or deposited, for example by filtration, on one side of one or more secondary components such as a woven or nonwoven fabric, such as fabric, paper or regenerated cellulose, (iii) where a biofabricated component is coated or deposited on both sides of one or more secondary materials having top and bottom sides or inner and outer sides, or (iv) where a biofabricated 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.

(105) The biofabricated material once produced may be associated with the one or more secondary components to form a composite. A composite may be formed simultaneously with the biofabricated material, for example, a secondary component such as a particle or fiber may be mixed with precursors of a biofabricated material at any step in its production as described herein. For example, a particulate or fibrous secondary material can be mixed with collagen, collagen fibrils, crosslinked collagen fibrils, lubricated collagen fibrils, dehydrated collagen fibrils (including in powdered form), crosslinked, dehydrated and lubricated collagen fibrils, which are subsequently processed, along with the secondary material into a composite comprising the biofabricated material component. The secondary component can be coated with or embedded in the resulting biofabricated material. An example of this is the deposit of crosslinked collagen fibrils on filter paper and the subsequent dehydration and lubrication of the composite of the filter paper (a secondary component) and the biofabricated material deposited by filtration on one side of the paper. Precursors of the biofabricated material component may be coated or otherwise applied to surface(s) of a secondary component and then processed into a final biofabricated material, for example, by at least one of fibrillation of collagen, crosslinking collagen fibrils, dehydration of collagen fibrils or crosslinked collagen fibrils, and lubrication of collagen fibrils or crosslinked collagen fibrils.

(106) Alternatively, a biofabricated material component once produced, may be coated or laminated on at least one surface of a secondary component having a top and bottom surface or inner and outer surface. In some embodiments, one or more layers of a flat secondary material will be sandwiched between two layers of a biofabricated component which will form the external layers of a composite having the aesthetic qualities of the biofabricated component and strength, thickness or other properties conferred by the internally sandwiched secondary component.

(107) The composites of the invention may also contain layered structures, including alternating or a repeating series of one or more layers of the biofabricated and secondary components. These layers may appear in any order in a composite. Secondary component layers may be adjacent to each other or to biofabricated layers. Biofabricated layers may be adjacent to each other or to layers of one or more secondary components. Such composites may comprise adjacent or multiple layers of the biofabricated component with or without a non-collagenous secondary component. For example, multiple layers of a biofabricated component may be deposited on one side of a filter paper or mesh to increase the thickness of the biofabricated material content of a composite.

(108) The composites of the invention include, but are not limited to (i) those that involve dispersing, encapsulating, incorporating, depositing, or otherwise introducing at least one biofabricated material into or onto at least one porous, permeable, or absorptive secondary component; (ii) those that involve layering, laminating, depositing, coating or otherwise contacting at least one secondary component with at least one biofabricated material; or (iii) those that sandwiching, layering, laminating, coating, or otherwise covering a top and bottom surface or inner and outer surface of at least one secondary component with at least one biofabricated material.

(109) They can involve incorporating or embedding one or more secondary materials into a network of collagen fibrils, for example, by mixing the secondary materials with biofabricated material precursors or adding them during the preparation of a biofabricated material comprising a network of crosslinked collagen fibrils. Examples of such secondary materials that may be incorporated into a biofabricated material to produce a composite include particles, wires, fabrics, or three dimensional objects. Once the secondary component is incorporated into a precursor of the biofabricated material component, the mixture may then be further processed into a biofabricated material that embeds, encapsulates or incorporates the secondary material.

(110) These methods include coating or depositing a biofabricated material or a precursor of a biofabricated material, such as unfibrillated collagen, not crosslinked collagen fibrils, not dehydrated collagen fibrils or not lubricated collagen fibrils on a secondary component substrate, such as a woven or nonwoven fabric, paper, or regenerated cellulose. For example, depositing may be accomplished by filtering a solution or suspension of collagen fibrils or crosslinked collagen fibrils through secondary material that retains the collagen fibrils on one side, for example, filter paper. The deposited collagen fibrils may then be further processed into a biofabricated material that is incorporated into or on one side of the secondary material. In some embodiments, the material may be deposited on both sides of a substrate. In others two substrates each containing a layer of biofabricated material can be laminated together with the biofabricated material facing inward or outward. Preferably for the purpose of providing a leather-like aesthetic, the layers of biofabricated material will face outward.

(111) Biofabricated materials may be deposited or coated on two sides of a secondary material substrate to provide a leather-like aesthetic to the outward facing sides. Alternatively, the biofabricated material can form one or more inner layers of a composite with the secondary material facing outward.

(112) A composite material may be produced by attaching a biofabricated material once produced to one or more secondary components, for example, by coating or laminating the biofabricated material to at least one surface of a secondary component having a top and bottom surface or inner and outer surface.

(113) In some embodiments, a composite will be produced by sandwiching one or more layers of a flat secondary material between at least two external layers of a biofabricated component thus providing the aesthetic qualities of the biofabricated component and strength, thickness or other properties conferred by the internally sandwiched secondary component.

(114) The composites of the invention may be produced by alternating or repeating series of one or more layers of the biofabricated and secondary components. These layers may appear in any order in a composite. The method may comprise arranging secondary component layers adjacent to each other or to biofabricated layers. Biofabricated layers may be adjacent to each other or to layers of one or more secondary components. Such composites may comprise adjacent or multiple layers of the biofabricated component with or without a non-collagenous secondary component. For example, multiple layers of a biofabricated component may be deposited on one side of a filter paper or mesh to increase the thickness of the biofabricated material content of a composite.

(115) Specific embodiments of the composite materials of invention include, without limitation, the following.

(116) 1. A composite material comprising:

(117) (i) at least one porous, permeable, or absorptive secondary component, and

(118) at least one biofabricated 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 more, in the form of fibrils aligned for 100 μm or more of their lengths, or both; wherein said material contains no more than 40% by weight water; and wherein said material contains at least 1% of a lubricant; or

(119) (i) at least one porous, permeable, or absorptive secondary component, and

(120) at least one biofabricated material comprising a network of recombinant non-human collagen fibrils, wherein the collagen contains substantially no 3-hydroxyproline, and optionally, substantially no hydroxylysine; wherein said material contains no more than 25% by weight water; and wherein said material contains at least 1% of a lubricant; or

(121) (ii) at least one layer of a secondary component, and

(122) at least one layer of a biofabricated 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 more, in the faun of fibrils aligned for 100 μm or more of their lengths, or both; wherein said material contains no more than 40% by weight water; and wherein said material contains at least 1% of a lubricant.;

(123) (iii) at least one layer of a secondary component, and

(124) at least one biofabricated material comprising a network of recombinant non-human collagen fibrils, wherein the collagen contains substantially no 3-hydroxyproline, and optionally, substantially no hydroxylysine; wherein said material contains no more than 25% by weight water; and wherein said material contains at least 1% of a lubricant.; or

(125) (iv) at least one layer of a secondary component, and

(126) at least two external layers of at least one biofabricated material having a top and bottom surface, or inner and outer surface, 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 more, in the form of fibrils aligned for 100 μm or more of their lengths, or both; wherein said material contains no more than 40% by weight water; and wherein said material contains at least 1% of a lubricant; or

(127) (v) at least one layer of a secondary component, and

(128) at least two external layers of at least one biofabricated material having a top and bottom surface, or inner and outer surface, comprising a network of recombinant non-human collagen fibrils, wherein the collagen contains substantially no 3-hydroxyproline, and optionally, substantially no hydroxylysine; wherein said material contains no more than 25% by weight water; and wherein said material contains at least 1% of a lubricant.

(129) 2. The composite of embodiment 1 that is (i) or (ii), wherein the secondary component has a top and bottom surface or an inner and outer surface.

(130) 3. The composite according to embodiment 2, wherein the biofabricated material is only on or only incorporated into one of the top, bottom, inner or outer surfaces.

(131) 4. The composite according to embodiment 2, wherein the biofabricated material is on or incorporated into both the top and bottom surfaces or both the inner or outer surfaces.

(132) 5. The composite according to embodiment 2, wherein the secondary component is a paper, regenerated cellulose, fabric, or other nonwoven or woven fibrous material.

(133) 6. The composite of embodiment 1, wherein the secondary component comprises at least one resin, polymer, or plastic.

(134) 7. The composite of embodiment 1, wherein the secondary component comprises at least one fiber, bead, wire, particle, mesh, woven, or nonwoven.

(135) 8. The composite of embodiment 1, wherein the biofabricated material contains less than 1% by weight of actin, keratin, elastin, fibrin, albumin, globulin, mucin, mucinoids, noncollagen structural proteins, and/or noncollagen nonstructural proteins;

(136) 9. The composite of embodiment 1, wherein the biofabricated material comprises at least 1% of at least one crosslinker.

(137) 10. The composite according to embodiment 1, wherein the diameters of fibrils in the biofabricated material exhibit a substantially unimodal distribution wherein at least 70% of the diameters of the fibrils in the material distribute around a single mode of diameter.

(138) 11. The composite according to embodiment 1, wherein the biofabricated material comprises at least one lubricant is selected from the group consisting of at least one fat, biological, mineral or synthetic oil, sulfonated oil, polymer, and organofunctional siloxane.

(139) 12. The composite according to embodiment 1, wherein the biofabricated material has an elastic modulus between 100 kPa and 1,000 MPa, wherein the elastic modulus varies by no more than 20% when measured at right angles across identical lengths of the material and that has a tensile strength of ranging from 1 MPa to 100 MPa, wherein the tensile strength varies by no more than 20% when measured at right angles across identical lengths of the material.

(140) 13. The composite according to embodiment 1, wherein the biofabricated material further comprises a surface coating or surface finish; wherein the surface coating or surface finish is distributed uniformly throughout the material such that its concentration by weight in or on identical unit volumes of the material varies by no more than 20%.

(141) 14. The composite according to embodiment 1, wherein the biofabricated material further comprises a dye, stain, resin, polymer, pigment or paint, wherein the dye, stain, resin, pigment or paint is distributed uniformly throughout the material such that its concentration by weight in or on identical unit volumes of the material varies by no more than 20%.

(142) 15. The composite according to embodiment 1, wherein the biofabricated material further comprises at least one filler, wherein the filler is distributed uniformly throughout the material such that its concentration by weight in or on identical unit volumes of the material varies by no more than 20%.

Method of Making a Composite

(143) Specific embodiments of a method for making a composite according to the invention include, without limitation the following:

(144) 1. A method for making a composite material comprising:

(145) (i) dispersing, encapsulating, incorporating, depositing, or otherwise introducing at least one biofabricated material into or onto at least one porous, permeable, or absorptive secondary component; wherein the at least one biofabricated material comprises 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 more, in the form of fibrils aligned for 100 μm or more of their lengths, or both; wherein said material contains no more than 40% by weight water; and wherein said material contains at least 1% of a lubricant; or

(146) (ii) dispersing, encapsulating, incorporating, depositing, or otherwise introducing at least one biofabricated material into or onto at least one porous, permeable, or absorptive secondary component; wherein said and at least one biofabricated material comprises a network of recombinant non-human collagen fibrils, wherein the collagen contains substantially no 3-hydroxyproline, and optionally, substantially no hydroxylysine; wherein said material contains no more than 25% by weight water; and wherein said material contains at least 1% of a lubricant; or

(147) (iii) layering, laminating, depositing, coating or otherwise contacting at least one secondary component, which has a top and bottom surface or an inner and outer surface, with at least one biofabricated material that comprises 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 more, in the form of fibrils aligned for 100 μm or more of their lengths, or both; wherein said material contains no more than 40% by weight water; and wherein said material contains at least 1% of a lubricant; or

(148) (iv) layering, laminating, depositing, coating or otherwise contacting at least one secondary component, which has a top and bottom surface or an inner and outer surface, with at least one biofabricated material that comprises a network of recombinant non-human collagen fibrils, wherein the collagen contains substantially no 3-hydroxyproline, and optionally, substantially no hydroxylysine; wherein said material contains no more than 25% by weight water; and wherein said material contains at least 1% of a lubricant; or

(149) (v) sandwiching, layering, laminating, coating, or otherwise covering a top and bottom surface or an inner and outer surface of at least one secondary component with at least one biofabricated material that comprises 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 more, in the form of fibrils aligned for 100 μm or more of their lengths, or both; wherein said material contains no more than 40% by weight water; and wherein said material contains at least 1% of a lubricant; or

(150) (vi) sandwiching, layering, laminating, coating, or otherwise covering a top and bottom surface or inner and outer surface of at least one secondary component with at least one biofabricated material that comprises a network of recombinant non-human collagen fibrils, wherein the collagen contains substantially no 3-hydroxyproline, and optionally, substantially no hydroxylysine; wherein said material contains no more than 25% by weight water; and wherein said material contains at least 1% of a lubricant.

(151) 2. The method according to embodiment 1, wherein said method is (i) and wherein the at least one biofabricated material is produced by a process comprising in any order:

(152) fibrillating an aqueous solution or suspension of non-human collagen molecules into collagen fibrils,

(153) crosslinking said collagen fibrils by contacting them with at least one crosslinking agent,

(154) dehydrating the crosslinked collagen fibrils so that they contain less than 40% by weight water,

(155) lubricating by incorporating at least 1% by weight of at least one lubricant into said material.

(156) 3. The method according to embodiment 2, wherein said biofabricated material is produced by fibrillating recombinant collagen.

(157) 4. The method according to embodiment 1, wherein said method is (ii) and wherein the at least one biofabricated material is produced by a process comprising in any order:

(158) fibrillating an aqueous solution or suspension of recombinant non-human collagen molecules into collagen fibrils,

(159) crosslinking said collagen fibrils by contacting them with at least one crosslinking agent,

(160) dehydrating the crosslinked collagen fibrils so that they contain less than 25% by weight water,

(161) lubricating by incorporating at least 1% by weight of at least one lubricant into said material.

(162) 5. The method according to embodiment 4, wherein said fibrillating, crosslinking, dehydrating and/or lubricating is performed for a time and under conditions that produce less than 10% by weight of the collagen fibrils in the biofabricated material in the form of collagen fibers having a diameter of 5 μm or more, in the form of fibrils aligned for 100 μm or more of their lengths, or both.

(163) 6. The method according to embodiment 1, wherein said method is (i) or (ii) and wherein the biofabricated material is incorporated into or onto the at least one porous, permeable, or absorptive secondary component.

(164) 7. The method according to embodiment 1, wherein the secondary component comprises at least one resin, polymer, or plastic.

(165) 8. The method according to embodiment 1, wherein the secondary component comprises at least one fiber, bead, wire, particle, mesh, woven, or nonwoven.

(166) 9. The method according to embodiment 1, wherein the secondary component comprises at least one electrically conductive material, magnetic material, fluorescent material, bioluminescent material, phosphorescent material, or combinations thereof.

(167) 10. The method of embodiment 1, wherein the biofabricated material is produced by fibrillating non-human collagen molecules to produce fibrils by at least one of adjusting a salt concentration or adjusting a pH of an aqueous solution containing said collagen molecules.

(168) 11. The method of embodiment 1, wherein the biofabricated material is produced by crosslinking collagen fibrils by contacting them with at least one compound selected from the group consisting of an amine, carboxylic acid, sulfate, sulfite, sulfonate, aldehyde, hydrazide, sulfhydryl, diazirine, aryl, azide, acrylate, epoxide, phenol, chromium compound, vegetable tannin, and syntan.

(169) 12. The method according to embodiment 1, wherein the biofabricated material is produced by dehydrating the network of collagen fibrils by contacting them with an agent that removes bound water from collagen.

(170) 13. The method according to embodiment 1, comprising lubricating the collagen fibrils with least one lubricant selected from the group consisting of fat, biological, mineral or synthetic oil, cod oil, sulfonated oil, polymer, and organofunctional siloxane.

(171) 14. The method according to embodiment 1, wherein the biofabricated material is produced by uniformly distributing the lubricant on or throughout the biofabricated material such that the concentration of the lubricant in identical unit volumes of the material varies by no more than 20%.

(172) 15. The method according to embodiment 1, wherein the biofabricated material is produced by uniformly distributing a dye, stain, pigment, resin, polymer, or paint in or on it, wherein the concentration of the dye, stain, pigment, resin, polymer, or paint in identical unit volumes of the biofabricated material varies by no more than 20%.

(173) 16. The method according to embodiment 1, wherein the biofabricated material is produced by incorporating at least one filler into it.

Biofabricated Component of Composites

(174) In one embodiment, the biofabricated material component comprises a network of collagen fibers, such as a biofabricated material or biofabricated leather:

(175) (i) comprising a network of non-human collagen fibrils,

(176) wherein less than 5, 10, 15, 20, 25, 30, 35, or 40% by weight of the collagen fibrils in the material are in the form of collagen fibers having a diameter of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μm or more and/or are in the form of fibrils aligned for 100 μm or more of their lengths; wherein said material contains no more than 10, 20, 30, 40, 50, or 60% by weight water; wherein said material contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40% by weight of a lubricant; and wherein optionally, the material comprises a top and bottom surface or an inner and outer surface; or

(177) (ii) comprising a network of recombinant non-human collagen fibrils, wherein the collagen contains substantially no 3-hydroxyproline, and optionally, substantially no hydroxylysine; wherein said material contains no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60% by weight water; wherein the material contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40% of a lubricant; and wherein optionally, the material comprises a top and bottom surface or an inner and outer surface. Water content in this material is preferably no more than 25 to 40%. Lubricant content may be selected to match or not exceed the absorptive capacity of the biofabricated material for a lubricant. Such a material may comprise mammalian collagen, such as bovine Type I or Type III collagen. Preferably it will not contain hair, hair follicle(s), or fat(s) of an animal that naturally expresses the collagen molecules it contains. For example, it may contain less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% by weight of actin, keratin, elastin, fibrin, albumin, globulin, mucin, mucinoids, noncollagen structural proteins, and/or noncollagen nonstructural proteins found in conventional leather. It may be substantially free of other collagenous proteins, carbohydrates, nucleic acids, or lipids, or immunogens, antigens, or allergens found in a conventional leather, such as an animal that naturally expresses the collagen molecules in a biofabricated material. Alternative embodiments may incorporate 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% of one or more of actin, keratin, elastin, fibrin, albumin, globulin, mucin, mucinoids, noncollagen structural proteins, and/or noncollagen nonstructural proteins found in conventional leather.

(178) The collagen used to produce the fibrils in this material may be isolated from a natural source, preferably in a purified form, or it may be recombinantly produced or produced by chemical synthesis. Collagen generally contains 4-hydroxyproline. It may different in chemical structure from collagen obtained from a natural source, for example, if may contain a lower content of, or substantially no 3-hydroxyproline, and optionally, substantially no hydroxylysine, glycosylated or crosslinked amino acid residues, or other post-translational modifications of a collagen amino acid sequence. Alternatively, it may contain a higher content of hydroxylated amino acid residues, glycosylated residues, crosslinks or other chemical modifications.

(179) The biofabricated material component described above generally comprises a network of collagen fibrils which may exhibit a fibril density of 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 network of fibrils can confer a grain texture, such as a top grain texture, feel, or aesthetic on a biofabricated material or biofabricated leather. However, a biofabricated material can exhibit a porosity and other physical properties that are more uniform than a corresponding conventional leather which can be controlled or tuned by control of composition, fibril size, crosslinking and lubricating in a biofabricated product.

(180) In many embodiments, the biofabricated material component of a complex described above will have a top and bottom surface, or an inner and outer surface, comprising the collagen fibrils. One or more of these surfaces may be externally exposed. A single layer of biofabricated material can exhibit substantially identical grain and appearance on both of its sides, unlike conventional leather products where collagen fibril or fiber diameters increase for more inner layers of a hide.

(181) In other embodiments a biofabricated material component of a complex may be cast, molded or otherwise configured into a particular shape which can exhibit substantially uniform properties over its surface(s).

(182) The collagen fibrils in the biofabricated material component of a complex can be tuned to have a particular diameter. The distribution of fibril diameters may exhibit a substantially unimodal distribution, a bimodal distribution, a trimodal distribution or other multimodal distributions. Multimodal distributions may be composed of two or more different preparations of fibrils produced using different fibrillation conditions. In a substantially unimodal distribution >50, 60, 70, 80, 90, 95 or 99% of diameters of the fibrils distribute around a single mode. In bimodal distributions at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% of the fibrils will distribute around one mode. In trimodal and other multimodal distributions, generally, at least about 5, 10, 15, 20, 25, 30% or more (depending on the number of modes) of the fibril diameters will distribute around a mode.

(183) A biofabricated material component may contain fibrils where at least 50, 60, 70, 80, 90, 95, or 99% of the collagen fibrils have diameters between 1 nm and 1 μm. Fibril diameters may be determined by methods known in the art including by visual inspection of micrographs or electron micrographs, such as scanning or transmission electron micrographs. For example, the collagen fibrils may have a collective average or individual fibril diameter ranging from 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 μm (1 μm).

(184) The collagen fibrils in the biofabricated material component described above are usually crosslinked by contact with at least one agent that forms crosslinks between collagen fibrils. Such a crosslinker may be selected from one or more of an amine, carboxylic acid, sulfate, sulfite, sulfonate, aldehyde, hydrazide, sulfhydryl, diazirine, aryl, azide, acrylate, epoxide, phenol, chromium compound, vegetable tannin, and syntan.

(185) Crosslinking may be performed at a crosslinker concentration ranging from 1, 5, 10, 25, 50, 75 to 100 mM and may be conducted under conditions that uniformly expose collagen fibrils to the crosslinker so that the average number of crosslinks formed is uniform and varies by no more than 5, 10, 15, 20, 25, 30, 40, 45, or 50% in identical unit volumes of the material.

(186) A biofabricated 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 the collagen or collagen fibrils in the material. The crosslinker may be present in a covalently or non-covalently form, for example, it may be covalently bound to the collagen fibrils. A crosslinker may be uniformly present in the biofabricated material where its concentration by weight (or by mole) varies by no more than 5, 10, 15, 20, 25, 30, 40, 45, or 50% in identical unit volumes of the material.

(187) The biofabricated material or biofabricated leather component of a complex described above contains a lubricant. Not lubricated materials containing a network of collagen fibrils can be produced, such a precursor substrates for later lubrication, but can lack the flexible and other useful properties of a lubricated product. Lubricants may be incorporated in any amount that facilitates fibril movement or that confers leather-like properties such as flexibility, decrease in brittleness, durability, strength, increase resistance to fracture or tearing, or water resistance. A lubricant content can range from about 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 biofabricated leather.

(188) Lubricants used in the biofabricated component of a complex include, but are not limited to fats, biological, mineral or synthetic oils, cod oil, sulfonated oil, 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, amphiphilic polymers, fatty acids, modified fatty acids, nonionic hydrophilic polymers, nonionic hydrophobic polymers, poly acrylic acids, poly methacrylic, acrylics, natural rubbers, synthetic rubbers, resins, amphiphilic anionic polymer and copolymers, amphiphilic cationic polymer and copolymers and mixtures thereof as well as emulsions or suspensions of these in water, alcohol, ketones, and other solvents.

(189) Solutions or emulsions containing a lubricant may be employed as lubricants, for examples, resins and other hydrophobic lubricants may be applied as emulsions or in solvents suitable for dissolving them. Such solutions may contain any amount of the lubricant suitable for application to or incorporation into a biofabricated 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 a lubricant or the same, or a corresponding amount to volume of other ingredients, such as at least one aqueous solvent, such as water, alcohols, such C.sub.1-C.sub.6 alcohols, like ethanol, ketones, such as C.sub.1-C.sub.6 ketones, aldehydes, such as C.sub.1-C.sub.6 aldehydes, waxes, surfactants, dispersants or other agents. Lubricants may be in various forms, such as O/W or W/O emulsions, in aqueous or hydrophobic solutions, in sprayable form, or other forms suitable for incorporation or application to a biofabricated material.

(190) Lubricants can be distributed uniformly throughout a biofabricated material component such that the concentration of the lubricant in identical unit volumes of the material varies by no more than 5, 10, 15, 20, 35, 30, 40, or 50% and may be compounded or mixed into forms suitable for uniform application to or into a biofabricated material.

(191) Some embodiments of a biofabricated material component, or a complex that incorporates it along with a secondary component, will exhibit many advantageous properties similar to leather or new or superior properties compared to conventional leather.

(192) A biofabricated material component or a complex containing it can have an elastic modulus of at least 100 kPA. It can range from 100 kPa to 1,000 MPa as well as any intermediate value in 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.

(193) A biofabricated material component or a complex containing it can exhibit a uniform elasticity, wherein the elastic modulus varies 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 at other angles) across identical lengths or widths (or volumes or fixed cross-sectional areas) of the material.

(194) A biofabricated material component or a complex containing it may be stretchable and can be elongated 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 in a relaxed state. This range includes all intermediate values.

(195) In some embodiments, a biofabricated material component or a complex containing it can have a tensile strength of at least 1 kPA. It can range from 1 kPa to 100 MPa as well as any intermediate value in 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 embodiments will exhibit a uniform tensile strength, wherein the tensile strength varies 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 at other angles) across identical lengths or widths (or volumes or fixed cross-sectional areas) of the material.

(196) Some biofabricated material components or complexes containing them may exhibit tear strength or resistance of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150 or 200% more than that of a conventional top grain or other leather of the same thickness comprising the same type of collagen, e.g., bovine Type I or Type III collagen, processed using the same crosslinker(s) or lubricants. Some embodiments will exhibit a uniform tear resistance which varies 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 at other angles) across identical lengths or widths (or volumes or fixed cross-sectional areas) of the material. A biofabricated material may have a tear strength ranging from about 1 to 500 N, for example, 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 as well as any intermediate tear strength within this range.

(197) A biofabricated material component, or a composite containing it, may have a softness as determined by ISO 17235 of 2, 3, 4, 5, 6, 7, 8, 10, 11, 12 mm or more. v. Some embodiments will exhibit a uniform softness which varies by no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 100% when measured in otherwise identical unit areas or volumes of the biofabricated material.

(198) In other embodiments, a biofabricated material component, or composite containing it, exhibits a customized thickness to provide top grain like products without the requirement for corium backing. In some embodiments the material or composite will have a top and bottom surface or an inner and outer surface which have identical or substantially the same grain, grain texture, feel, and appearance. Other embodiments of a biofabricated material component or a complex incorporating it are embossed with a pattern, distressed, or printed, stained or painted. Other embodiments of the biofabricated material component or complex containing it have a surface coating or surface finish, which may be distributed uniformly on or throughout the material such that its concentration by weight in identical unit volumes or over unit areas of the material varies by no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50%. Some embodiments of the biofabricated material component or complex containing it may contain a dye, stain, resin, polymer, pigment or paint, optionally, wherein the dye, stain, resin, polymer, pigment or paint is distributed uniformly throughout the material such that its concentration by weight in identical unit volumes or on unit areas of the material or complex varies by no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50%.

(199) Certain embodiments of the biofabricated material component described above may contain fillers as well as other substances or components incorporated into the network of collagen fibrils. For example, some embodiments will contain a filler, such as at least one of polymeric microsphere(s), bead(s), fiber(s), wire(s), or organic salt(s) as a secondary component. These can be selected so as to control the organization of the dehydrated collagen fibril network by keeping the fibrils spaced apart during drying. A filler may be soluble under some conditions or otherwise in a form that permits it removal from a biofabricated material after drying or other processing.

(200) Other embodiments include secondary components of at least one woven or nonwoven material incorporated into the network of collagen fibrils or a network of collagen fibers incorporated into the nonwoven or woven material.

(201) In some embodiments the biofabricated material component or complex incorporating it will be incorporated into other products such as footwear, clothing, sportswear, uniforms, wallets, watchbands, bracelets, luggage, upholstery, or furniture.

Method for Making Biofabricated Component

(202) The method according to the invention includes, but is not limited to, the following embodiments of a method for making a biofabricted material component.

(203) A method for making:

(204) (i) a biofabricated material component comprising a network of non-human collagen fibrils, wherein less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 or 40% by weight of the collagen fibrils in the material are in the form of collagen fibers having a diameter of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μm or more and/or are in the form of fibrils aligned for 25, 50, 100, 150, 200, 250, 300, 350 or 400 μm or more of their lengths; wherein said material contains no more than 10, 15, 20, 25, 30, 35,40, 45, 50, 55 or 60% by weight water; and wherein said material contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40% of a lubricant, comprising in any order: fibrillating an aqueous solution or suspension of non-human collagen molecules into collagen fibrils, crosslinking said collagen fibrils by contacting them with at least one crosslinking agent, dehydrating the crosslinked collagen fibrils so that they contain less than 40% by weight water, incorporating at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40% by weight of at least one lubricant into said material, and, optionally, casting, molding, or otherwise forming said material that comprises a top and bottom surface or an inner and outer surface; or

(205) (ii) a biofabricated material component comprising a network of recombinant non-human collagen fibrils, wherein the collagen contains substantially no 3-hydroxyproline, and optionally, substantially no hydroxylysine; wherein said material contains no more than 10, 15, 20, 25, 30, 35,40, 45, 50, 55 or 60% by weight water; and wherein said material contains at least 1% of a lubricant comprising in any order: fibrillating an aqueous solution or suspension of non-human collagen molecules into collagen fibrils, rosslinking said collagen fibrils by contacting them with at least one crosslinking agent, dehydrating the crosslinked collagen fibrils so that they contain no more than 5, 10, 15, 20 or 25% by weight water, and incorporating at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, or 50% by weight of at least one lubricant into said material, and, optionally, casting, molding, or otherwise forming said material that comprises a top and bottom surface or an inner and outer surface.

(206) The collagen or collagenous material for use in this method may comprise mammalian collagen, such as bovine Type I, Type III collagen or the other types and sources of collagens or collagenous proteins described herein. It may be obtained from a mammal or other animal or, in some embodiments expressed recombinantly by Escherichia coli, Bacillus subtilis, or another bacterium; by Pichia, Saccharomyces, or another yeast or fungi; by a plant cell; by an insect cell or by a mammalian cell.

(207) Collagen for use in the methods disclosed herein may be isolated from cells, such as those described above, that are cultured in vitro, such as from cultured mammalian or animal cells. Alternatively, collagen or collagenous proteins may be obtained by other means, such as by chemical synthesis. It may different in chemical structure from collagen obtained from a natural source, for example, if may contain a lower content of, or substantially no hydroxylysine or 3-hydroxyproline, glycosylated or crosslinked amino acid residues, or other post-translational modifications of a collagen amino acid sequence. Alternatively, it may contain a higher content of hydroxylated amino acid residues, glycosylated residues, crosslinks or other chemical modifications.

(208) Preferably a collagen will not contain hair, hair follicle(s), or fat(s) of an animal that naturally expresses the collagen molecules it contains as these can detract from its uniformity, strength and aesthetic properties. For example, it may contain less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% by weight of actin, keratin, elastin, fibrin, albumin, globulin, mucin, mucinoids, noncollagen structural proteins, and/or noncollagen nonstructural proteins found in conventional leather. It may be substantially free of other collagenous proteins, carbohydrates, nucleic acids, or lipids, or immunogens, antigens, or allergens found in a conventional leather, such as an animal that naturally expresses the collagen molecules in a biofabricated material.

(209) In some embodiments a collagen or collagen-like material may be purified to substantial homogeneity or may have a degree of purity not inconsistent with its ability to form fibrils, for example, it may contain 25, 30, 40, 50, 60, 70, 80, 90, 95 or 99% by weight collagen based on its total protein content or based on its total weight. Mixtures of different types of collagen or collagens from different biological sources may be used in certain embodiments to balance the chemical and physical properties of collagen fibrils or to produce a mixture of fibrils having complementary properties. Such mixtures may contain 1, 5, 10, 25, 50, 75, 95, or 99% by weight of a first collagen and 99, 95, 90, 75, 50, 25, 10 or 1% by weight of a second, third, or subsequent collagen component. These ranges include all intermediate values and ratios of collagens where the total collagen content of all collagen components by weight is 100%.

(210) The methods disclosed herein can provide a biofabricated material component having substantially uniformly distributed fibrils, crosslinked fibrils, dehydrated fibrils and/or lubricated fibrils. For example, the fibrils may be distributed throughout the material so that the concentration by weight (or by number or average numbers) of the collagen fibrils in identical unit volumes of the material varies by no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50%.

(211) In some embodiments the biofabricated material component will be produced by staking the material after the crosslinking, dehydrating and/or lubricating.

(212) In the embodiments described herein, a collagen solution or suspension is fibrillated, for example, by adjusting a salt concentration of the solution or suspension, by adjusting its pH, for example, raising the pH of an acidic solution of collagen, or both. In some embodiments, fibrillation may be facilitated by including a nucleating agent. Salts used for fibrillation include but are not limited to phosphate salts and chloride salts, such as Na.sub.3PO.sub.4, K.sub.3PO.sub.4, KCl, and NaCl. Salt concentration during fibrillation may be adjusted to range from 10 mM to 2M, or pH may be adjusted to pH 5.5, 6.0, 6.5, 7.0, 8.0 or more with an acid, a base, or a buffer. Salt concentration and pH may be simultaneously adjusted to induce or promote fibrillation. In certain embodiments of the methods described herein an aqueous solution or suspension of collagen molecules having a pH below pH 6.0 can be fibrillated by adjusting the pH to pH 6.0 to 8.0.

(213) In some embodiments of the methods described herein, the collagen fibrils will be crosslinked during a process of their formation or after completion of fibrillation. Crosslinking may be performed concurrently with incorporation of a secondary component.

(214) In other embodiments, collagen fibrils are crosslinked by contacting them with at least one amine, carboxylic acid, sulfate, sulfite, sulfonate, aldehyde, hydrazide, sulfhydryl, diazirine, aryl, azide, acrylate, epoxide, phenol, chromium compound, vegetable tannin, and syntan.

(215) One or more crosslinkers may be added at a concentration ranging from 1 mM to 100 mM, for example at a concentration 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.

(216) The time, temperature and other chemical and physical conditions of crosslinking may be selected to provide a particular degree of crosslinking among the collagen fibrils so that the resulting crosslinked fibrils contain a particular degree of one or more different crosslinkages. A resulting crosslinked fibril preparation may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10% or more of a crosslinking agent based on the weight of the crosslinking agent and the weight of the collagen or on the weight of a crosslinked network of collagen fibrils, such as a hydrogel. The crosslinker may be covalently- or non-covalently bound to the collagen fibrils. The numbers of crosslinks between or among collagen molecules, tropocollagen, or fibrils in identical unit volumes of the material after crosslinking, or an average number of crosslinks between collagen molecules, tropocollagen, or collagen fibrils, may vary by no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50%.

(217) The methods described herein require a dehydration or dewatering 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 incorporation of a secondary component.

(218) In some embodiments, dehydrating involves contacting a network of collagen fibrils with acetone, syntan, or other agent that removes bound water from collagen. In other embodiments, some water may be removed from a fibril preparation or crosslinked fibril preparation by filtration or evaporation and water remaining associated with the network of collagen fibrils then removed using a solvent such as acetone or other chemical agents that remove water.

(219) The methods described herein generally require lubrication of the network of collagen fibrils produced. Lubrication may take place during fibrillation, crosslinking, of dehydration, or during any of these steps, or after one or more of these steps is substantially complete. Lubrication may be performed concurrently with incorporation of a secondary component.

(220) In some embodiments lubrication will involve contacting a network of crosslinked collagen fibrils with one or more lubricants such as fats, biological, mineral or synthetic oils, cod oil, sulfonated oil, polymers, organofunctional siloxanes, and other agent used for fatliquoring conventional leather; or mixtures thereof.

(221) In other embodiments, lubricant(s) will be applied using methods that facilitate uniform lubrication of a dehydrated crosslinked network of collagen fibrils, so that the concentration of the lubricant by weight in identical unit volumes of the material varies by no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50%. Such application may occur by dip-coating, spray-coating, vapor deposition, spin-coating, Doctor Blade coating, brush coating as well as other known coating or deposition methods.

(222) In further embodiments of the methods described herein, a surface coating or surface finish is applied to a biofabricated material. While these may be applied to a surface of a material comprising a network of collagen fibrils during the various steps of the preparation of a biofabricated material, they will generally be applied to a crosslinked, dehydrated and lubricated product. The uniform lubrication made possible by the methods described herein facilitates the successful uniform application and adherence of such coatings or finishes.

(223) In other embodiments, the methods described herein can include incorporating or contacting a biofabricated material during the various steps of its preparation or after it has been crosslinked, dehydrated and lubricated with other functional ingredients including, but not limited to a dye, stain, pigment, resin, polymer, or paint. In further embodiments, these functional ingredients may be applied or incorporated under conditions that uniformly distribute these agents on or throughout the material so that their concentration by weight in identical unit volumes of the material varies by no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50%.

(224) In other embodiments, the method described herein involves incorporating a filler of secondary component into a biofabricated material during the various steps of its preparation or after it has been crosslinked, dehydrated and lubricated. Generally, these fillers are incorporated prior to dehydration, for example, during fibrillation or crosslinking. Such fillers include, but are not limited to polymeric microspheres, beads, fibers, wires, or organic salts.

(225) Some embodiments of the methods described above will involve incorporating into or onto a biofabricated material during or after its preparation at least one woven or nonwoven material. For example, by filtering crosslinked fibrils using a woven or nonwoven paper or fabric material. Other embodiments involve incorporating a biofabricated material during or after its preparation into at least one woven or nonwoven material.

(226) Commercial embodiments of the method involving incorporating a biofabricated material into products such as footwear, clothing, sportswear, uniforms, wallets, watchbands, bracelets, luggage, upholstery, furniture, or other industrial, commercial or consumer products.

(227) The following non-limiting Examples are illustrative of the present invention. The scope of the invention is not limited to the details described in these Examples.

EXAMPLE 1

Controlling the Thickness of Biofabricated Leather

(228) The thickness of the biofabricated material used in a composite may be controlled by adjusting collagen content. Hydrogels of extracted bovine type I collagen were formed at different collagen concentrations and volumes to produce dried collagen materials of different thicknesses. Collagen was dissolved in 0.01N HCl at either 5 g/L or 9 g/L, then 1 part 10× PBS was added to 9 parts dissolved collagen to induce collagen fibrillation and gel formation.

(229) Solutions of either 0.8 L or 1.6 L of the fibrillating collagen were then cast into molds and incubated at 25° C. to allow hydrogel formation. The 0.8 L solution produced a gel of 1.5 cm thickness while the 1. 7 L solution produced a gel of 3.0 cm thickness. These gels were dehydrated and lubricated in acetone, then dried and mechanically staked into a leather like material. The thickness of the final dried material correlated with the total amount of collagen in the starting hydrogel.

(230) The thickness of biofabricated leather was controlled by varying its total collagen content. Samples A, B and C were produced using 4, 7.2 or 14.4 gr of collagen, respectively, in a volume (hydrated gel area) of 525 cm.sup.2. Biofabricated leathers were produced from each sample by crosslinking, lubricating and dewatering As shown in Table 1, increasing the content of collagen in the gels increased the thickness of the resulting biofabricated leather.

(231) TABLE-US-00001 TABLE 1 Gel Total Leather Gel Density Gel Volume Thickness Collagen Thickness Sample (g/L) (L) (cm) (g) (mm) A 5 0.8 1.5 4 0.1 B 9 0.8 1.5 7.2 0.2 C 9 1.6 3.0 14.4 1.1

EXAMPLE 2

Production of Biofabricated Leather from Type I Collagen

(232) The biofabricated component of the composites described herein may be produced from Type I collagen.

(233) Type I collagen was purchased from Wuxi Biot Bio-technology Company, ltd. (Medical Collagen Sponge). The collagen was isolated from bovine tendon by acid treatment followed by pepsin digestion, and was purified by size exclusion chromatography, frozen and lyophilized.

(234) The lyophilized protein (4.1 g) was dissolved in 733 ml 0.01 N HCL using an overhead mixer. After the collagen was adequately dissolved, as evidenced by a lack of solid collagen sponge in the solution (at least 1 hr mixing at 1600 rpm), 82 uL of the tanning agent Relugan GTW was added to the solution followed by 81 mL of a 10× PBS, pH 11.2 to raise the pH of the solution to 7.2.

(235) The solution was then mixed for 3 min before pouring the solution into a silicon mold. The collagen solution was incubated in the silicon mold for 2 hrs at 25° C. to allow the collagen to fibrillate into a viscoelastic hydrogel.

(236) Plateau of rheological properties along with solution opacity (as measured by absorbance of 425 nm light) indicated that fibrillation was complete at this point and the presence of collagen fibrils was confirmed with scanning electron microscopy (FIG. 3) and transmission electron microscopy (FIG. 4).

(237) The fibrillated collagen hydrogel was removed from the molds and placed in 700 mL of acetone in a plastic jar and shaken on an orbital shaker at 40 rpm at 25° C. The hydrogel was dehydrated by refreshing the acetone after an overnight incubation followed by 5× 1 hr washes and another overnight incubation. Acetone was refreshed after each wash to remove water from the gel.

(238) Following acetone dehydration, the collagen gel was incubated in a fat liquor solution containing 20% (v/v) of either cod liver oil or castor oil in 80% acetone or ethanol, respectively, overnight while shaking at 40 rpm.

(239) Following incubation in the fat liquor solution, the collagen gel was dried at 37C. After drying, the material became soft and leather-like or a biofabricated leather. Excess oil can be removed to improve the leather-like aesthetic of the materials.

(240) Sample weights and mechanical analysis confirmed penetration of the oils into the fibrillar gel. By dissolving the oils in good solvents, the oils were able to penetrate the fibrillar collagen network as evidenced by an increase in dry weight of the materials as well as a decrease in the elastic modulus of the material compared to hydrogels that we not dehydrated or fat liquored in solvent.

(241) The biofabricated leather had a grain texture on both the top and bottom surfaces and consistently absorbed dyes on both the top and bottom surfaces.

EXAMPLE 3

Production of Biofabricated Leather from Type III Collagen

(242) The biofabricated component of the composites described herein may be produced using Type III collagen.

(243) A solution of recombinant collagen type III at 2.5 mg/ml in 0.01 N HCl (FibroGen, Inc.) was fibrillated by adding 1 part of a 200 mM of sodium phosphate solution (22 mL), pH 11.2 to 9 parts of the collagen solution (200 mL) to increase the pH to 7 and stirred 2 hours at room temperature.

(244) Fibrillation was confirmed by measuring 400 nm absorbance of the solution over time.

(245) After fibrillation, the fibrils were tanned by adding Relugan GTW (2% w/w offer on the collagen) to the fibril suspension and mixing for 30 min.

(246) 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 10 mg/ml fibril pellet was further centrifuged using an ultra-centrifuge at 21,000 RPM for 30 minutes yielding a fibril gel with a concentration of ˜40-50 mg/ml.

(247) The physical properties of the fibril gel were assessed with a rheometer.

(248) The storage modulus and complex viscosity demonstrate a mostly elastic material.

(249) This fibril gel was then dried in a food dehydrator set to 37° C. for 18 hrs.

(250) After drying, the material was dyed and retanned by incubating in a solution of Lowepel acid black dye (2% w/w offer on the collagen) and Lubritan WP (20% w/w offer on the collagen).

(251) The material was drummed in this solution and squeezed to ensure penetration of dye and syntan into the material. The material was then finally dried and staked to produce a leather-like material.

EXAMPLE 4

Production of Biofabricated Leather from Type III Collagen

(252) The biofabricated component of the composites described herein may be produced using Type II collagen.

(253) Recombinant collagen type III was purchased from Fibrogen, Inc. The collagen was supplied at a concentration of 2.5 mg/mL in 0.01N HCl.

(254) To initiate the assembly of collagen fibrils, 1 part 200 mM Na.sub.2HPO.sub.4, pH 11.2 (100 mL) was added to 9 parts of the stock collagen type III solution at room temperature to bring the solution to pH 7.2. The solution was mixed at 1600 rpm for 1 hr using an overhead mixer.

(255) After 1 hr of stirring, the collagen fibrils were reacted with Relugan GTW which was added to the solution at a 2% (w/w) offer on the mass of the collagen. The solution was mixed at 1600 rpm for 1 hr using an overhead mixer.

(256) Lipoderm A1 and Tanigan FT were then added to the solution at offers of 80% (w/w) each on the mass of the collagen. The solution was mixed at 1600 rpm for 30 min using an overhead mixer. The pH of the solution was then lowered to 4 using a 10% (v/v) formic acid solution. The solution was mixed at 1600 rpm for 30 min using an overhead mixer.

(257) 144 mL of the solution was then filtered through a 47 mm Whatman no. 1 membrane using a Buchner funnel attached to a vacuum pump (pressure of −27 in Hg) and a rubber dam on top of the Buchner funnel. Vacuum was pulled for 18 hrs.

(258) The concentrated fibril tissue was then allowed to dry under ambient conditions and hand staked for 30 min by rolling, bending and pulling the material to produce a leather-like material.

EXAMPLE 5

Expancel

(259) Type I bovine collagen, isolated from bovine tendon by acid treatment followed by pepsin digestion and purified by size exclusion chromatography, frozen and lyophilized, was purchased from Wuxi Biot Bio-technology co., Ltd. (Medical Collagen Sponge).

(260) Using an overhead mixer, 10 gr of the lyophilized collagen protein was dissolved by mixing at 1,600 rpm in 1 L of 0.01N HCl, pH 2, for at least one hour until no solid collagen sponge was present.

(261) 111.1 ml of 200 mM sodium phosphate (pH adjusted to 11.2 with sodium hydroxide) was then added to raise the pH of the collagen solution to 7.2.

(262) The pH 7.2 collagen solution was then stirred for 10 minutes and 0.1 ml of a 20% Relugan GTW (BASF) as a crosslinker, which was 2% on the weight of collagen, was added to produce crosslinked collagen fibrils.

(263) The crosslinked collagen fibrils were then mixed with 5 ml of 20% Tanigan FT (Lanxess) and stirred for one hour.

(264) Subsequently, 1 gr of Expancel Microspheres 461 WE 20 d36 (AkzoNobel), which is 10% of the weight of the collagen) and 40 ml of Truposol Ben (Trumpler), which is 80% of the weight of the collagen, were added and stirred for an additional hour using an overhead stirrer.

(265) The pH of the solution was the reduced to pH 4.0 by addition of 10% formic acid and stirred for an hour.

(266) After the reduction in pH, 150 ml of the solution was filtered through 90 mm Whatman No. 1 membrane using a Buchner funnel attached to a vacuum pump at a pressure of −27 mmHg.

(267) The concentrated fibril tissue was then allowed to dry under ambient conditions and hand staked for 30 minutes by rolling, bending and pulling the material to produce a leather-like material. This material may be incorporated into the composites described herein.

EXAMPLE 6

Titanium Dioxide (White Pigment)

(268) Type I bovine collagen was purchased from Wuxi Biot Bio-technology co., Ltd. (Medical Collagen Sponge). This source of collagen is type I collagen isolated from bovine tendon by acid treatment followed by pepsin digestion and purified by size exclusion chromatography, frozen and lyophilized. The lyophilized protein (10 grams) was dissolved in 1 L of 0.01N HCl, pH 2 using an overhead mixer. After the collagen was adequately dissolved, as evidenced by a lack of solid collagen sponge in the solution (at least 1 hr mixing at 1,600 rpm), 111.1 ml of 200 millimolar sodium phosphate (pH adjusted to 11.2 with sodium hydroxide) to raise the pH of the solution to 7.2. The resulting collagen solution was stirred for 10 minutes and 0.1 ml of a 20% Relugan GTW (BASF) crosslinker solution, which was 2% on the weight of collagen.

(269) To the crosslinked collagen fibril solution was added 5 mls of 20% Tanigan FT (Lanxess) was added followed by stirring for one hour.

(270) Following Tanigan-FT addition, 1 gr Expancel Microspheres (10% on the weight of collagen) 461 WE 20 d36 (AkzoNobel), 40 mis (80% on the weight of collagen) of Truposol Ben (Trumpler) and 2 mls (10% on the weight of collagen) of PPE White HS a pa (Stahl) was added and stirred for additional hour using an overhead stirrer.

(271) The pH of the solution was reduced to 4.0 using 10% formic acid and stirred for an hour.

(272) After pH change, 150 ml of the solution was filtered through 90 mM Whatman No. 1 membrane using a Buchner funnel attached to a vacuum pump at a pressure of −27 mmHg.

(273) The concentrated fibril tissue was then allowed to dry under ambient conditions and hand staked for 30 minutes by rolling, bending and pulling the material to produce a leather-like material. This material may be incorporated into the composites described herein.

EXAMPLE 7

Hycar Resin (26552)

(274) Bovine collagen was purchased from Wuxi Biot Bio-technology co., Ltd. (Medical Collagen Sponge). This source of collagen is type I collagen isolated from bovine tendon by acid treatment followed by pepsin digestion and purified by size exclusion chromatography, frozen and lyophilized.

(275) The lyophilized protein (10 grams) was dissolved in 1 litre of 0.01N HCl, pH 2 using an overhead mixer. After the collagen was adequately dissolved, as evidenced by a lack of solid collagen sponge in the solution (at least 1 hr mixing at 1600 rpm), 111.1 ml of 200 mM sodium phosphate (pH adjusted to 11.2 with sodium hydroxide) to raise the pH of the solution to 7.2.

(276) The resulting collagen solution was stirred for 10 minutes and 0.1 ml of a 20% Relugan GTW (BASF) crosslinker solution, which was 2% of the weight of the collagen, tanning agent solution was added.

(277) To the crosslinked collagen fibril solution was added 5 mls of 20% Tanigan FT (Lanxess) was added and stirred for one hour. Following Tanigan-FT addition, 1 gram Expancel Microspheres (10% on the weight of collagen) 461 WE 20 d36 (AkzoNobel), 40 mls (80% on the weight of collagen) of Truposol Ben (Trumpler) and 2 mls (10% on the weight of collagen) of PPE White HS a pa (Stahl) was added and added and stirred for additional hour using an overhead stirrer.

(278) The pH of the solution was reduced to 4.0 using 10% formic acid and a variety of offers of Hycar Resin 26552 (Lubrizol) was added and stirred for an additional hour. Following pH change and resin addition 150 ml of the solution was filtered through 90 millimeter Whatman No. 1 membrane using a Buchner funnel attached to a vacuum pump at a pressure of −27 mmHg. To facilitate activation, the Hycar Resin 26552 is mixed with the fibril solution and heated at 50° C. for 2 hrs.

(279) The concentrated fibril tissue was then allowed to dry under ambient conditions and hand staked for 30 minutes by rolling, bending and pulling the material to produce a leather-like material. This material may be incorporated into the composites described herein.

(280) The addition of resin lead to improved mechanical properties as shown below in FIG. 1.

(281) After pH change, 150 ml of the solution was filtered through 90 millimeter Whatman No. 1 membrane using a Buchner funnel attached to a vacuum pump at a pressure of −27 mmHg. The solution immediately formed a green precipitate and was unable to be filtered.

(282) TABLE-US-00002 Example Substrates Crosslinker Dehydrater Lubricant Result 5 Type I collagen + Relugan Tanigan Truposol Leather- Expancel GTW FT like microspheres material 6 Type I collagen + Relugan Tanigan ″ Leather- Expancel GTW FT like microspheres material 7 Type I collagen + Relugan Tanigan ″ Leather- Expancel GTW FT like microspheres material, better mechanical properties

(283) After Relugan is a retanning agent based on polymer, resin or aldehyde. Tanigan is a sulfone-based syntan. Truposol Ben is a fatliquor for chrome-free leather. Lipoderm Liquor A1 is a fatliquor based on long chain alcohol, paraffin, anionic surfactants, in water Hycar Resin 26552: formaldehyde-free acrylic based emulsion.

EXAMPLE 8

Encapsulated Carbon Fibers

(284) Bovine collagen was purchased from Wuxi Biot Bio-technology co., Ltd. (Medical Collagen Sponge). This source of collagen is type I collagen isolated from bovine tendon by acid treatment followed by pepsin digestion and purified by size exclusion chromatography, frozen and lyophilized. The lyophilized protein (4.1 g) was dissolved in 733 mL of 0.01N HCl, pH 2 using an overhead mixer. After the collagen was adequately dissolved, as evidenced by a lack of solid collagen sponge in the solution (at least 1 hr mixing at 1,600 rpm), 82 uL of the tanning agent Relugan GTW was added to the solution followed by 81 mL of a 10× PBS, pH 11.2 to raise to pH of the solution to 7.2. The solution was mixed for 3 min, then poured into a mold containing a secondary material of 0.25 inch chopped carbon fibers. Carbon fibers were purchased from Fibre Glast Developments Corp. The carbon fibers were mixed in the collagen solution to disperse the fibers throughout the collagen matrix. The collagen solution was incubated in the silicon mold for 2 hrs at 25° C. to allow the collagen to fibrillate into a viscoelastic hydrogel, encapsulating the carbon fibers.

(285) The fibrillated collagen hydrogels with encapsulated carbon fibers were removed from the molds and dehydrated in a series of acetone solutions (5×1 hr at 25° C., 40 rpm). Following acetone dehydration, the collagen gel was incubated in a fat liquor solution containing 20% (v/v) cod liver oil in 80% acetone overnight while shaking at 40 rpm. Following incubation in the cod liver oil solution, the collagen gel was dried at 37° C. The fibrillated collagen hydrogel was removed from the molds and placed in 700 mL of acetone in a plastic jar and shaken on an orbital shaker at 40 rpm at 25° C. The hydrogel was dehydrated by refreshing the acetone after an overnight incubation followed by 5×1 hr washes and another overnight incubation. Acetone was refreshed after each wash to remove water from the gel. Following acetone dehydration, the collagen gel was incubated in a fat liquor solution containing 20% (v/v) of either cod liver oil or castor oil in 80% acetone or ethanol, respectively, overnight while shaking at 40 rpm. Following incubation in the fat liquor solution, the collagen gel was dried at 37 C. After drying, the material becomes soft and leather-like. Further, the carbon fibers are encapsulated within the tanned and fat liquored collagen network and can be handled without delaminating or pulling out of the biofabricated leather.

EXAMPLE 9

Layered Non-Woven

(286) Bovine collagen was dissolved as in Example 8. Once the collagen was dissolved, as evidenced by a lack of solid collagen sponge in the solution (at least 1 hr mixing at 1600 rpm), 0.2 g of Lowepel acid black dye dissolved in 5 mL DI water was added dropwise to the stirring collagen solution. The dye was mixed for 1 hr @ 1600 rpm to allow dye fixation to collagen. 82 uL of the tanning agent Relugan GTW was then added to the solution followed by 81 mL of a 10× PBS, pH 11.2 to increase to pH of the solution to 7.2. The solution was mixed for 3 min and integrated with a secondary material of wool nonwoven felt using a vacuum technique. Wool felts were purchased from US Felts and treated with 1M hydroxylamine, lg/L triton n-57 surfactant, pH 8 overnight @ 50° C. to remove surface lipids and increase wettability and reactivity of the wool fibers. 60 mL of the collagen precursor solution was pulled into the wool felt under house vacuum. A gradient of dye was visible from the top surface of the felt to the bottom. Following integration with the collagen solution, the wool felt was laid topside down onto a freshly cast collagen precursor solution. The collagen and wool felt was incubated for 2 hrs @ 25° C. to allow fibrillation. After fibrillation, the material was dried in a dehydrator @ 37 C. The dried material was staked into a soft, leather-like material with wool backing.

EXAMPLE 10

Embedded Fabrics with Photoluminescent Patterns

(287) Qdots functionalized with a primary amine and PEG spacer were purchased from Sigma. The Qdots were diluted 1:10 in a collagen precursor solution (5 wt % col type I, 1× PBS, 0.02 uL GTW/mg col) chilled on ice. The Qdot/collagen solution was then screen printed onto a secondary material of silk woven fabric in the shape of an “M”. The Qdot/collagen screen printed fabric was incubated for 1 hr @ RT before encapsulating the fabric in a collagen gel. As in Example 2, the collagen precursor solution (5 mg/mL col type I, 1× PBS, 0.02 uL GTW/mg col) was cast into a silicon mold 3 min after adding the PBS and the fabric was placed in the middle of the collagen solution. The solution was incubated at 25° C. for 1 hr to allow fibrillation and then the gel with encapsulated fabric was dehydrated in a series of acetone, followed by fat liquoring in cod oil/acetone and drying. After drying and staking, the material was exposed to a UV light source to illuminate the embedded Qdot “M”.

EXAMPLE 11

Embedded Three-Dimensional Objects

(288) Qdots functionalized with a primary amine and PEG spacer were purchased from Sigma. The Qdots were diluted 1:10 in a Slygard 184 polydimethylsiloxane (PDMS) base followed by mixing the Qdot/base 10:1 with a curing agent. After mixing, the Qdot/base/curing agent solution was cast into a mold in the shape of an “M”. The PDMS “M” was cured overnight at 40° C. then removed from the mold to produce an elastomeric and photoluminescent “M”. As in Example 2, the collagen precursor solution (5 mg/mL col type I, 1× PBS, 0.02 uL GTW/mg col) was cast into a silicon mold 3 min after adding the PBS and the PDMS “M” was placed in the middle of the collagen solution. The solution was incubated at 25° C. for 1 hr to allow fibrillation and then the gel with encapsulated fabric was dehydrated in a series of acetone, followed by fat liquoring in cod oil/acetone and drying (see Example 2 for details). After drying and staking, the encapsulated three-dimensional “M” produced a tactile pattern on the surface of the biofabricated leather in the shape of the “M”. In addition, the material was exposed to a UV light source to illuminate the embedded Qdots in thE PDMS “M”.

EXAMPLE 12

Wool Felt Composite

(289) The process of Example 9 is repeated with wool felt and the collagen precursor solution of Example 6. A composite leather is formed.

EXAMPLE 13

Lycra® Composite

(290) A 3″ by 3″ sample of the leather of Example 2 is laminated with a 3″ by 3″ polyester-polyurethane copolymer felt (Lycra®) with a holt melt adhesive at 50° C. A leather-secondary material backed composite is formed.

EXAMPLES 14-20

(291) As shown by the Examples 14-20 below, the biofabricated material of the invention can be successfully applied or integrated in to secondary components to produce strong leather-like composites.

EXAMPLE 14

Spacer Fabric

(292) Bovine collagen was purchased from Wuxi Biot Bio-technology co., Ltd. (Medical Collagen Sponge). This source of collagen is type I collagen isolated from bovine tendon by acid treatment followed by pepsin digestion and purified by size exclusion chromatography, frozen and lyophilized. The lyophilized protein (10 grams) was dissolved in 1 litre of 0.01N HCl, pH 2 using an overhead mixer.

(293) After the collagen was adequately dissolved, as evidenced by a lack of solid collagen sponge in the solution (at least 1 hr mixing at 1,600 rpm), 111.1 millilitres of 200 millimolar sodium phosphate (pH adjusted to 11.2 with sodium hydroxide) to raise the pH of the solution to 7.2. The resulting collagen solution was stirred for 10 minutes and 0.1 millilitres of a 20% Relugan GTW (BASF) (2% on the weight of collagen) tanning agent solution was added.

(294) To the crosslinked collagen fibril solution was added 5 mls of 20% Tanigan FT (Lanxess) was added and stirred for one hour.

(295) Following Tanigan-FT addition, 1 gram Expancel Microspheres (10% on the weight of collagen) 461 WE 20 d36 (AkzoNobel) and 40 mls (80% on the weight of collagen) of Truposol Ben (Trumpler) was added and added and stirred for additional hour using an overhead stirrer. The pH of the solution was changed to 4.0 using 10% formic acid and stirred for an hour.

(296) After pH change a 75 mm disc of a 100% polyester 3D spacer fabric was cut out and placed on top of a 90 mm Whatman no. 1 membrane, a thin layer of high vacuum grease (Dow Corning) was applied around the rim of the membrane to hold down the material whilst filtering.

(297) 150 mL of the solution was then filtered through the textile and Whatman no. 1 membrane using a Buchner funnel attached to a vacuum pump (pressure of −27 inHg). Vacuum was pulled for 40 mins.

(298) The concentrated fibril tissue was then allowed to dry in a humidity chamber at 20° C. at 65%. When the concentrated fibril tissue had reached 20% moisture it was pressed in a carver press 50° C. for 10 mins at 1 metric tonne of pressure and hand staked for 30 min by rolling, bending and pulling the material to produce a leather-like material.

(299) The spacer fabric remained integrated into the fibril tissue, resulting in a leather-like material that had an exposed fabric back on one side and an embossed pattern on its surface created by the embedded textile. The material was finished with a high performance coating, routinely used in the footwear industry.

EXAMPLE 15

(300) The procedure of Example 2 was repeated substituting the 75 mm disc for smaller sections that are zonally integrated into the end material.

EXAMPLE 16

Polyester Mesh Netting

(301) Bovine collagen was purchased from Wuxi Biot Bio-technology co., Ltd. (Medical Collagen Sponge). This source of collagen is type I collagen isolated from bovine tendon by acid treatment followed by pepsin digestion and purified by size exclusion chromatography, frozen and lyophilized. The lyophilized protein (10 grams) was dissolved in 1 litre of 0.01N HCl, pH 2 using an overhead mixer.

(302) After the collagen was adequately dissolved, as evidenced by a lack of solid collagen sponge in the solution (at least 1 hr mixing at 1600 rpm), 111.1 millilitres of 200 millimolar sodium phosphate (pH adjusted to 11.2 with sodium hydroxide) to raise the pH of the solution to 7.2.

(303) The resulting collagen solution was stirred for 10 minutes and 0.1 millilitres of a 20% Relugan GTW (BASF) (2% on the weight of collagen) tanning agent solution was added.

(304) To the crosslinked collagen fibril solution was added 5 mls of 20% Tanigan FT (Lanxess) was added and stirred for one hour. Following Tanigan-FT addition, 1 gram Expancel Microspheres (10% on the weight of collagen) 461 WE 20 d36 (AkzoNobel) and 40 mls (80% on the weight of collagen) of Truposol Ben (Trumpler) was added and added and stirred for additional hour using an overhead stirrer. The pH of the solution was changed to 4.0 using 10% formic acid and stirred for an hour.

(305) After pH change a 75 mm disc of a polyester mesh netting was cut out and placed on top of a 90 mm Whatman no. 1 membrane, a thin layer of high vacuum grease (Dow Corning) was applied around the rim of the membrane to hold down the material whilst filtering. 150 mL of the solution was then filtered through the textile and Whatman no. 1 membrane using a Buchner funnel attached to a vacuum pump (pressure of −27 inHg). Vacuum was pulled for 40 mins.

(306) The concentrated fibril tissue was then allowed to dry in a humidity chamber at 20° C. at 65%.

(307) When the concentrated fibril tissue had reached 20% moisture it was pressed in a carver press 50° C. for 10 mins at 1 metric tonne of pressure and hand staked for 30 min by rolling, bending and pulling the material to produce a leather-like material.

(308) The fabric was removed 15 mins into staking, resulting in a double-sided grain with a different textured surface, and aesthetic, on each side of the material. The material was finished with a high performance coating, routinely used in the footwear industry.

EXAMPLE 17

Polyester Textile

(309) The procedure of Example 3 is repeated with the additional step of laminating a 100% polyester technical textile to one side of the material.

EXAMPLE 18

Coating

(310) Bovine collagen was purchased from Wuxi Biot Bio-technology co., Ltd. (Medical Collagen Sponge). This source of collagen is type I collagen isolated from bovine tendon by acid treatment followed by pepsin digestion and purified by size exclusion chromatography, frozen and lyophilized. The lyophilized protein (10 grams) was dissolved in 1 litre of 0.01N HCl, pH 2 using an overhead mixer.

(311) After the collagen was adequately dissolved, as evidenced by a lack of solid collagen sponge in the solution (at least 1 hr mixing at 1600 rpm), 111.1 millilitres of 200 millimolar sodium phosphate (pH adjusted to 11.2 with sodium hydroxide) to raise the pH of the solution to 7.2.

(312) The resulting collagen solution was stirred for 10 minutes and 0.1 millilitres of a 20% Relugan GTW (BASF) (2% on the weight of collagen) tanning agent solution was added.

(313) To the crosslinked collagen fibril solution was added 5 mls of 20% Tanigan FT (Lanxess) was added and stirred for one hour. Following Tanigan-FT addition, 1 gram Expancel Microspheres (10% on the weight of collagen) 461 WE 20 d36 (AkzoNobel) and 40 mls (80% on the weight of collagen) of Truposol Ben (Trumpler) was added and added and stirred for additional hour using an overhead stirrer. The pH of the solution was changed to 4.0 using 10% formic acid and stirred for an hour.

(314) After pH change 150 mL of the solution was then filtered through a 90 mm Whatman no. 1 membrane using a Buchner funnel attached to a vacuum pump (pressure of −27 inHg). Vacuum was pulled for 40 mins. The concentrated fibril tissue was then allowed to dry in a humidity chamber at 20° C. at 65%.

(315) When the concentrated fibril tissue had reached 20% moisture it was pressed in a carver press 50° C. for 10 mins at 1 metric tonne of pressure and hand staked for 30 min by rolling, bending and pulling the material to produce a leather-like material.

(316) The material was finished with a high performance coating, routinely used in the footwear industry. The finished material was then glued over three stripes of leather board to create a three dimensional surface texture and aesthetic.

EXAMPLE 19

Polyester Mesh Netting

(317) Bovine collagen was purchased from Wuxi Biot Bio-technology co., Ltd. (Medical Collagen Sponge). This source of collagen is type I collagen isolated from bovine tendon by acid treatment followed by pepsin digestion and purified by size exclusion chromatography, frozen and lyophilized. The lyophilized protein (10 grams) was dissolved in 1 litre of 0.01N HCl, pH 2 using an overhead mixer. After the collagen was adequately dissolved, as evidenced by a lack of solid collagen sponge in the solution (at least 1 hr mixing at 1600 rpm), 111.1 millilitres of 200 millimolar sodium phosphate (pH adjusted to 11.2 with sodium hydroxide) to raise the pH of the solution to 7.2.

(318) The resulting collagen solution was stirred for 10 minutes and 0.1 millilitres of a 20% Relugan GTW (BASF) (2% on the weight of collagen) tanning agent solution was added.

(319) To the crosslinked collagen fibril solution was added 5 mls of 20% Tanigan FT (Lanxess) was added and stirred for one hour. Following Tanigan-FT addition, 1 gram Expancel Microspheres (10% on the weight of collagen) 461 WE 20 d36 (AkzoNobel) and 40 mls (80% on the weight of collagen) of Truposol Ben (Trumpler) was added and added and stirred for additional hour using an overhead stirrer. The pH of the solution was changed to 4.0 using 10% formic acid and stirred for an hour.

(320) After pH change three strips (each 10 mm wide) of 100% polyester mesh netting fabric was cut out and placed horizontally (with a 5 mm gap in between each piece) on top of a 90 mm Whatman no. 1 membrane, a thin layer of high vacuum grease (Dow Corning) was applied around the rim of the membrane to hold down the material whilst filtering.

(321) 150 mL of the solution was then filtered through the textile and Whatman no. 1 membrane using a Buchner funnel attached to a vacuum pump (pressure of −27 inHg). Vacuum was pulled for 40 mins.

(322) The concentrated fibril tissue was then allowed to dry in a humidity chamber at 20° C. at 65% and when it had reached 20% moisture it was placed in an oven at 50° C. for 2 hours. The mesh netting fabric remained integrated into the fibril tissue, resulting in a fabric-backed material that had a fabric embossed pattern on its surface created by the embedded textile.

(323) The contraction of the concentrated fibril tissue around the mesh netting created a three-dimensional end material that self-assembled; this process can be controlled to create a desired end shape.

EXAMPLE 20

Polyester Mesh Netting

(324) Bovine collagen was purchased from Wuxi Biot Bio-technology co., Ltd. (Medical Collagen Sponge). This source of collagen is type I collagen isolated from bovine tendon by acid treatment followed by pepsin digestion and purified by size exclusion chromatography, frozen and lyophilized. The lyophilized protein (4.1 g) was dissolved in 733 mL of 0.01N HCl, pH 2 using an overhead mixer.

(325) After the collagen was adequately dissolved, as evidenced by a lack of solid collagen sponge in the solution (at least 1 hr mixing at 1600 rpm), 82 uL of the tanning agent Relugan GTW was added to the solution followed by 81 mL of a 10× PBS, pH 11.2 to raise to pH of the solution to 7.2.

(326) The solution was mixed for 3 min, then poured into a mold containing a piece of 100% polyester mesh netting (measuring 75 mm×200 mm) that was pinned into place—suspended 5 mm above the bottom of the mold.

(327) The collagen solution was incubated in the silicon mold for 2 hrs at 25° C. to allow the collagen to fibrillate into a viscoelastic hydrogel, 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 jar and shaken on an orbital shaker at 40 rpm at 25° C.

(328) The hydrogel was dehydrated by refreshing the acetone after an overnight incubation followed by 5×1 hr washes and another overnight incubation. Acetone was refreshed after each wash to remove water from the gel. Following acetone dehydration, the collagen gel was incubated in lubricating solution containing 20% (v/v) of either cod liver oil or castor oil in 80% acetone or ethanol, respectively, overnight while shaking at 40 rpm. Following incubation in the fat liquor solution, the collagen gel was dried at 37° C.

(329) After drying, the material becomes soft and leather-like. Further, the encapsulated mesh netting creates a doubled sided textured grain surface, which can be modified almost infinitely depending on the type, and structure, of the fabric embedded.

Interpretation of Description

(330) Terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. For example, as used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and/or “comprising,” when used in this specification, specify the presence of stated features, steps, operations, elements, and/or components, but do not preclude 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 items and may be abbreviated as “/”.

(331) Spatially relative terms, such as “under”, “below”, “lower”, “over”, “upper” and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures is inverted, elements described as “under” or “beneath” other elements or features would then be oriented “over” the other elements or features. Thus, the exemplary term “under” can encompass both an orientation of over and under. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Similarly, the terms “upwardly”, “downwardly”, “vertical”, “horizontal” and the like are used herein for the purpose of explanation only unless specifically indicated otherwise.

(332) Although 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 indicates otherwise. These terms may be used to distinguish one feature/element from another feature/element. Thus, a first feature/element discussed below could be termed a second feature/element, and similarly, a second feature/element discussed below could be termed a first feature/element without departing from the teachings of the present invention.

(333) Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising” means various components can be co jointly employed in the methods and articles (e.g., compositions and apparatuses including device and methods). For example, the term “comprising” will be understood to imply the inclusion of any stated elements or steps but not the exclusion of any other elements or steps.

(334) As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word “substantially”, “about” or “approximately,” even if the term does not expressly appear. The phrase “about” or “approximately” may be used when describing magnitude and/or position to indicate that the value and/or position described is within a reasonable expected range of values and/or positions. For example, a numeric value may have a value that is +/−0.1% of the stated value (or range of values), +/−1% of the stated value (or range of values), +/−2% of the stated value (or range of values), +/−5% of the stated value (or range of values), +/−10% of the stated value (or range of values), etc. Any numerical range recited herein is intended to include all sub-ranges subsumed therein.

(335) When a feature or element is herein referred to as being “on” another feature or element, it can be directly on the other feature or element or intervening features and/or elements may also be present. In contrast, when a feature or element is referred to as being “directly on” another feature or element, there are no intervening features or elements present. It will also be understood that, when a feature or element is referred to as being “connected”, “attached” or “coupled” to another feature or element, it can be directly connected, attached or coupled to the other feature or element or intervening features or elements may be present. In contrast, when a feature or element is referred to as being “directly connected”, “directly attached” or “directly coupled” to another feature or element, there are no intervening features or elements present. Although described or shown with respect to one embodiment, the features and elements so described or shown can apply to other embodiments. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed “adjacent” another feature may have portions that overlap or underlie the adjacent feature.

(336) Although various illustrative embodiments are described above, any of a number of changes may be made to various embodiments without departing from the scope of the invention as described by the claims. For example, the order in which various described method steps are performed may often be changed in alternative embodiments, and in other alternative embodiments one or more method steps may be skipped altogether. Optional features of various device and system embodiments may be included in some embodiments and not in others. Therefore, the foregoing description is provided primarily for exemplary purposes and should not be interpreted to limit the scope of the invention as it is set forth in the claims.

(337) The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. As mentioned, other embodiments may be utilized and derived there from, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term “invention” merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is, in fact, disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.

INCORPORATION BY REFERENCE

(338) All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference, especially referenced is disclosure appearing in the same sentence, paragraph, page or section of the specification in which the incorporation by reference appears.