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(11) EP 3 205 754 A1

(12) EUROPEAN PATENT APPLICATION

(43) Date of publication: (51) Int Cl.: 16.08.2017 Bulletin 2017/33 D01F 4/00 (2006.01) D06C 29/00 (2006.01) D06M 10/00 (2006.01) D01D 5/00 (2006.01) (2006.01) (21) Application number: 17156318.2 C08H 1/06

(22) Date of filing: 15.02.2017

(84) Designated Contracting States: (72) Inventors: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB • Marga, Francoise Suzanne GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO Brooklyn, NY New York 11220 (US) PL PT RO RS SE SI SK SM TR • Jakab, Karoly Robert Designated Extension States: Brooklyn, NY New York 11220 (US) BA ME • Purcell, Brendan Designated Validation States: Brooklyn, NY New York 11220 (US) MA MD • Williamson, David Brooklyn, NY New York 11220 (US) (30) Priority: 15.02.2016 US 201662295438 P 15.02.2016 US 201662295444 P (74) Representative: J A Kemp 14 South Square (71) Applicant: Modern Meadow, Inc. Gray’s Inn Brooklyn, NY 11200 (US) London WC1R 5JJ (GB)

(54) METHOD FOR MAKING ELECTROCOMPACTED

(57) Biofabricated made by electrocompac- These electrocompacted leathers may have leather-like tion. Described herein are biofabricated leather materials properties and may be processed as native leather and derived from electrocompacted collagen networks. used to form leather goods. EP 3 205 754 A1

Printed by Jouve, 75001 PARIS (FR) 1 EP 3 205 754 A1 2

Description taraldehyde or oxazolidine compounds), syntans (syn- thetic tannins, using aromatic polymers), and the like. CROSS REFERENCE TO RELATED APPLICATIONS [0006] Natural leather is typically prepared in three main parts: preparatory stages, , and crusting. [0001] This patent application claims priority to U.S. 5 Surface coating may also be included. The preparatory provisional patent application no. 62/295,438, titled stages prepare the /skin for tanning, and unwanted "ELECTROSPUN LEATHER AND METHODS OF FAB- raw skin components are removed. The preparatory RICATION OF ELECTROSPUN LEATHER," filed on stages may include: preservation, soaking (rehydrating), February 15, 2016; and U.S. provisional patent applica- liming, de-hairing, de-fleshing (removing subcutaneous tion no. 62/295,444, titled "ELECTROCOMPACTED10 material), splitting, re-liming, (to remove de- LEATHER AND METHODS OF FABRICATION OF hairing and liming chemicals), bating (protein proteoly- ELECTROCOMPACTED LEATHER," filed on February sis), degreasing, frizzing, bleaching, pickling (changing 15, 2016. Each of these is herein incorporated by refer- pH), de-pickling, etc. ence in its entirety. [0007] Tanning is performed to convert proteins in the 15 hide/skin into a stable material that will not putrefy, while INCORPORATION BY REFERENCE allowing the material to remain flexible. Chromium is the most commonly used tanning material. The pH of the [0002] All publications and patent applications men- skin/hide may be adjusted (e.g., lowered, e.g. to pH tioned in this specification are herein incorporated by ref- 2.8-3.2) to enhance the tanning; following tanning the pH erence in their entirety to the same extent as if each in- 20 may be raised ("basification" to a slightly higher level, dividual publication or patent application was specifically e.g., pH 3.8-4.2). and individually indicated to be incorporated by refer- [0008] Crusting refers to the post-tanning treatment ence. that may include coloring (dying), thinning, drying or hy- drating, and the like. Examples of crusting techniques FIELD 25 include: wetting (rehydrating), sammying (drying), split- ting (into thinner layers), shaving, neutralization (adjust- [0003] This invention relates to engineered/biofabri- ing pH to more neutral level), retanning, dyeing, fatliq- cated leather materials that mimic naturally-derived ma- uoring, filling, stuffing, stripping, whitening, fixation of un- terials. In particular, this invention is directed towards bi- bound chemicals, setting, conditioning, softening, buff- ofabricated leather materials formed by an electrocom- 30 ing, etc. paction or electrospinning method. [0009] In practice, the process of converting animal skin into leather may include sequential steps such as: BACKGROUND unhairing/dehairing, liming, deliming and bating, pickling, tanning, neutralizing/Dyeing and Fat liquoring, drying [0004] Leather is used in a vast variety of applications, 35 and finishing. The dehairing process may chemically re- including furniture upholstery, clothing, shoes, luggage, move the hair (e.g., using an alkali solution), while the handbag and accessories, and automotive applications. liming step (e.g., using an alkali and sulfide solution) may Currently, skins of animals are used as raw materials for further complete the hair removal process and swell natural leather. However, skins from livestock pose en- ("open up") the collagen. During tanning, the skin struc- vironmental concerns because raising livestock requires 40 ture may be stabilized in the "open" form by replacing enormous amounts of feed, pastureland, water, and fos- some of the collagen with complex ions of chromium. sil fuel. Livestock also produces significant pollution for Depending on the compounds used, the color and texture the air and waterways. In addition, use of animal skins of the leather may change. Tanned leather may be much to produce leather is objectionable to socially conscious more flexible than an untreated hide, and also more du- individuals. The global leather industry slaughters more 45 rable. than a billion animals per year. Most of the leather comes [0010] In practice, the process of converting animal from countries with no animal welfare laws or have laws skin into leather may include sequential steps such as: that go largely or completely unenforced. Leather pro- unhairing/dehairing, liming, deliming and bating, pickling, duced without killing animals would have tremendous tanning, neutralizing/Dyeing and Fat liquoring, drying fashion novelty and appeal. 50 and finishing. The dehairing process may chemically re- [0005] Natural leather is typically a durable and flexible move the hair (e.g., using an alkali solution), while the material created by the tanning of animal and liming step (e.g., using an alkali and sulfide solution) may skin, often hide. Tanning is generally understood further complete the hair removal process and swell to be the process of treating the skins of animals to pro- ("open up") the collagen. During tanning, the skin struc- duce leather. Tanning may be performed in any number 55 ture may be stabilized in the "open" form by replacing of well-understood ways, including vegetable tanning some of the collagen with complex ions of chromium. (e.g., using tannin), chrome tanning (chromium salts in- Depending on the compounds used, the color and texture cluding chromium sulfate), aldehyde tanning (using glu- of the leather may change. Tanned leather may be much

2 3 EP 3 205 754 A1 4 more flexible than an untreated hide, and also more du- lagen fibrils, fibers, and fiber bundles, its complex as- rable. sembly is achieved in vivo during development and is [0011] Skin, or animal hide, is formed primarily of col- critical in providing mechanical support to the tissue while lagen, a fibrous protein. Collagen is a generic term for a allow for cellular motility and nutrient transport. family of at least 28 distinct collagen types; animal skin 5 [0014] In animal hide, slight variations in fibrous colla- is typically type 1 collagen (so the term collagen is typi- gen organization are observed with the age and species cally assumed to be type 1 collagen), although other of the animal resulting in differences in the physical prop- types of collagen may be used in forming leather. Colla- erties of the tissue (and differences in the resulting leath- gens are characterized by a repeating triplet of amino er). Variations in collagen organization are also observed 10 acids, -(Gly-X-Y) n-, so that approximately one-third of the through the thickness of the hide. The top grain side of amino acid residues are in collagen are glycine. X is often hide is composed of a fine network of collagen fibrils while proline and Y is often hydroxyproline. Thus, the structure deeper sections (corium) are composed of larger fiber of collagen may consist of twined triple units of peptide bundles (FIG. 2). The smaller fibril organization of the chains of differing lengths. Different animals may pro- grain layer gives rise to a soft and smooth leather aes- duce different amino acid compositions of the collagen, 15 thetic while the larger fiber bundle organization of deeper which may result in different properties (and differences regions givesrise to a rough andcourse leather aesthetic. in the resulting leather). Collagen fiber monomers may The porous, fibrous organization of collagen allows mol- be produced from alpha-chains of about 1050 amino ac- ecules to more easily penetrate, stabilize, and lubricate ids long, so that the triple helix takes the form of a rod of the hide during leather tanning. The combination of the about 300 nm long, with a diameter of 1.5 nm. In the20 innate collagen organization in hide along with the mod- production of extracellular matrix by fibroblast skin cells, ifications achieved through tanning give rise to the desir- triple helix monomers may be synthesized and the mon- able strength, drape and aesthetic properties of leather. omers may self-assemble into a fibrous form. These triple [0015] Manyattempts havebeen madethroughout his- helices may be held together by salt links, hydrogen tory to imitate leather with a variety of synthetic materials. bonding, hydrophobic bonding, and covalent bonding. 25 As mentioned to earlier, there is a strong demand for Triple helices can be bound together in bundles called alternatives to leather as leather production involves the fibrils, and fibril bundles come together to create fibers slaughter of animals, which carries with it a large envi- (FIG. 1). These structures have a characteristic banded ronmental impact to raise and process. The increasing appearance due to the staggered overlap monomers. demand for leather products also promotes stockyard The distance between bands is approximately 67 nm for 30 practices and factory farming where mistreatment of an- Type 1 collagen. Fibers typically divide and join with each imals has been documented. As a result, the quality and other throughout a layer of skin. Variations of the availability of leather continues to decrease as planetary crosslinking or linking may provide strength to the mate- resources become ever more strained. rial. Fibers may have a range of diameters depending on [0016] Attempts to create synthetic leather have all the type of animal hide. In addition to type I collagen, skin 35 come up short in reproducing leather’s unique set of prop- (hides) may include other types of collagen as well, in- erties. Examples of synthetic leather materials include cluding type III collagen (reticulin), type IV collagen, and Clarino, Naugahyde, Corfam, , amongst oth- type VII collagen. ers. They are made of various chemical and polymer in- [0012] The various types of collagen exist throughout gredients, including polyvinyl chloride, polyurethane, ni- the mammalian body. For example, besides being the 40 trocellulose coated cotton cloth, polyester, or other nat- main component of skin and animal hide, Type I collagen ural cloth or fiber materials coated with a synthetic poly- also exists in cartilage, tendon, vascular ligature, organs, mer. These materials are assembled using a variety of muscle, and the organic portion of bone. Successful ef- techniques, often drawing from chemical and textile pro- forts have been made to isolate collagen from various duction approaches, including non-woven and advanced regions of the mammalian body in addition to 45 the spinning processes. While many of these materials have hide/skin. Decades ago, researchers found that at neutral found use in footwear, upholstery, and apparel applica- pH, acid-solubilized collagen aggregated into fibrils com- tions, they have fallen short for luxury application, as they posed of the same cross-striated patterns observed in cannot match the breathability, handfeel, or aesthetic native collagen. (Schmitt F.O. J. Cell. Comp Physiol. properties that make leather so unique and beloved. To 1942;20:11) This lead to use of collagen being used in 50 date, no alternative leather-like materials have been tissue engineering and a variety of biomedical applica- made from collagen or collagen-like proteins, and there- tions. In more recent years, collagen has been harvested fore these materials lack the chemical composition and from bacteria and yeast using recombinant techniques. structure of collagen that produces a leather aesthetic. [0013] Regardless of the type of collagen, all are The abundance of acidic and basic amino acid side formed and stabilized through a combination of electro- 55 groups along the collagen polypeptide chain, along with static interactions involving salt bridges, hydrogen bond- its organization into a strong yet porous, fibrous structure ing, Van der Waals interactions, and covalent bonding allow modification through tanning processes and pro- often catalyzed by enzymatic reactions. For Type I col- duces the desirable strength, softness and aesthetic of

3 5 EP 3 205 754 A1 6 leather. [0023] For example, a method of making an electro- [0017] The top grain surface of leather is often regard- compacted leather material may include: applying a so- edas themost desirable dueto its softtexture and smooth lution of non-human, monomeric collagen in an aqueous surface. As discussed previously, the grain is a highly buffer onto an electrocompaction surface, wherein the porous network of organized collagen fibrils. An organ- 5 solution is substantially free of collagen fibers and fibril ized collagen fibril is arranged as endogenous collagen bundles; compacting the collagen into a dense network to have lacunar regions and overlapping regions. See, with an electrical field; inducing fibrillation of the collagen e.g., FIG. 1. The strength of the collagen fibril, microscale to form collagen fibrils; stabilizing the fibrillar collagen porosity, and density of fibrils in the grain may allow tan- network; incorporating lubricant in the collagen network; ningagent penetration to stab ilize andlubricate the fibrils, 10 dyeing and applying a surface finish on the collagen net- producing a soft, smooth and strong material that people work; and removing water from the stabilized network desire. and drying the collagen network. [0018] Leather derived from animal mainly consists of [0024] Any appropriate collagen monomers may be collagen type I when hides are obtained from adult ani- used. As used herein a collagen monomer may refer to mals, and significant proportion of collagen types III and 15 tropocollagen, e.g., a triple helical tropocollagen, which IV, together with collagen type I when skins are derived may be polymerized to form fibrils, secondary- and terti- from young animals. The composition and ratio of colla- atry- structures, as illustrated in FIG. 1. Any type of col- gen types affect the physical properties of leather, a lagen may be used, including in particular Type III colla- younger hide with proper tanning provides the softest gen (non-human) or combinations of collagen subtypes leather. 20 (e.g., Type III and type I). The collagen monomers may [0019] The top grain surface of leather is often regard- be polymerized into dimers, trimers and higher order ol- edas themost desirable dueto its softtexture and smooth igomers prior to compaction and fibrillation. surface. As discussed previously, the grain is a highly [0025] In any of the methods described herein, the col- porous network of organized collagen fibrils. An organ- lagen fibrils formed as part of the method may be stabi- ized collagen fibril is arranged as endogenous collagen 25 lized by adding a crosslinking agent (e.g., an aldehyde, to have lacunar regions and overlapping regions. See, such as gluteraldehyde) to the aqueous solution to sta- e.g., FIG. 1. The strength of the collagen fibril, microscale bilize the collagen fibrils. The fibrillated collagen may be porosity, and density of fibrils in the grain may allow tan- stabilized through chromium, aldehyde or vegetable tan- ningagent penetration to stab ilize andlubricate the fibrils, nin based tanning processes. producing a soft, smooth and strong material that people 30 [0026] As part of any of the methods described herein, desire. the water may be removed from the collagen fibrils after [0020] It would be desirable to manufacture leathers forming and electrocompacing and/or electrospiining. (e.g., by engineering/biofabrication) that can be control- For example a material that displaces the water may be lably and reproducibly provide leather having desired used, by reacting the collagen fibrils with a dewatering properties, and in particular leather that may be made 35 agent to displace water bound to the collagen fibrils with without the need to kill animals. Described herein are the dewatering agent. The dewatering agent may be de- engineered/biofabricated leathers and methods of mak- watering and coalescing agent, such as a syntan (e.g., ing them that may address the issues raised above. a sulfonated condensation product of an aromatic com- pound), which may displace the water bound to the col- SUMMARY 40 lagen fibrils. Alternatively or additionally, water may be removed from the fibrillated collagen through solvent ex- [0021] In general, described herein are methods of changes with solvents such as acetone, ethanol, or die- forming a biofabricated leather material. In particular, de- thyl ether. scribed herein are methods of forming a biofabricated [0027] In general, the collagen monomer solution may leather material by electrocompaction or electrospinning. 45 be free or substantially free of collagen fibers and fibril Although both methods (electrospinning and electrocom- bundles. The collagen monomers may be recombinant. paction) are described and illustrated herein separately, The collagen (and therefore the resulting fibrils) may be it should be understood that two methods may be com- modified to promote chemical or physical crosslinking bined, and some or all of the steps or components (e.g., between collagen fibrils. The collagen fibrils may be sta- the starting materials, further processing steps, etc.) de- 50 bilizedas a network throughincorporating molecules with scribed for one may be applied to the either technique. di, tri and multifunctional reactive groups such as chro- [0022] For example, described herein are methods for mium, amine, carboxylic acid, sulfate, sulfite, sulfonate, making an electrocompacted leather material by: apply- aldehyde, hydrazide, sulfhydryl, diazirine, aryl-azide, ing a solution of non-human, monomeric collagen onto acrylate, epoxide, or phenol. These may be used with or an electrocompaction surface; compacting the protein in- 55 without the additional (e.g., in a recombinant collagen) to a dense network with an electrical field; inducing fibril- the incorporation of reactive groups for any of these add- lation of the protein; incorporating lubricant in the net- ed to the collagen. work; and removing water from the network. [0028] Any method for fibrillating the collagen may be

4 7 EP 3 205 754 A1 8 used, including the use of salt and pH. For example, fi- collagen. brillation may be induced through the addition of salts [0034] Tanning (e.g., stabilizing the electrospun net- such as sodium phosphate, potassium phosphate, po- work) may include incorporating one or more of a chro- tassium chloride and sodium chloride, and/or through a mium based, aldehyde based, epoxide based, or sulfo- pH shift following the addition of acids or bases such as 5 NHS based crosslinkers into the dissolved collagen so- sodium carbonate, sodium bicarbonate and sodium hy- lution prior to electrospinning. For example, tanning may droxide. Alternatively or additionally, fibrillation may be include stabilizing the electrospun network using a chro- induced(or aided)through the incorporation of nucleation mium- or aldehyde-based tanning process. agents such as collagen microgels, microparticles, nan- [0035] Any of these methods may include incorporat- oparticles, and natural and synthetic microfibers. 10 ing a lubricant into the unwoven electrospun network of [0029] In any of the methods described herein one or collagen through a fatliquoring processes. The lubricant more spacing agent may be included, such as a bead, (e.g., oils such as fish oils, etc.) may be incorporated fiber or the like. while the collagen is in solution before or after electros- [0030] Any of the methods described herein may in- pinning or electrocompaction, before fibrillation, after fi- clude a drying step instead or in addition to displaying 15 brillation, etc. the water in the fibrils. For example, any of these methods [0036] Any of these methods may include dying the may include drying the fibrillated collagen through air or unwoven electrospun network of collagen. Drying may vacuum drying to remove some of the water. For exam- be done in air (including in a humidified chamber), by ple, at least 80% of the water is removed from the fibril- pulling a vacuum, compressing the material, or some lated collagen. 20 combination thereof. During or after drying, the method [0031] Lubricants (e.g., fat liquors), dyes, tanning may include staking (e.g., mechanically manipulating) agents, spacer material, or the like may be incorporated the material. For example, the material may be mechan- into the material prior to drying and/or after drying. Thus, ically manipulated midway or partway through the drying. the resulting material may have a uniform distribution of The material may be dried to a final water content of lubricants, tanning agent(s), dyes, spacing materials25 between about 10% and about 25% (e.g., between 15% (e.g., microspheres), etc. throughout the entire volume and 20%, etc.). of material (sheet) that is formed. [0037] Any of these methods may also include applying [0032] The resulting formed material may have fibrils a finish to the surface of the unwoven electrospun net- that are 1 nm to 1 mm in diameter. The fibrils may be 100 work of collagen. For example, any of these methods nm to 1 mm in length. The fibrils may be arranged in a 30 may include retanning and finishing the unwoven elec- tangled (disordered) network throughout the entire vol- trospun network of collagen. ume, unlike traditional leather. For example, the fibril net- [0038] The biofabricated leathers described herein work may lack higher order fiber and fiber bundle organ- may be used in place of natural leather. For example, ization throughout the entire formed material (e.g., the the biofabricated leather (e.g., formed by electrospinning entire thickness). The fibril density may be, e.g., 5 mg/cc 35 and/or electrocompaction) may be used to form one or to 500 mg/cc. Virtually any thickness may be formed in more of a: watchstrap, footwear, wallet, jewelry, belt, this manner, including thickness of between about 0.05 glove, handbag, briefcase, piece of luggage, upholstery mm to 2 mm or greater (e.g., 5mm, 6mm, 7 mm, 8 mm, for furniture or transportation, or clothing article. 9 mm, 10mm, etc.). [0033] Also described are methods of forming an elec- 40 BRIEF DESCRIPTION OF THE DRAWINGS trospun leather material, by electrospinning a collagen solution to form an unwoven collagen network and tan- [0039] ning the electrospun protein. For example, a method of forming an electrospun leather material may include: cul- FIG. 1 is a drawing showing the composition of col- turing collagen-secreting cells; harvesting collagen from 45 lagen in a hierarchical fashion. 1) shows each colla- the cells into a collagen solution; forming an unwoven gen molecule and how they are packed with respect network of collagen by electrospinning the collagen so- to neighboring collagen molecules; 2) shows lution; and tanning the unwoven electrospun network of stacked collagen that make up collagen fibers; 3) collagen. As previously mentioned, any of the steps used shows a cross-section of the collagen fibrils; 4) for electrocompaction described herein may be used with 50 shows collagen fibers; and 5shows bundles of col- electrospinning instead and any of the steps used for lagen fibers. electrospinning may be used for electrocompaction. For FIG. 2 is a picture showing the composition of buffalo example, any of these methods may include culturing hide. The top grain layer and the corium layer un- protein-secreting cells and harvesting collagen from the derneath are shown and the relative amount of col- cells to form the collagen (e.g., collagen monomer) so- 55 lagen fiber bundles are indicated. lution. Alternatively or additionally, any of these methods FIGS. 3A-3C illustrate the electrocompaction of col- may include acquiring collagen from an animal or plant lagen. FIG. 3A shows a compaction cell with the sil- source and forming a collagen solution from the acquired iconespacers. FIG. 3B showsan assembled cellwith

5 9 EP 3 205 754 A1 10 collagen solution between the plates before and after cross-linked and cross-linked samples. assembly. FIG. 3C shows compacted and fibrillated FIG. 11 is a table illustrating weights in an example collagen matrix, forming a gel. The gels become of different electrospun leathers that are ether cross- opaque after fibrillation. linked or non-cross-linked before and after fatliquor- FIGS. 4A and 4B is a transmission electron micro- 5 ing. graphs (TEM)of electrocompactedcollagen material FIGS. 12A-12D illustrate mechanical properties of (e.g., which may be referred to as a hydrogel) before electrospun leather fabricated as described herein (FIG. 4A) and after (FIG. 4B) fibrillation. The pres- before and after fat liquoring in ethanol/cod oil (e.g., ence of periodic dark bands in FIG. 4B indicate the 80/20). presence of fibrils. 10 FIGS. 13A and 13B show mechanical properties of FIGS. 5A and 5B show a scanning electron micro- electrospun leather before and after fatliquoring in graphs (SEM) of bovine grain (FIG. 5A, on the bot- ethanol/casterol oil (80/20). FIG. 13A shows elon- tom)and electrocompacted collagen (FIG.5B) at two gate percent and FIG. 13B shows tensile strength. magnifications. The dense fibrillar network indicates assembly of collagen into a network similar to the 15 DETAILED DESCRIPTION grain of genuine leather. FIG. 5A is adapted from "Structure of bovine skin and hair root," Matthias [0040] In general, described herein are methods of Wagner and David Bailey, TFL. forming biofabricated leather materials from a solution FIG. 6 is a strain-stress graph of the dried electro- of, e.g., monomeric, collagen by elctrocompaction and/or compacted collagen material following fatliquoring 20 electrospinning. The resulting material may be referred with different materials. Lipoderm A1 fatliquor pro- to as electrocompacted leather material or electrospun vides more internal lubrication than cod liver oil, in- leather. Also described herein are the resulting materials, dicated by the increase in both tensile strength and which may be structurally (including ultrastructurally) elongation, preventing fibers to stick, known to result and/or compositionally distinct from native (e.g., "natural" in a brittle material. 25 leathers) and other man-made leather materials. FIG. 7A illustrates the generic technique for electro- [0041] Reconstituted collagen networks have been spinning, showing the use of appositive high voltage widely employed in biomedical applications, regenera- load to spin a solution of material (e.g. collagen) onto tive medicine and tissue engineering, however to date a target. such reconstituted collagen networks have not been FIG. 3B shows another illustration of electrospinning 30 made into leathers that may be used in place of natural to form fibers that may be deposited onto a target. leather. For example, collagen may be obtained from an- FIGS. 8A-8D illustrate prior art examples of networks imal tissues known to be abundant in collagen type I of fibers obtained by electrospinning. In FIG. 8A (such as tendons and skin) via acid extraction and/or (adapted from Zhou J, Sui F, Yao M et al., Neural enzymatic digestion, purified and stored in a weak acidic Regeneration Research, 2013 8(16): 1455-1464) 35 solution, consisting of mixtures of monomers, dimers and oriented (on left) and random (right) deposition of trimers of the collagen triple helix. Fibrillogenesis may be collagen nano-fibers is shown. FIGS. 8B-8D show initiated by adjusting the pH and ionic strength of the example of mono (FIG. 8B) and bi-layered (FIG. 8C solution; the monomers either self-assemble into fibrils and larger magnification view in FIG. 8D) collagen and/or connect through chemical crosslinkers forming a fibers are shown. 40 fibril matrix. Fibril formation is not restricted to full length, FIG. 9A illustrates one example of a collagen net- extracted collagen monomers only: recombinant colla- work formed by electrospinning at two magnifica- gen and collagen-like proteins (often truncated) fibrillate tions. Any appropriate thickness of collagen network in a similar way when pH and ionic strength is optimized. may be formed in this manner. This method has a disadvantage in manufacturing gels FIG.9B illustrates a cross-linked (by additionof DHT) 45 of high concentration and orientation of fibers, collagen electrospun collagen network at two magnifications. concentration in animal hide is around 200-300 mg/ml. FIG. 9C is a table comparing the stability of non- (or To attain such density further processing may be re- pre-) cross-linked and cross-linked collagen net- quired, such as drying and mechanical compaction as works such as those shown in FIGS. 9A and 9B in described herein. various solvents. 50 [0042] Collagen matrices (including hydrogels) of high FIG. 9D shows exemplary fiber dimensions (diame- density and orientation may be manufactured via isoe- ter, planar density and thicknesses) for the electro- lectric focusing. This method may rely on the concentra- spun collagen networks (forming electrospun leath- tion of ampholytic molecules such as collagen by manip- er) as described herein. Any appropriate range of ulating the electrochemical environment of collagen in a collagen fiber dimensions may be formed. 55 solution. Briefly, collagen may be extracted from animal FIG. 10 shows exemplary images of electrospun col- tissues as mentioned before, purified, then dialyzed lagen networks (forming electrospun leather mate- against deionized water, removing salts and acids. The rials) before, during and after fatliquoring in non- solutionmay be poured betweentwo linear or planar elec-

6 11 EP 3 205 754 A1 12 trodes and an electric field and current is applied across onto an electrocompaction surface; compacting the col- the collagen solution, resulting in a pH gradient. The col- lagen into a dense network with an electrical field; induc- lagen molecules may then migrate and accumulate either ing fibrillation of the collagen; stabilizing the fibrillar col- in a narrow string (linear electrodes) or in a plane (planar lagen network; removing water from the collagen (includ- electrodes) at a pH value corresponding to their isoelec- 5 ing from the entire network and/or displacing water bound tric point. The duration of the migration and the thickness to the collagen fibrils); incorporating lubricant in the col- of the assembling collagen layer may depend on the volt- lagen network; dyeing and applying a surface finish on age applied, as well as the distance between the plates. the collagen network; and drying the collagen network. When migration ceases (indicated by a more or less sta- [0048] In any of these methods the protein monomers ble current) the resulted collagen structure is removed 10 (e.g., collagen monomers) may be polymerized into dim- andplaced in an aqueous environmentwhere adjustment ers, trimers and higher order oligomers prior to compac- of pH and ionic strength will promote fibrillogenesis and tion and fibrillation. The fibrillation may be induced crosslinking yielding in a highly compacted, dense colla- through the addition of salts such as sodium phosphate, gen matrix. potassium phosphate, potassium chloride and sodium [0043] Without substantial modification, such materi- 15 chloride. Fibrillation may be induced through a pH shift als (e.g., gels) may be suitable for research and devel- following the addition of acids or bases such as sodium opment performing basic research in cell and matrix bi- carbonate, sodium bicarbonate and sodium hydroxide. ology (including tissue engineering and regenerative Fibrillation may be induced through the incorporation of medicine), however they are not usually appropriate, nucleation agents such as collagen microgels, micropar- withoutsignificant further processing, to form leather. De- 20 ticles, nanoparticles, and natural and synthetic micro fib- scribed herein are leather-like material (engineered ers. leather/biofabricated leather) and methods for forming [0049] Collagen fibrils may be chemically modified to such leather-like materials for consumer use using elec- promote chemical or physical crosslinking between col- trocompacted and/or electrospun collagen networks. lagen fibrils. Stabilization of the fibrillar collagen network 25 may be accomplished through incorporating molecules Electrocompaction with di, tri and multifunctional reactive groups such as chromium, amine, carboxylic acid, sulfate, sulfite, sul- [0044] The biomaterials described herein that possess fonate, aldehyde, hydrazide, sulfhydryl, diazirine, aryl- leather-like properties, may be formed from an electro- azide, acrylate, epoxide, or phenol. compacted and fibrillated collagen which is further cross- 30 [0050] Any of these electrocompacted leathers may be linked and lubricated at the structural fibril level, similar fixed, e.g. by cross-linking the collagen. For example, the to tanned natural leather. This approach produces leath- fibrillated collagen may be stabilized through chromium, er-like material similar to a leather grain, offering tuna- aldehyde or vegetable tannin based tanning processes bility across a wide range of properties (structure, known in the leather industry. strength, elongation, density, etc.) and it is not restrictive 35 [0051] Any of these electrocompacted leathers may al- to collagen type I found in animal hide, a limitation of the so have a reduced water content (e.g., may be dehydrat- traditional leather industry. ed) compared to native leather. For example, the water [0045] For example, in general a method for making content of the fibrillated collagen may be greater than an electrocompacted leather material may include: ap- 90% (w/w), which may be reduced to less than 15% (e.g., plying a solution of dissolved protein in aqueous buffer 40 less than 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, onto an electrocompaction surface; compacting the pro- 14%, 15%, etc.) of the final leather. Thus, water may be tein into a dense network with an electrical field; inducing removed from the collagen material through solvent ex- fibrillation of the protein; removing water from the net- changes with solvents such as acetone, ethanol, or die- work; and incorporating lubricant in the network. The pro- thyl ether. The water may be removed through air or vac- tein may be any appropriate protein, including collagen 45 uum drying. For example, at least 75%, 77%, 78%, 79%, (any of the known 28+ types of collagen and variations 80%, 81%, 82%, 83%, 84%, 85%, etc. of the water may thereof) and non-collagen proteins that may form (e.g., be removed. by self-assembly) fibrils, such as, e.g., keratin, chitin, etc. [0052] Any of these electrocompacted leathers may in- [0046] Thus, a method for making an electrocompact- clude a lubricant (e.g., by fat liquoring to add the lubri- ed leather material may include: applying a solution of 50 cant). The lubricant may be any hydrophobic material, dissolved collagen in aqueous buffer onto an electrocom- such as a lubricating fat and/or oil that may be incorpo- paction surface; compacting the collagen into a dense rated into the material. network with an electrical field; inducing fibrillation of the [0053] Any of these materials may be dyed, retanned, collagen network; removing water from the collagen net- finished and dried through processes known in the leath- work; and incorporating lubricant in the collagen network. 55 er industry. [0047] For example, a method for making an electro- [0054] In some variations, an electrocompacted leath- compacted leather material, the method comprising: ap- er material may be fabricated from a matrix of electro- plying a solution of dissolved collagen in aqueous buffer compacted and cross-linked collagen fibrils as described

7 13 EP 3 205 754 A1 14 herein and may include any of the 28 types of collagen rent across the two plates (FIG. 3B), voltage and current proteins isolated from animal tissues or variants thereof. wasmonitored with multimeters connected in parallel and For example, collagen produced by expressing recom- in series to the plates. The electrical parameters were binant DNA in bacteria, plant, yeast, or mammalian cells chosen to result in an electric field of 1-25 kV and current may be used as the source collagen. The collagen or 5 density of 0.5-10 A/m2. The duration of the electrocom- collagen-like proteins may be produced through chemi- paction process depended on the voltage applied and cal peptide synthesis technologies. In other variations, it plate separation: increasing the electrical field strength is not necessary to form a hydrogel (or may not be ac- was achieved either by increasing the voltage or decreas- curate to refer to the resulting fibrils of collagen as a "hy- ing the separation between the plates. The closer the drogel"). 10 plates were, the higher the pH gradient become, provid- [0055] The collagen (e.g., in some cases a matrix of ing thus the capability to control layer thickness. The pro- collagen fibrils) may be transformed into a leather-like cedure was repeated by removing excess water from the material by removing water from the material, or in some top of the already compacted layer and replacing it with cases displacing the water from the fibrils of collagen stock collagen solution. Thus, sequential compaction (e.g., using a dewetting agent), and incorporating15 steps controlled the thickness of the final material. The crosslinking agents, fats, or oils to stabilize the collagen resulting collagen sheets (FIG. 3C) consisted of closely network. The final water content of the material may be associated, dense, entangled collagen monomers where between 25 and 10 % (e.g., between 15% and 20%, etc.) no D-banding was observed (FIG. 4A) The collagen (w/w). sheet was then incubated in the presence of PBS for a [0056] The material of the fibril network may be a po- 20 minimum of 2 hrs. to fibrillate it. The presence of fibers rous network of collagen or collagen-like fibrils. The fibrils was confirmed by transmission electron microscopy may be between about 1 nm to 1 mm in diameter. The (FIG. 4B), whereas the dense porous fibril network was fibrils may be between about 100 nm to 1 mm in length. visualized with scanning electron microscopy (FIG. 5). The fibril network may lack higher order fiber and fiber [0059] The fibrillated and compacted collagen was de- bundle organization. The fibril density may be between 25 hydrated in a series of acetone solutions (3 x 1hr at 25°C). about 5 mg/cc to 500 mg/cc (e.g., between 10 mg/cc to Following acetone dehydration, the collagen material 400 mg/cc, between 20 mg/cc to 300 mg/cc, between was incubated in a fat liquor solution containing either 100 mg/cc to 400 mg/cc, or any subrange thereof). The 20% (v/v) cod liver oil or 20% (v/v) Lipoderm A1 fatliquor thickness of of the material may be between about 0.05 in 80% acetone overnight while shaking at 40rpm. Fol- mm to 10 mm (e.g., between 0.5 mm to 5 mm, greater 30 lowing incubation in the fatliquor solution, the material than 1 mm, etc.); this thickness may be the thickness of was dried at room temperature overnight. Mechanical the dried material. The elongation at break may be be- analysis confirmed penetration of the cod oil into the ma- tween 0% to 300%. The tensile strength may be between terial, preventing fibril-fibril sticking during drying (FIG. 6). about 1 MPa to 100 MPa. The elastic modulus may be between about 1 kPa to 100 MPa. 35 ELECTROSPINNING [0057] The finished leather-like material may be used in any product where native leather is used, such as a [0060] Although collagen networks have been pro- watchstrap, footwear, wallet, jewelry, belt, glove, hand- duced as materials for biomedical applications such as bag, briefcase, piece of luggage, upholstery for furniture forming implant for use in a body, very little has been or transportation, or clothing article. 40 done regarding forming collagen materials for use as du- rable, attractive and wearable fabrics such as leather, Examples which raise a number of very different concerns and is- sues compared to biomedical materials. For example, [0058] Commercially available collagen type I was ob- when forming collagen structures for biomedical applica- tained from bovine skin via acetic acid extraction and 45 tions, monomers of the collagen triple helix have been pepsin digestion was purchased as a 6 mg/ml solution extracted from animal tissue, such as bovine dermis, re- in 0.1 N hydrochloric acid. This stock solution ∼(50 ml) solubilized in acidic solution and then electrospun. The was dialyzed against 5 gl of deionized water overnight. basic principle of the electrospinning is illustrated in Silicone spacers of 1 or 2 mm diameter (in this example, FIGS. 7A-7B. In the initial basic technology, fibers were the spacers are spherical, however other shapes and 50 deposited at a low rate and only in a random manner. dimensions may be used) were arranged on a stainless Lately several advances have been achieved: the fibers steel or graphite plate to enclose a 2.5 x 5 cm area, open formed by electrospinning can now be deposited accord- on one side (FIG. 3A). The dialyzed solution was poured ing to a defined orientation (FIG. 8A), the density and into this mold and covered with a second plate, enclosing diameters of fibers can be modified during the process an approximately 1.25 ml volume between the two plates. 55 to form multi-layered material (FIG. 8B), and the method The silicone spacers also served as electrical insulators may be scaled (see, e.g., http://www.sncfibers.com; and between the plates. A stable, variable voltage source with currentlimiting capabilitywas supplying theelectrical cur-

8 15 EP 3 205 754 A1 16 http://arsenalmedical.com/technology/axiocore-drug- unwoven electrospun network of collagen through a fat delivery-platform). liquoring processes. [0068] In general, any of these electrospun leathers [0061] For example, twisted, continuous nanofiber may also be dyed and may therefore include a dying step. yarns may be formed having diameters of -100 microm- 5 For example, any of these methods may include dying eters. Described herein are methods for forming an en- the unwoven electrospun network of collagen. Similarly, gineered leather (e.g., biofabricated leather) using elec- these materials may be finished by applying a finish to trospinning, as well an engineered/biofabricated electro- the surface of the unwoven electrospun network of col- spun leathers made using these techniques. The result- lagen. For example, the method may include retanning ing leather may be distinguishable from native leather, 10 (e.g., a second tanning/crosslinking step) and finishing but may have the same gross properties, look and feel the unwoven electrospun network of collagen. Any of (texture) of native leather including grossly mimicking the these methods may include using the formed material to dermis structure (grain and corium) of native leather. create a leather product. For example, any of these meth- [0062] In general, described herein are electrospun ods may include forming from the unwoven electrospun leather materials and methods of making them. For ex- 15 network of collagen one or more of a: watchstrap, foot- ample, a method of forming an electrospun leather ma- wear, wallet, jewelry, belt, glove, handbag, briefcase, terial may include: electrospinning a protein solution piece of luggage, upholstery for furniture or transporta- (e.g., a collagen solution) to form an unwoven (e.g., col- tion, or clothing article. lagen) network and tanning the electrospun protein. [0063] For example, a method of forming an electro- 20 Example 2 spun leather material may include: culturing collagen- secreting cells; harvesting collagen from the cells into a [0069] FIGS. 9A-9D illustrate one example of an elec- collagen solution; forming an unwoven network of colla- trospun leather fabricated as described herein. In this gen by electrospinning the collagen solution; tanning the example, Type I collagen was isolated from bovine skin unwoven electrospun network of collagen. 25 by acid/basic style extraction method was frozen and [0064] In any of these variations, collagen may be used lyophilized. The lyophilized protein was dissolved in acid- as described herein. Other proteins and particularly fibril ic solution before being electrospun. The fiber orientation or fiber-assembling proteins, may be used additionally was random as observed by SEM (FIG. 9A). This elec- or alternatively, such as keratins, chitins, etc. trospun network was cross-linked to improve stability. A [0065] In any of these examples, the protein solution 30 dehydrothermal cross linker (DHT; FIG. 9B) was used. (e.g., collagen) solution may be formed by a cell-cultured One noticeable effect was the resistance in aqueous so- system, or from an animal or plant source (including cul- lutions (FIG. 9C). The DHT cross-linked network re- tured extracts from animals or plants that are cultured). mained intact while the un-cross-linked material dis- For example, any of these methods may include culturing solved in water and saline solutions. The formed net- a protein-secreting cells and harvesting collagen from 35 works were measured. The characteristics of the fibers the cells into the collagen solution. Alternatively or addi- that constituted the networks were evaluated by SEM tionally, any of these methods may include acquiring col- imaging (FIG. 9D). lagen from an animal or plant source and forming a col- [0070] The electrospun collagen sheets were incubat- lagen solution from the acquired collagen. ed in a fat liquor solution containing 20% (v/v) castor oil [0066] In general, tanning includes cross-liking of the 40 in 80% ethanol overnight while shaking at 40rpm. Fol- protein that is being electrospun. In particular, tanning lowing incubation in the castor oil solution, the electro- includes cross-linking of collagen. Tanning may com- spun sheets were dried overnight at 37°C in a dehydrator. prise stabilizing the electrospun network by incorporating The samples were imaged before, during and after the oneor more of a chromiumbased, aldehyde based, epox- fat liquoring process was completed (FIG. 10). After dry- ide based, or sulfo-NHS based crosslinkers into the dis- 45 ing, the weight and mechanical properties of the material solved collagen solution prior to electrospinning. For ex- were measured and compared to the ones of the untreat- ample, tanning may include stabilizing the electrospun ed sheets. (Shown in FIGS. 11 and 12A-12D). Weight network using a chromium- or aldehyde-based tanning measurements and mechanical analysis confirmed pen- process. Surprisingly, the cross-linking agent (tanning etration of the castor oil into the fibrillar collagen network, agent) may be added to the solution before it is electro- 50 preventing fibril-fibril sticking during drying (FIGS. 13A- spun. 13B). [0067] Any of the electrospun leathers described here- in may include a lubricant; the lubricant may be added Example 3 bya fatliquoring process. Insome variations, the lubricant (e.g., a hydrophobic material such as an oil or fat) may 55 [0071] Type I collagen isolated from bovine skin by ac- be added during the electrospinning process or after the id/basic style extraction method was frozen and lyophi- network has been formed. For example, any of these lized. The lyophilized protein was dissolved in acidic so- methods may include incorporating a lubricant into the lution before being electrospun. In this example, the di-

9 17 EP 3 205 754 A1 18 ameter and orientation were controlled to achieve multi- addition to the orientation depicted in the figures. For layered network with distinctive properties in each layer. example, if a device in the figures is inverted, elements The bottom layer was composed of larger bundles of fib- described as "under" or "beneath" other elements or fea- ers (to mimic the corium) while the top layer was made tures would then be oriented "over" the other elements of smaller fibers in random orientation. 5 or features. Thus, the exemplary term "under" can en- [0072] The electrospuncollagen multi-layered network compass both an orientation of over and under. The de- was incubated in a fat liquor solution containing 20% (v/v) vice may be otherwise oriented (rotated 90 degrees or castor oil in 80% ethanol overnight while shaking at at other orientations) and the spatially relative descriptors 40rpm. Following incubation in the castor oil solution, the used herein interpreted accordingly. Similarly, the terms electrospun sheets were dried overnight at 37oC in a 10 "upwardly", "downwardly", "vertical", "horizontal" and the dehydrator. The multi-layered networks described herein like are used herein for the purpose of explanation only may have a higher mechanical strength, and/or lower unless specifically indicated otherwise. water solubility than the monolayers (even thick monol- [0076] Although the terms "first" and "second" may be ayers) describe in Example 2, above. used herein to describe various features/elements (in- [0073] When a feature or element is herein referred to 15 cluding steps), these features/elements should not be as being "on" another feature or element, it can be directly limited by these terms, unless the context indicates oth- on the other feature or element or intervening features erwise. These terms may be used to distinguish one fea- and/or elements may also be present. In contrast, when ture/element from another feature/element. Thus, a first a feature or element is referred to as being "directly on" feature/element discussed below could be termed a sec- another feature or element, there are no intervening fea- 20 ond feature/element, and similarly, a second feature/el- tures or elements present. It will also be understood that, ement discussed below could be termed a first feature/el- when a feature or element is referred to as being "con- ement without departing from the teachings of the nected", "attached" or "coupled" to another feature or el- present invention. ement, it can be directly connected, attached or coupled [0077] Throughout this specification and the claims to the other feature or element or intervening features or 25 which follow, unless the context requires otherwise, the elements may be present. In contrast, when a feature or word "comprise", and variations such as "comprises" and element is referred to as being "directly connected", "di- "comprising" means various components can be co-joint- rectly attached" or "directly coupled" to another feature ly employed in the methods and articles (e.g., composi- or element, there are no intervening features or elements tions and apparatuses including device and methods). present. Although described or shown with respect to 30 For example, the term "comprising" will be understood one embodiment, the features and elements so de- to imply the inclusion of any stated elements or steps but scribed or shown can apply to other embodiments. It will not the exclusion of any other elements or steps. also be appreciated by those of skill in the art that refer- [0078] As used herein in the specification and claims, ences to a structure or feature that is disposed "adjacent" including as used in the examples and unless otherwise another feature may have portions that overlap or under- 35 expressly specified, all numbers may be read as if pref- lie the adjacent feature. aced by the word "about" or "approximately," even if the [0074] Terminology used herein is for the purpose of term does not expressly appear. The phrase "about" or describing particular embodiments only and is not intend- "approximately" may be used when describing magni- ed to be limiting of the invention. For example, as used tude and/or position to indicate that the value and/or po- herein, the singular forms "a", "an" and "the" are intended 40 sition described is within a reasonable expected range to include the plural forms as well, unless the context of values and/or positions. For example, a numeric value clearly indicates otherwise. It will be further understood may have a value that is +/- 0.1% of the stated value (or that the terms "comprises" and/or "comprising," when range of values), +/- 1% of the stated value (or range of used in this specification, specify the presence of stated values), +/- 2% of the stated value (or range of values), features, steps, operations, elements, and/or compo- 45 +/- 5% of the stated value (or range of values), +/- 10% nents, but do not preclude the presence or addition of of the stated value (or range of values), etc. Any numer- one or more other features, steps, operations, elements, ical values given herein should also be understood to components, and/or groups thereof. As used herein, the include about or approximately that value, unless the term "and/or" includes any and all combinations of one context indicates otherwise. For example, if the value or more of the associated listed items and may be ab- 50 "10" is disclosed, then "about 10" is also disclosed. Any breviated as "/". numerical range recited herein is intended to include all [0075] Spatially relative terms, such as "under", "be- sub-ranges subsumed therein. It is also understood that low", "lower", "over", "upper" and the like, may be used when a value is disclosed that "less than or equal to" the herein for ease of description to describe one element or value, "greater than or equal to the value" and possible feature’s relationship to another element(s) or feature(s) 55 ranges between values are also disclosed, as appropri- as illustrated in the figures. It will be understood that the ately understood by the skilled artisan. For example, if spatially relative terms are intended to encompass dif- the value "X" is disclosed the "less than or equal to X" as ferent orientations of the device in use or operation in well as "greater than or equal to X" (e.g., where X is a

10 19 EP 3 205 754 A1 20 numerical value) is also disclosed. It is also understood stantially free of collagen fibers and fibril bun- that the throughout the application, data is provided in a dles; numberof different formats,and that this data, represents compacting the collagen into a dense network endpoints and starting points, and ranges for any com- with an electrical field; bination of the data points. For example, if a particular 5 inducing fibrillation of the collagen to form col- data point "10" and a particular data point "15" are dis- lagen fibrils; closed, it is understood that greater than, greater than or stabilizing the fibrillar collagen network; equal to, less than, less than or equal to, and equal to 10 incorporating lubricant in the collagen network; and 15 are considered disclosed as well as between 10 and and 15. It is also understood that each unit between two 10 removing water from the stabilized network. particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also dis- 2. The method of claim 1, wherein the collagen mono- closed. mers are polymerized into dimers, trimers and higher [0079] Although various illustrative embodiments are order oligomers prior to compaction and fibrillation. described above, any of a number of changes may be 15 made to various embodiments without departing from the 3. The method of claim 1 or claim 2, wherein stabilizing scope of the invention as described by the claims. For comprises adding a crosslinking agent to stabilize example, the order in which various described method the collagen fibrils. steps are performed may often be changed in alternative embodiments, and in other alternative embodiments one 20 4. The method of any preceding claim, further compris- or more method steps may be skipped altogether. Op- ing reacting the collagen fibrils with a dewatering tional features of various device and system embodi- agent to displace water bound to the collagen fibrils ments may be included in some embodiments and not with the dewatering and coalescing agent. in others. Therefore, the foregoing description is provided primarily for exemplary purposes and should not be in- 25 5. The method of claim 4, wherein the dewatering agent terpreted to limit the scope of the invention as it is set is a sulfonated condensation product of an aromatic forth in the claims. compound. [0080] The examples and illustrations included herein show, by way of illustration and not of limitation, specific 6. The method of any preceding claim, wherein fibrilla- embodiments in which the subject matter may be prac- 30 tion is induced through the addition of salts such as ticed. As mentioned, other embodiments may be utilized sodium phosphate, potassium phosphate, potassi- and derived there from, such that structural and logical um chloride and sodium chloride. substitutions and changes may be made without depart- ing from the scope of this disclosure. Such embodiments 7. The method of any preceding claim, wherein fibrilla- of the inventive subject matter may be referred to herein 35 tion is induced through a pH shift following the addi- individually or collectively by the term "invention" merely tion of acids or bases such as sodium carbonate, for convenience and without intending to voluntarily limit sodium bicarbonate and sodium hydroxide. the scope of this application to any single invention or inventive concept, if more than one is, in fact, disclosed. 8. The method of any preceding claim, wherein stabi- Thus, although specific embodiments have been illus- 40 lization of the fibrillar collagen network is accom- trated and described herein, any arrangement calculated plished through incorporating molecules with di, tri to achieve the same purpose may be substituted for the and multifunctional reactive groups such as chromi- specific embodiments shown. This disclosure is intended um,amine, carboxylic acid, sulfate, sulfite, sulfonate, to cover any and all adaptations or variations of various aldehyde, hydrazide, sulfhydryl, diazirine, aryl- embodiments. Combinations of the above embodiments, 45 azide, acrylate, epoxide, or phenol. andother embodimentsnot specificallydescribed herein, will be apparent to those of skill in the art upon reviewing 9. The method of any preceding claim, wherein the fi- the above description. brillated collagen is stab ilized through chromium, al- dehyde or vegetable tannin based tanning process- 50 es. Claims 10. The method of any preceding claim, wherein water 1. A method for making an electrocompacted leather is removed from the fibrillated collagen through sol- material, the method comprising: vent exchanges with solvents such as acetone, eth- 55 anol, or diethyl ether. applying a solution of non-human, monomeric collagen in an aqueous buffer onto an electro- 11. The method of any preceding claim, wherein water compaction surface, wherein the solution is sub- is removed from the fibrillated collagen through air

11 21 EP 3 205 754 A1 22

or vacuum drying.

12. The method of any preceding claim, wherein lubri- cating fats and oils are uniformly incorporated into the material. 5

13. The method of any preceding claim, wherein the col- lagen monomers are recombinant collagen.

14. The method of any preceding claim, wherein the fibril 10 network lacks higher order fiber and fiber bundle or- ganization, and/or wherein the fibril density is 5 mg/cc to 500 mg/cc

15. The method of any preceding claim, wherein the15 thickness is 0.05 mm to 2 mm.

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

• US 62295438 A [0001] • US 62295444 A [0001]

Non-patent literature cited in the description

• SCHMITT F.O. J. Cell. Comp Physiol., 1942, vol. 20, •ZHOUJ; SUI F ; YAO M et al. Neural Regeneration 11 [0012] Research, 2013, vol. 8 (16), 1455-1464 [0039]

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