Scaffold material for artificial dermal layer

Abstract

Disclosed herein are compositions useful as artificial dermal layer scaffold materials. Methods of making and processing scaffold materials for artificial dermal layers are disclosed herein. Disclosed herein are scaffold materials for supporting immortalized fibroblasts.

Description

[0001] Cross-reference

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 400,704, filed Aug. 24, 2022, and U.S. Provisional Patent Application No. 63 / 515,982, filed Jul. 27, 2023, each of which is hereby incorporated by reference in its entirety.

[0003] Brief description of the invention

[0004] In some embodiments, compositions are disclosed herein that comprise a scaffold in contact with an extracellular matrix comprising collagen, wherein the scaffold can comprise a non-woven needled material. In some embodiments, compositions are disclosed herein that comprise a scaffold in contact with an extracellular matrix comprising collagen, wherein the scaffold can comprise a three-dimensional woven material. In some embodiments, compositions are disclosed herein that comprise a scaffold in contact with an extracellular matrix comprising collagen, wherein the scaffold can comprise polycaprolactone (PCL), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polyethylene terephthalate (PET), nylon, polyethylene (PE), polyethylene furandicarboxylate (PEF), polypropylene (PP), polyvinyl alcohol (PVA), cotton, bast fiber, viscose, modal, lyocell, plant-based protein fiber, bio-based material, viscose, cellulose, alginate fiber, thermoplastic starch, or any combination thereof. In some embodiments, compositions are disclosed herein that comprise a scaffold that is at least partially coated and in contact with an extracellular matrix comprising collagen, wherein the scaffold is at least partially coated with a coating comprising: basement membrane matrix, vitronectin, fibronectin, proteins extracted from soybeans, proteins extracted from peas, proteins extracted from corn, synthetically produced peptides, RNA-binding glycine-rich (RBG) proteins, polylysine, synthetic proteins, RGD peptides, polylysine, polyarginine, polyornithine, recombinant proteins, oligomers, polymers, or any combination thereof. In some embodiments, compositions are disclosed that comprise a dissolvable scaffold in contact with an extracellular matrix comprising collagen, wherein the scaffold is in contact with a solvent for the dissolvable scaffold. In some embodiments, the scaffold can comprise polycaprolactone (PCL), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polyethylene terephthalate (PET), nylon, polyethylene (PE), polyethylene furandicarboxylate (PEF), polypropylene (PP), polyvinyl alcohol (PVA), cotton, bast fiber, viscose, modal, lyocell, plant-based protein fiber, bio-based material, viscose, cellulose, alginate fiber, thermoplastic starch, or any combination thereof. In some embodiments, the scaffold can include a non-woven configuration that produces a first porosity. In some embodiments, the first porosity can comprise an average pore size of about 30 - 70 μm. In some embodiments, the first porosity can comprise an average pore size of about 80 - 120 μm. In some embodiments, the first porosity can comprise an average pore size of about 50 μm or about 100 μm. In some embodiments, the scaffold can comprise a second porosity produced by needling.In some embodiments, the scaffold may be at least partially coated with a coating comprising matrigel, vitronectin, fibronectin, a protein extracted from soybeans, a protein extracted from peas, a protein extracted from corn, a synthetically produced peptide, an RNA-binding glycine-rich (RBG) protein, polylysine, a synthetic protein, an RGD peptide, polylysine, polyarginine, polyornithine, a recombinant protein, an oligomer, a polymer, or any combination thereof. In some embodiments, the scaffold is at least partially coated with a coating comprising matrigel, vitronectin, fibronectin, a protein extracted from soybeans, a protein extracted from peas, a protein extracted from corn, a synthetically produced peptide, an RNA-binding glycine-rich (RBG) protein, polylysine, a synthetic protein, an RGD peptide, polylysine, polyarginine, polyornithine, a recombinant protein, an oligomer, a polymer, GTMAC, a carbohydrate-binding module, a cellulose-binding domain, a starch-binding domain, or a combination thereof. In some embodiments, the scaffold is at least partially coated with a carbohydrate-binding module. In some embodiments, the carbohydrate-binding module is a cellulose-binding domain. In some embodiments, the scaffold comprises cellulose. In some embodiments, the carbohydrate-binding module is a starch-binding module. In some embodiments, the scaffold comprises starch. In some embodiments, the carbohydrate-binding module is associated with an enzyme. In some embodiments, the enzyme does not hydrolyze the scaffold. In some embodiments, the coating may comprise a modification. In some embodiments, the modification may include a reduction modification, an addition modification, or a combination thereof. In some embodiments, the modification comprises hydrolysis. In some embodiments, the modification exposes chemically reactive groups comprising hydroxyl, carboxylic acid, ketone, or a combination thereof. In some embodiments, the modification comprises oxidation. In some embodiments, the oxidation is performed with sodium periodate. In some embodiments, the scaffold comprises an amine, a carboxylic acid, a sulfate, an aldehyde, a hydrazide, a thiol, a diaziridine, an aryl-azide, an acrylate, or an epoxide. In some embodiments, the scaffold is at least partially coated with a coating comprising GTMAC. In some embodiments, the scaffold comprises a primary amine. In some embodiments, at least partially coating with a coating comprising GTAMC increases the surface charge of the scaffold. In some embodiments, the collagen is associated with the scaffold. In some embodiments, the collagen is associated with the scaffold by non-specific adsorption. In some embodiments, the collagen is associated with the scaffold by van der Waals interactions. In some embodiments, the collagen is associated with the scaffold by hydrogen bonding. In some embodiments, the collagen is associated with the scaffold by depletion interactions. In some embodiments, the collagen is associated with the scaffold by electrostatic interactions. In some embodiments, the modification of the scaffold increases the strength of non-specific adsorption of collagen to the scaffold. In some embodiments, the collagen is linked to the scaffold by covalent interactions.In some embodiments, the scaffold comprises a carbodiimide or N-hydroxysuccinimide ester (NHS ester). In some embodiments, the collagen comprises a carbodiimide or N-hydroxysuccinimide ester (NHS ester). In some embodiments, the carbodiimide is N,N′-dicyclohexylcarbodiimide (DCC) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). In some embodiments, the collagen associates with the scaffold by EDC / NHS coupling. In some embodiments, the scaffold comprises an azide or an alkyne. In some embodiments, the collagen comprises an azide or an alkyne. In some embodiments, the collagen associates with the scaffold by click chemistry reaction. In some embodiments, the scaffold comprises a Michael donor or a Michael acceptor. In some embodiments, the collagen comprises a Michael donor or a Michael acceptor. In some embodiments, the collagen associates with the scaffold by coupling of the Michael donor and the Michael acceptor. In some embodiments, the Michael donor comprises an enolate. In some embodiments, the Michael acceptor comprises an α,β-unsaturated carbonyl. In some embodiments, the scaffold comprises a thiol or a maleimide. In some embodiments, the collagen comprises a thiol or a maleimide. In some embodiments, the collagen associates with the scaffold by coupling of the thiol and the maleimide. In some embodiments, the collagen associates with the scaffold in the presence of (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (sulfo-SMCC), wherein the scaffold and the collagen comprise thiols. In some embodiments, the collagen associates with the scaffold by the Maillard reaction.

[0005] In some embodiments, the compositions provided herein comprise a scaffold. In some embodiments, the scaffolds disclosed herein can be dissolvable scaffolds, where the scaffold can be contacted with a solvent for the dissolvable scaffold. In some embodiments, the temperature of the solvent is below the boiling point of the solvent. In some embodiments, the solvent can be at a temperature of about 40°C to about 50°C. In some embodiments, the scaffold can comprise a thermoplastic polymer. In some embodiments, the thermoplastic polymer can comprise polyvinyl alcohol (PVA), and wherein the solvent can comprise water. In some embodiments, the thermoplastic polymer comprises polyvinyl alcohol (PVA) or polyvinyl alcohol (PVOH). In some embodiments, the thermoplastic polymer comprises polylactic acid (PLA). In some embodiments, the solvent comprises water. In some embodiments, the solvent comprises an organic solvent. In some embodiments, the organic solvent comprises acetone, benzylamine, or ethyl acetate. In some embodiments, the scaffold can comprise polylactic acid (PLA), and wherein the solvent can comprise benzyl, ethyl, haloalkane, or a combination thereof. In some embodiments, the solvent can comprise benzylamine. In some embodiments, the scaffold degrades and dissolves over time by hydrolysis. In some embodiments, the scaffold degrades over time by hydrolysis in a neutral aqueous solution. In some embodiments, the scaffold comprises polyhydroxyalkanoate (PHA), polyhydroxybutyrate (PHB), polycaprolactone (PCL), polylactic acid (PLA), or oxidized alginate. In some embodiments, the solvent comprises a solubilizing agent. In some embodiments, the solubilizing agent comprises ethylenediaminetetraacetic acid (EDTA). In some embodiments, the scaffold comprises alginate. In some embodiments, the solubilizing agent comprises a strong acid. In some embodiments, the strong acid includes hydrochloric acid, nitric acid, hydroiodic acid, perchloric acid, chloric acid, or a combination thereof. In some embodiments, the solubilizing agent comprises a strong base. In some embodiments, the strong base includes lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, strontium hydroxide, barium hydroxide, or a combination thereof. In some embodiments, the solubilizing agent comprises an oxidizing agent. In some embodiments, the oxidizing agent partially or completely degrades the scaffold. In some embodiments, the oxidizing agent is sodium periodate.

[0006] In some embodiments, the compositions provided herein comprise a scaffold. In some embodiments, the scaffolds disclosed herein can include bio-based materials and the solvent can include an enzyme that degrades the bio-based materials. In some embodiments, the scaffold can comprise cellulose and the solvent can comprise cellulase. In some embodiments, the scaffold comprises an ester-containing polymer. In some embodiments, the solvent comprises an esterase. In some embodiments, the solvent comprises a lipase. In some embodiments, the scaffold comprises calcium alginate. In some embodiments, the solvent comprises an alginate lyase.

[0007] In some embodiments, the compositions provided herein comprise a scaffold. In some embodiments, the scaffolds disclosed herein may comprise a three-dimensional woven material. In some embodiments, the three-dimensional woven material may comprise a spacer fabric. In some embodiments, the spacer fabric may comprise a front side in contact with a filler and a back side in contact with the filler, wherein the filler separates the front side from the back side. In some embodiments, the separation of the front side from the back side by the filler may create gaps to allow the entry of nutrients, removal of waste, cell attachment, cell growth, or any combination thereof. In some embodiments, the three-dimensional woven material may comprise a pile weave. In some embodiments, the three-dimensional woven material may include terry, frieze, velvet, corduroy, velveteen, or any combination thereof. In some embodiments, the scaffold may comprise a multi-layer scaffold material. In some embodiments, the multi-layer scaffold material may comprise a combination of different shape factors. In some embodiments, the different shape factors may include non-woven materials, woven materials, needle-punched materials, three-dimensional structures, or any combination thereof. In some embodiments, the scaffold may comprise a three-layer composite material, wherein the three-layer composite material may comprise two outer layers and one inner layer. In some embodiments, the two outer layers may include surface layer characteristics, and the inner layer may include bulk properties. In some embodiments, the multi-layer may include multi-layer thin materials. In some embodiments, the multi-layers may be fused together, held together by entanglement, laminated together, stitched together, adhered together, woven together, printed together, or any combination thereof. In some embodiments, the compositions disclosed herein may further comprise isolated animal cells in contact with the scaffold. In some embodiments, the isolated animal cells may be isolated animal fibroblasts or fibroblast-like cells. In some embodiments, the extracellular matrix may be produced by the isolated animal fibroblasts or fibroblast-like cells. In some embodiments, the isolated animal cells may be immortalized isolated animal cells. In some embodiments, the immortalized isolated animal cells may grow beyond the Hayflick limit. In some embodiments, the immortalized isolated animal cells may grow through about 40 cell divisions, about 50 cell divisions, or about 60 cell divisions. In some embodiments, the isolated animal cells may be bovine or porcine cells. In some embodiments, the isolated animal cells may include human cells. In some embodiments, the compositions disclosed herein may be at least partially decellularized. In some embodiments, the at least partially decellularized composition may comprise substantially no intact cells in the composition.In some embodiments, the scaffold further comprises polyglycolic acid (PGA), polybutylene succinate (PBS), bioabsorbable synthetic polymers, cellulose, cellulose acetate, acrylics, fibers, linen, rayon, velvet, modacrylic, olefin polyester, saran, vinylon, wool, jute, hemp, bamboo, linen fabric, or any combination thereof. In some embodiments, the scaffold may further comprise bio-based nylon, bio-based PET, bio-based PEF, bio-based polylactic acid (PLA), or any combination thereof. In some embodiments, the scaffold may comprise nylon 1,6, nylon 4,6, nylon 510, nylon 5,6, nylon 5,12, nylon 6, nylon 6,6, nylon 11, nylon 10.10, nylon 12, or any combination thereof. In some embodiments, the scaffold may include bast fibers, where the bast fibers may include flax, hemp, linen, jute, ramie, kenaf, sisal, or any combination thereof. In some embodiments, the scaffold may have a thickness of from about 0.1 mm to about 4 mm. In some embodiments, the scaffold may have a thickness of from about 1 mm to about 3 mm. In some embodiments, the scaffold may have a thickness of about 1 mm. In some embodiments, the scaffold may have a thickness of about 2 mm. In some embodiments, the scaffold may have a thickness of about 3 mm. In some embodiments, the scaffold may include fibers having a decitex of about 6.7 decitex. In some embodiments, the scaffold may comprise fibers having a diameter of from about 1 μm to about 100 μm. In some embodiments, methods of making the compositions disclosed herein are provided, which include inoculating isolated animal fibroblasts or fibroblast-like cells onto a scaffold to form a composition. In some embodiments, the scaffold may comprise a thermoplastic polymer, which may subsequently be substantially removed from the extracellular matrix prior to tanning. In some embodiments, the thermoplastic polymer may comprise polyvinyl alcohol (PVA), and the methods disclosed herein may include substantially removing PVA by contacting PVA with water at a temperature of from about 18 °C to about 90 °C prior to tanning. In some embodiments, the thermoplastic polymer may comprise polylactic acid (PLA), and the methods disclosed herein may include substantially removing PLA by contacting PLA with a solvent to remove PLA. In some embodiments, the solvent may comprise benzyl, ethyl, haloalkane, benzylamine, or any combination thereof. In some embodiments, the scaffold may include a dissolvable scaffold, which may be substantially removed by contacting the scaffold with a solvent. In some embodiments, methods of making a scaffold may include needling the scaffold using a needling machine to entangle the fibers in the nonwoven scaffold material. In some embodiments, the knitting machine may include barbed knitting needles. In some embodiments, needling may create pores in the scaffold material.In some embodiments, a method of preparing a scaffold can include at least partially coating the scaffold with a coating comprising: basement membrane matrix, vitronectin, fibronectin, a protein extracted from soybeans, a protein extracted from peas, a protein extracted from corn, a synthetically produced peptide, an RNA-binding glycine-rich (RBG) protein, polylysine, a synthetic protein, an RGD peptide, polylysine, polyarginine, polyornithine, a recombinant protein, an oligomer, a polymer, or any combination thereof.

[0008] In some embodiments, methods of preparing any of the compositions provided herein are disclosed, which include at least partially coating a scaffold with a coating comprising a basement membrane matrix, vitronectin, fibronectin, a protein extracted from soybeans, a protein extracted from peas, a protein extracted from corn, a synthetically produced peptide, an RNA-binding glycine-rich (RBG) protein, polylysine, a synthetic protein, an RGD peptide, polylysine, polyarginine, polyornithine, a recombinant protein, an oligomer, a polymer, GTMAC, a carbohydrate-binding module, a cellulose-binding domain, a starch-binding domain, or a combination thereof. In some embodiments, at least partially coating the scaffold with GTMAC provides primary amines on the scaffold. In some embodiments, the carbohydrate-binding module is associated with an enzyme. In some embodiments, the enzyme does not hydrolyze the scaffold. In some embodiments, the carbohydrate-binding module is a cellulose-binding domain. In some embodiments, the carbohydrate-binding module is a starch-binding domain. In some embodiments, the scaffold comprises a modification. In some embodiments, the modification includes adding an amine, a carboxylic acid, a sulfate, an aldehyde, a hydrazide, a thiol, a diazirine, an aryl-azide, an acrylate, or an epoxide. In some embodiments, the modification includes hydrolysis, oxidation, enzymatic digestion, acidification, or alkalinization. In some embodiments, the modification exposes one or more reactive groups that include a hydroxyl group, a carboxylic acid, a ketone, or a combination thereof. In some embodiments, the method further includes inoculating the scaffold with isolated animal fibroblasts or fibroblast-like cells to form a composition. In some embodiments, the composition comprises collagen. In some embodiments, the collagen, fibroblasts, and / or fibroblast-like cells are associated with the scaffold. In some embodiments, the collagen is associated with the scaffold by specific adsorption. In some embodiments, specific adsorption includes an interaction with a carbohydrate-binding module. In some embodiments, the carbohydrate-binding module comprises a cellulose-binding domain or a starch-binding domain. In some embodiments, the collagen, fibroblasts, and / or fibroblast-like cells are associated with the scaffold by non-specific adsorption. In some embodiments, the collagen is associated with the scaffold by non-specific adsorption. In some embodiments, the isolated animal fibroblasts and / or fibroblast-like cells are associated with the scaffold by non-specific adsorption. In some embodiments, the non-specific adsorption includes van der Waals interactions. In some embodiments, the non-specific adsorption includes hydrogen bonding. In some embodiments, the non-specific adsorption includes depletion interactions. In some embodiments, the non-specific adsorption includes electrostatic interactions. In some embodiments, the modification of the scaffold increases the non-specific adsorption of collagen, isolated animal fibroblasts, and / or fibroblast-like cells to the scaffold. In some embodiments, the collagen, isolated animal fibroblasts, and / or fibroblast-like cells are associated with the scaffold by covalent interactions.In some embodiments, the collagen associates with the scaffold through covalent interactions. In some embodiments, the isolated animal fibroblasts and / or fibroblast-like cells are linked to the scaffold through covalent interactions. In some embodiments, the scaffold comprises carbodiimide or N-hydroxysuccinimide ester (NHS ester). In some embodiments, the collagen, isolated animal fibroblasts and / or fibroblast-like cells comprise carbodiimide or N-hydroxysuccinimide ester (NHS ester). In some embodiments, the collagen associates with the scaffold through EDC / NHS coupling. In some embodiments, the fibroblasts and / or fibroblast-like cells associate with the scaffold through EDC / NHS coupling. In some embodiments, the scaffold comprises azide or alkyne. In some embodiments, the collagen, isolated animal fibroblasts and / or fibroblast-like cells comprise azide or alkyne. In some embodiments, the collagen associates with the scaffold through click chemistry reaction. In some embodiments, the collagen associates with the scaffold through click chemistry reaction. In some embodiments, the fibroblasts and / or fibroblast-like cells associate with the scaffold through click chemistry reaction. In some embodiments, wherein the scaffold comprises a Michael donor or a Michael acceptor. In some embodiments, the collagen, fibroblasts and / or fibroblast-like cells comprise a Michael donor or a Michael acceptor. In some embodiments, the collagen associates with the scaffold through the coupling of a Michael donor and a Michael acceptor. In some embodiments, the fibroblasts and / or fibroblast-like cells associate with the scaffold through the coupling of a Michael donor and a Michael acceptor. In some embodiments, the Michael donor comprises an enolate. In some embodiments, the Michael acceptor comprises an α,β-unsaturated carbonyl. In some embodiments, the scaffold comprises thiol or maleimide. In some embodiments, the collagen, fibroblasts and / or fibroblast-like cells comprise thiol or maleimide. In some embodiments, the collagen is linked to the scaffold through the coupling of thiol and maleimide. In some embodiments, the fibroblasts and / or fibroblast-like cells associate with the scaffold through the coupling of thiol and maleimide. In some embodiments, the collagen associates with the scaffold in the presence of sulfo-SMCC. In some embodiments, both the scaffold and the collagen comprise thiol. In some embodiments, the collagen, fibroblasts and / or fibroblast-like cells associate with the scaffold through the Maillard reaction. In some embodiments, the collagen associates with the scaffold through the Maillard reaction.

[0009] In some embodiments, a method is disclosed herein that includes transplanting the compositions disclosed herein onto a patient in need of a skin graft for treating skin loss or injury. In some embodiments, the patient may have a laceration, contusion, injury, sore, burn, wound, surgical wound, surgically excised skin, necrotic skin, or any combination thereof. In some embodiments, a method is disclosed herein that includes tanning the compositions disclosed herein to produce cruelty-free leather. In some embodiments, a method is disclosed herein that uses the cruelty-free leather disclosed herein as an alternative to traditional leather in leather products. Leather products comprising the cruelty-free leather disclosed herein are disclosed. In some embodiments, the leather products may include watchbands, belts, braces, packaging, shoes, boots, footwear, gloves, clothing, bags, clutches, wallets, coin purses, billfolds, key cases, credit card holders, pen cases, backpacks, cases, money clips, saddles, harnesses, whips, luggage, travel goods, rucksacks, briefcases, document cases, attache cases, business cases, pet supplies, holsters, collars, hunting and fishing supplies, gun cases, cutlery cases, pistol holsters, stationery, writing pads, book covers, camera cases, eyeglass cases, cigarette cases, cigar cases, jewelry boxes, cell phone cases, sports goods, balls, basketballs, soccer balls, footballs, or any combination thereof. In some embodiments, the clothing may include tops, bottoms, outerwear, or any combination thereof. In some embodiments, the bag may include a handbag with or without a shoulder strap. In some embodiments, the luggage may include trunks, suitcases, travel bags, beauty cases, toilet kits, or any combination thereof. In some embodiments, a method is disclosed herein that uses the compositions disclosed herein for treating a disease or disorder. In some embodiments, kits are disclosed that comprise the compositions disclosed herein or the leather products disclosed herein.

[0010] The present disclosure relates to a method that includes: 1) inoculating isolated animal fibroblasts or fibroblast-like cells onto a scaffold to form a cell layer on the scaffold, where the scaffold comprises polycaprolactone (PCL), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polyethylene terephthalate (PET), nylon, polyethylene (PE), polyethylene furanoate (PEF), polypropylene (PP), polyvinyl alcohol (PVA), cotton, bast fiber, viscose, modal, lyocell, plant-based protein fiber, alginate fiber, thermoplastic starch, or any combination thereof; 2) growing the cells to produce a composition comprising an extracellular matrix; and 3) tanning the composition comprising the extracellular matrix to form cruelty-free leather. In some embodiments, methods of using the cruelty-free leather disclosed herein as an alternative to traditional leather in leather products are disclosed. Leather products comprising the cruelty-free leather disclosed herein are disclosed. In some embodiments, the leather products can include watch straps, belts, braces, packaging, shoes, boots, footwear, gloves, clothing, bags, handbags, wallets, coin purses, billfolds, key cases, credit card holders, pen cases, backpacks, boxes, money clips, saddles, harnesses, whips, luggage, travel goods, canvas backpacks, briefcases, document cases, attache cases, business cases, pet supplies, leather cases, collars, hunting and fishing supplies, gun cases, cutlery cases, holsters, stationery, writing pads, book covers, camera cases, glasses cases, cigarette cases, cigar cases, jewelry boxes, phone cases, sports goods, balls, basketballs, footballs, rugby balls, or any combination thereof. In some embodiments, the clothing can include tops, bottoms, outerwear, or any combination thereof. In some embodiments, the bag can include a handbag with or without a shoulder strap. In some embodiments, the luggage can include large suitcases, carry-on suitcases, travel bags, makeup cases, toiletry bags, or any combination thereof. In some embodiments, methods of using the compositions disclosed herein for treating a disease or disorder are disclosed. In some embodiments, kits are disclosed that comprise a composition or a leather product as disclosed herein.

[0011] Incorporated by reference

[0012] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0013] Brief description of the drawings

[0014] The novel features of the invention are set forth with particularity in the appended claims. The features and advantages of the invention will be better understood from the following detailed description, which sets forth exemplary embodiments that utilize the principles of the invention, as well as the accompanying drawings::

[0015] Figure 1 A shows a microscopic image of unstained fibroblasts attached to a single-strand nylon scaffold under shaking conditions. Figure 1 B shows a microscopic image of unstained fibroblasts attached to a double-strand nylon scaffold under shaking conditions. Figure 1 C shows a microscopic image of unstained fibroblasts attached to a 40:60 bamboo / cotton scaffold under shaking conditions. Figure 1 D shows a microscopic image of fibroblasts attached to a single-strand cotton scaffold material under shaking conditions. Figure 1 E shows a microscopic image of calcein-AM stained fibroblasts attached to a single-strand nylon scaffold under shaking conditions. Figure 1 F shows a microscopic image of calcein-AM stained fibroblasts attached to a double-strand nylon scaffold under shaking conditions. Figure 1 G shows a microscopic image of calcein-AM stained fibroblasts attached to a 40:60 bamboo / cotton scaffold under shaking conditions. Figure 1 H shows a microscopic image of calcein-AM stained fibroblasts attached to a single-strand cotton scaffold under shaking conditions. Figure 1 I shows a microscopic image of Hoescht dye stained fibroblasts attached to a single-strand nylon scaffold under shaking conditions. Figure 1 J shows a microscopic image of Hoescht dye stained fibroblasts attached to a double-strand nylon scaffold under shaking conditions. Figure 1 K shows a microscopic image of Hoescht dye stained fibroblasts attached to a 40:60 bamboo / cotton hybrid scaffold material under shaking conditions. Figure 1 L shows a microscopic image of Hoescht dye stained fibroblasts attached to a single-strand cotton scaffold under shaking conditions.

[0016] Figure 2 A shows the scanning data of the raw skin grown on a photo-perforated PLA scaffold with a pore size of 55 μm. Figure 2 B shows the scanning data of the raw skin grown on a PLA scaffold with a pore size of 55 μm. Figure 2 C shows the scanning data of the raw skin grown on a PLA scaffold with a pore size of 80 μm. Figure 2 D shows the raw skin grown on a PLA scaffold with a pore size of 100 μm.

[0017] Figure 3 A shows the histological image of a 5-μm cross-section of the tissue stained with a trichrome staining method, where the scaffold is PLA with an average pore size of about 100 μm. Figure 3Figure B shows a histological image of a 5-μm cross-section of tissue stained with a three-color staining method, where the scaffold is PLA with an average pore size of approximately 50 μm.

[0018] Figure 4A Figure shows a schematic diagram of an experiment conducted to determine the effect of PLA scaffolds immersed in FBS on cell attachment and proliferation. Figure 4B Figure shows the cell / biopsy results of samples grown in a PET control not immersed in serum, compared with the cell / biopsy results of PLA immersed in serum and not immersed in serum.

[0019] Figure 5 Figure shows the percentage of cells on PLA and Lyocell scaffolds, which are unmodified, conjugated with pea protein, and conjugated with wheat protein, determined by MTT assay after 1 day and 4 days, where NT = untreated, Pea = pea protein, Wheat = wheat protein.

[0020] Figure 6 Figure A shows a fluorescence microscopic image of fibroblasts grown on an unmodified PLA scaffold for 1 day. Figure 6 Figure B shows a fluorescence microscopic image of fibroblasts grown on a pea-protein-conjugated PLA scaffold for 1 day. Figure 6 Figure C shows a fluorescence microscopic image of fibroblasts grown on a wheat-protein-conjugated PLA scaffold for 1 day. Figure 6 Figure D shows a fluorescence microscopic image of fibroblasts grown on an unmodified Lyocell scaffold for 1 day. Figure 6 Figure E shows a fluorescence microscopic image of fibroblasts grown on a pea-protein-conjugated Lyocell scaffold for 1 day. Figure 6 Figure F shows a fluorescence microscopic image of fibroblasts grown on a wheat-protein-conjugated Lyocell scaffold for 1 day.

[0021] Figure 7 Figure A and Figure 7 Figure B show histological images of 5-μm cross-sections of tissue stained with a three-color staining method on scaffolds with markers highlighting dense tissue, fibers, cell nuclei, and lower-density tissue.

[0022] Figure 8 Figure A shows a histological image of a 5-μm cross-section of natural cowhide stained with a three-color staining method, marking the granular and dermal positions. Figure 8 Figure B shows a histological image of a 5-μm cross-section of the top layer of cowhide (grain side) stained with a three-color staining method.

[0023] Figure 9A shows a fluorescence microscopic image of fibroblasts magnified 4-fold after the addition of Calcein AM. Live cells exhibit green fluorescence, and the arrow highlights the cell spreading, indicating the affinity for the scaffold. Figure 9 B shows a fluorescence microscopic image of fibroblasts magnified 10-fold after the addition of Calcein AM. Live cells exhibit green fluorescence, and the arrow highlights the cell spreading, indicating the affinity for the scaffold. The second arrow highlights the fibers under white light illumination.

[0024] Figure 10 A shows a fluorescence microscopic image of fibroblasts magnified 10-fold after the addition of Calcein AM, making the live cells exhibit green fluorescence. The cell spreading and attachment highlight the good affinity for the scaffold. Figure 10 B shows a fluorescence microscopic image of fibroblasts magnified 10-fold after the addition of Calcein AM, making the live cells exhibit green fluorescence. The cell aggregation and clustering indicate a poor affinity for the scaffold.

[0025] Figure 11 Shows a histological image of a 5-μm cross-section of the tissue stained with trichrome staining, where the markings indicate the slide panorama, scale bar, cursor position, and tissue thickness.

[0026] Figure 12 Shows the remaining glucose levels measured in fresh medium, bamboo / cotton 40:60, single-strand nylon, double-strand nylon, and single-strand cotton scaffolds under static and shaking conditions during the last 4 feedings from day 10 to day 17 of tissue culture.

[0027] Figure 13 Shows the basic physical data of the tissue of VL1 (blue) on 100 gsm (blue), 50 gsm (red), 55-μm pore size light-perforated (55LP) (green), pressed PET (PPET) (purple), and PPET scaffolds. The physical data include the harvested thickness, skin thickness, harvested weight, raw material weight, skin weight, and calculated tissue-only weight.

[0028] Figure 14 A and Figure 14 B show histological images of 5-μm cross-sections of the tissue grown on a 55-μm pore size light-perforated scaffold stained with trichrome staining.

[0029] Figure 15 A, Figure 15 B, Figure 15 C, Figure 15 D, Figure 15 E and Figure 15 F show histological images of 5-μm cross-sections of the tissue grown on a 50-gsm scaffold stained with trichrome staining.

[0030] Figure 16 A,Figure 16 B, Figure 16 C, Figure 16 D, Figure 16 E and Figure 16 F show histological images of 5-μm cross-sections of tissue grown on 100 gsm scaffolds stained with a trichrome stain.

[0031] Figure 17 A shows a fluorescence microscopy image of fibroblasts grown on an unmodified PLA scaffold after 4 days. Figure 17 B shows a fluorescence microscopy image of fibroblasts grown on a pea protein-conjugated PLA scaffold after 4 days. Figure 17 C shows a fluorescence microscopy image of fibroblasts grown on a wheat protein-conjugated PLA scaffold after 4 days. Figure 17 D shows fibroblasts grown on an unmodified Lyocell scaffold after 4 days. Figure 17 E shows fibroblasts grown on a pea protein-conjugated Lyocell scaffold after 4 days. Figure 17 F shows fibroblasts grown on a wheat protein-conjugated Lyocell scaffold after 4 days.

[0032] Figure 18 Shows the cell viability of fibroblasts grown on unmodified Lyocell (Lyo NT), pea protein-modified Lyocell (Lyo Pea) or wheat protein-modified Lyocell (Lyo Wheat), and pea protein-modified PLA (PLA Pea) or wheat protein-modified PLA (PLA Wheat), which was normalized to the viability of fibroblasts grown on unmodified PLA (PLA NT) on day 1 (blue) and day 4 (red).

[0033] Figure 19 Shows the relative cell viability of fibroblasts grown on unmodified Lyocell (Lyo), Lyocell conjugated with pea protein by cold adsorption (Lyo pea cold), Lyocell conjugated with pea protein by a single autoclave cycle (Lyo pea auto 1), Lyocell conjugated with pea protein by 2 autoclave cycles (Lyo pea auto2), and Lyocell conjugated with pea protein by 4 autoclave cycles (Lyo pea auto 4) relative to fibroblasts grown on unmodified PLA at 1 day (blue) and 4 days (red).

[0034] Figure 20 A shows a fluorescence microscopy image of fibroblasts grown on an unmodified PLA scaffold after 1 day. Figure 20 B shows a fluorescence microscopy image of fibroblasts grown on an unmodified Lyocell scaffold after 4 days. Figure 20C shows the fluorescence microscopic image of fibroblasts grown on a pea protein-conjugated Lyocell scaffold for 4 days, where the pea protein was conjugated by autoclaving. Figure 20 D shows the fluorescence microscopic image of fibroblasts grown on a pea protein-conjugated PLA scaffold for 4 days. Figure 20 E shows the fluorescence microscopic image of fibroblasts grown on an oxidized Lyocell scaffold for 4 days. Figure 20 F shows the fluorescence microscopic image of fibroblasts grown on an oxidized Lyocell scaffold for 4 days, which has been functionalized with pea protein.

[0035] Figure 21 Shows the relative cell viability of fibroblasts grown on pea protein-conjugated PLA (PLA Pea), pea protein-conjugated Lyocell (autoclaved Lyocell Pea), oxidized and pea protein-conjugated Lyocell (oxidized Lyocell Pea), oxidized Lyocell, and unmodified Lyocell scaffolds on the 1st day (blue) and 4th day (red) compared to the cells grown on unmodified PLA.

[0036] Figure 22 Shows the average collagen concentration of biopsies at seeding densities of 125k cells / cm 2 250k cells / cm 2 500k cells / cm 2 and 1M cells / cm 2 after 24 hours or 96 hours of digestion.

[0037] Figure 23 Shows the collagen concentration (wet weight) of tissues at seeding densities of 62.5k cells / cm 2 125k cells / cm 2 500k cells / cm 2 after 24 hours (blue) or 96 hours (orange) of digestion.

[0038] Figure 24 Shows the collagen concentration per unit wet weight in tissues at seeding densities of 500k cells / cm 2 125k cells / cm 2 60k cells / cm 2 and 30k cells / cm 2 after 24 hours (orange) and 96 hours (blue) of digestion.

[0039] Figure 25 A shows the fluorescence microscopic image of fibroblasts that have successfully attached to the scaffold and spread out. Figure 25 B shows the fluorescence microscopic image of fibroblasts that have not successfully attached to the scaffold and have aggregated.

[0040] Figure 26 Shows the glucose concentration (g / L) of cell cultures after 1 day (blue), 3 days (red), and 7 days (green) of growth on control scaffolds and nylon, PLA, 50:50 PLA:Bioco PLA, cotton, trilobal viscose, conventional viscose, and Lyocell scaffold materials.

[0041] Figure 27 Shows the collagen content (blue) and biopsy (red) normalized to protein content of tissues grown on PET scaffolds and PLA, PVOH, PET, Lyocell with nylon mesh, 3.3 dtex Lyocell, 1.7 dtex Lyocell, 6.7 dtex Lyocell, and Lyocell with alginate scaffolds in hPL medium.

[0042] Figure 28 Shows tanned hides produced from the growth of tissues on various scaffolds. From left to right in the top row, the tissues are hPL PET, FBS PET, PVOH (absent), PLA, and in the bottom row are 1.7 dtex Lyocell, 3.3 dtex Lyocell, 6.7 dtex Lyocell, alginate, and nylon mesh.

[0043] Figure 29 Shows a control hide not exposed to high temperature (left), a hide exposed to 95 °C to remove the scaffold after tanning (middle), and a hide exposed to 95 °C to remove the scaffold before tanning (right).

[0044] Figure 30 A shows the hide before (left) and after (right) tanning after treatment with benzylamine. Figure 30 B shows the hide before (left) and after (right) tanning after treatment with ethyl acetate. Figure 30 C shows the hide untreated with benzylamine or ethyl acetate.

[0045] Figure 31 Shows the collagen concentration (blue) and total protein concentration (red) of biopsy tissues grown on 55 μm pore size scaffolds (55 - 1), 55 μm pore size optically perforated (55 - LP - 1) scaffolds, 80 μm pore size scaffolds (80 - 1), and 100 μm pore size scaffolds (100 - 1).

[0046] Figure 32 Shows the collagen content (normalized by volume) after 24 hours (blue) or 96 hours (red) of digestion, which is from tissues grown on pressed controls (pressed PET), Thinsulate, cut - line towels, cut - line spacers, fibertex, double - pressed PET, smooth - faced laminates, rough - faced laminates, autoclaved laminates, and only adsorbable scaffold materials.

[0047] Figure 33 Shows the potential standardization factors of tissues grown on compression controls (compressed PET), Thinsulate, cut towel, cut spacer, fibertex, double-layer compressed PET, smooth surface laminate, rough surface laminate, autoclaved laminate, and adsorbable-only scaffold materials, including DNA (μg), thickness (mm*5), and weight (mg*10).

[0048] Figure 34 Shows the thickness, weight, hydroxyproline content, and DNA content of tissues grown on compression control (PET) (blue) and Fibertex (red).

[0049] Figure 35 Shows the collagen content of biopsy tissues grown on compression control (PET) (blue) and Fibertex (red) after 4 weeks or 8 weeks of growth and after 24 hours or 96 hours of digestion.

[0050] Figure 36 Shows the histological image of a 5-μm cross-section of tissue grown on a compressed PET scaffold stained with trichrome.

[0051] Figure 37 Shows the histological image of a 5-μm cross-section of tissue grown on a double-layer compressed PET scaffold stained with trichrome.

[0052] Figure 38 Shows the histological image of a 5-μm cross-section of tissue grown on a knitted towel-like material stained with trichrome.

[0053] Figure 39 A and Figure 39 B show the histological image of a 5-μm cross-section of tissue stained with trichrome, showing a flat, aligned collagen arrangement.

[0054] Figure 40 A and Figure 40 B show the histological image of a 5-μm cross-section of tissue grown on a knitted towel-like material stained with trichrome.

[0055] Figure 41 A and Figure 41 B show images of the knitted towel-like material on which tissues can grow.

[0056] Figure 42 Shows the histological image of a 5-μm cross-section of tissue grown on Vicryl and a compressed laminate scaffold stained with trichrome.

[0057] Figure 43Shows a histological image of a 5μm cross-section of tissue grown on a Vicryl and coarsely pressed laminated scaffold stained with trichrome staining.

[0058] Figure 44 Shows a histological image of a 5μm cross-section of tissue grown on a Vicryl and autoclaved pressed laminated scaffold stained with trichrome staining.

[0059] Figure 45 Shows a histological image of a 5μm cross-section of tissue grown on a Fibertex scaffold stained with trichrome staining.

[0060] Figure 46 Shows a histological image of a 5μm cross-section of tissue grown on a Thinsulate scaffold stained with trichrome staining.

[0061] Figure 47 Shows a histological image of a 5μm cross-section of tissue grown on a Vicryl-only scaffold stained with trichrome staining. Detailed description of the invention

[0063] Several aspects will be described below and described with reference to example applications. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the features described herein. The features described herein may be practiced without one or more of the specific details or using other methods. The features described herein are not limited by the order of the acts or events described, as some acts may occur in a different order and / or concurrently with other acts or events. Additionally, not all of the acts or events shown are required to implement the methods in accordance with the features described herein.

[0064] The terms used herein are for the purpose of describing particular situations only and are not intended to be limiting. As used herein, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" are also intended to include the plural forms. Additionally, to the extent that the terms "comprises", "comprising", "has", "having", "contains", or variants thereof are used in the detailed description and / or claims, these terms are intended to be inclusive in a manner similar to the term "including".

[0065] In the present disclosure, the term "about" or "approximately" may refer to a range of up to 10% of a given value. In the present disclosure, the term "substantially" means something that can be accomplished to a great extent or degree.

[0066] As used herein, the term "fibroblast" may include connective tissue cells present in the skin and tendons of the body. Fibroblasts can be obtained from a biopsy of the skin or tendon. In addition to the skin and tendon, fibroblasts are also commonly present in many tissues and organs. A fibroblast can be a type of biological cell that synthesizes the extracellular matrix and collagen. A fibroblast can be a class of biological cells that produce the structural framework (matrix) of animal tissues and play a key role in wound healing. A fibroblast can be the most common connective tissue cell in animals. Fibroblasts can comprise the main part of a tissue layer grown in culture, which can be tanned into leather.

[0067] As used herein, the term "fibroblast-like cell" may refer to fibroblasts that have been grown in culture, immortalized, or a combination thereof. Fibroblast-like cells can comprise cells that have differentiated into cells having a morphology, phenotype, or a combination thereof that is substantially similar to that of fibroblasts. Fibroblast-like cells can comprise genetic or phenotypic alterations from fibroblasts while still expressing gene expression that is substantially similar to that of fibroblasts. Fibroblast-like cells can have characteristics similar to those of fibroblasts, such as the ability to synthesize collagen, the extracellular matrix, the structural matrix (matrix) of animal tissues, or any combination thereof. Compared to fibroblasts, fibroblast-like cells can produce a substantially similar level of collagen.

[0068] As used herein, the term "pluripotent stem cell" may refer to any precursor cell that has the ability to form any adult somatic cell except for the placenta.

[0069] As used herein, the term "embryonic stem cell" or "ES cell" or "ESC" may refer to a precursor cell that has the ability to form any adult somatic cell.

[0070] As used herein, the term "induced pluripotent stem cell" or "iPS cell" or "iPSC" may refer to a type of pluripotent stem cell that is artificially derived from a non-pluripotent cell (e.g., an adult somatic cell). Induced pluripotent stem cells have the same ability to form any adult somatic cell as embryonic stem cells, but may not be derived from an embryo.

[0071] As used herein, the term "isolated" may refer to a cell that has been removed from an animal or human body. Isolated cells can be grown in culture or in vitro. Isolated cells can be in contact with other isolated cells, scaffolds, culture media, or a combination thereof.

[0072] As used herein, the term "decellularized" or "decellularized" may refer to the removal of cells from a cell layer. The term "at least partially decellularized" may refer to the removal of at least some cells from a cell layer. "Decellularized" may refer to the process of removing cells to form a decellularized cell layer, and can be achieved by methods such as salting or using detergents.

[0073] As used herein, the term "cruelty-free leather" may refer to the leather materials described herein, which can be used as leather materials for any mammal or non-mammal. The disclosure herein can be used for human and non-human mammals, such as members of non-human primates and bovine, sheep, porcine, equine, canine, and feline species, as well as rodents, such as mice, rats, and guinea pigs, members of the rabbit family including rabbits, fish including sharks and rays, birds including ostriches, and reptiles including lizards, snakes, and crocodiles. In some embodiments, the cruelty-free leather may include artificial leather. In some embodiments, the cruelty-free leather may include environmentally friendly leather, plastic-free leather, or any combination thereof. In some embodiments, the cruelty-free leather may include tanned artificial cell layers, tanned at least partially decellularized cell layers, or a combination thereof. In some embodiments, the cell layer or at least partially decellularized cell layer may include a dermal layer, an epidermal layer, at least partially decellularized dermal layer, at least partially decellularized epidermal layer, or any combination thereof. In some embodiments, the mammalian cruelty-free leather that can be formed may depend on the cell sources used in the invention described herein, such as keratinocytes and fibroblasts. For example, when bovine keratinocytes and fibroblasts can be used to form a skin equivalent, bovine cruelty-free leather can be formed.

[0074] Cruelty-free leather composition

[0075] In some embodiments, compositions are disclosed herein that include scaffolds for culturing cells. In some embodiments, the scaffold may provide a matrix or support for cell attachment, growth, production of extracellular matrix, or any combination thereof. In some embodiments, the scaffolds and cell compositions disclosed herein can be tanned to produce cruelty-free leather. In some embodiments, the methods or compositions disclosed herein can be used to produce skin equivalents. In some embodiments, the compositions disclosed herein can be transplanted onto a patient in need for treating a disease or disorder. In some embodiments, cells, cell layers, stratified structures, or cruelty-free leather can be seeded onto the scaffold. In some embodiments, the scaffold may include a substrate. In some embodiments, the scaffold may provide a certain degree of stiffness (e.g., tear resistance), elasticity, or both. In some embodiments, the cruelty-free leather may include a part or the whole of the scaffold. In some embodiments, the cruelty-free leather may not include the scaffold. In some embodiments, after assisting in forming layers in the cruelty-free leather, the scaffold can be removed from the final cruelty-free leather product. In some embodiments, the scaffold included in the cruelty-free leather may degrade over time. In some embodiments, the scaffold can be degradable, biodegradable, bioabsorbable, resorbable, or any combination thereof.

[0076] In some embodiments, the scaffold may comprise a three-dimensional woven material. In some embodiments, the three-dimensional woven material may comprise a spacer fabric. In some embodiments, the spacer fabric may comprise a plurality of faces. In some embodiments, the plurality of faces may comprise a front face and a back face. In some embodiments, the faces may comprise a woven material. In some embodiments, a filler may separate the front face from the back face. In some embodiments, the filler may keep the front face and the back face at a certain distance. In some embodiments, the filler may provide a gap to allow the entry of nutrients, the removal of waste, cell growth, or any combination thereof.

[0077] In some embodiments, the scaffold may be made of natural materials, synthetic materials, or any combination thereof. In some embodiments, the scaffold may comprise a substrate. In some embodiments, the substrate may comprise a substrate for cell growth. In some embodiments, the scaffold may be formed using a mesh made of a bioabsorbable synthetic polymer. In some embodiments, the scaffold may be formed by attaching a nylon mesh to a silicon film. In some embodiments, the scaffold may comprise a bilayer structure of a collagen sponge and a silicon sheet. In some embodiments, the scaffold may be formed using a terminal collagen sponge. In some embodiments, the scaffold may be made in the form of a sheet. In some embodiments, the scaffold may be formed by matching collagen sponges with different pore sizes. In some embodiments, an acellular dermal matrix (ADM) may be formed using at least partially acellular fibrin glue, allogeneic skin, or a combination thereof.

[0078] Scaffold material

[0079] In some embodiments, the present disclosure provides compositions that include a scaffold in contact with a collagen-containing extracellular matrix, wherein the scaffold can include a nonwoven needled material. In some embodiments, the present disclosure provides compositions that include a scaffold in contact with a collagen-containing extracellular matrix, wherein the scaffold can include a three-dimensional woven material. In some embodiments, the present disclosure provides compositions that include a scaffold in contact with an extracellular matrix that includes collagen, wherein the scaffold can include polycaprolactone (PCL), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polyethylene terephthalate (PET), nylon, polyethylene (PE), polyethylene furandicarboxylate (PEF), polypropylene (PP), polyvinyl alcohol (PVA), cotton, bast fiber, viscose, modal, lyocell, plant-based protein fiber, bio-based material, viscose, cellulose, alginate fiber, thermoplastic starch, or any combination thereof. In some embodiments, the scaffold can include polycaprolactone. In some embodiments, the scaffold can include polylactic acid. In some embodiments, the scaffold can include poly(lactic-co-glycolic acid). In some embodiments, the scaffold can include polyethylene terephthalate. In some embodiments, the scaffold can include nylon. In some embodiments, the scaffold can include polyethylene. In some embodiments, the scaffold can include polyethylene furandicarboxylate. In some embodiments, the scaffold can include polypropylene. In some embodiments, the scaffold can include polyvinyl alcohol. In some embodiments, the scaffold can include cotton. In some embodiments, the scaffold can include bast fiber. In some embodiments, the scaffold can include viscose. In some embodiments, the scaffold can include modal. In some embodiments, the scaffold can include lyocell. In some embodiments, the scaffold can include plant-based protein fiber. In some embodiments, the scaffold can include bio-based material. In some embodiments, the scaffold can include cellulose. In some embodiments, the scaffold can include alginate fiber. In some embodiments, the scaffold can include thermoplastic starch.

[0080] The methods and compositions provided herein can provide design advantages over the use of natural leather. In some embodiments, the choice of scaffold material, as described in Example 13, can affect the physical properties of the resulting material. In some embodiments, the choice of scaffold material can affect the tear strength (e.g., average double tear strength) of the resulting material. In some embodiments, the choice of scaffold material can result in a stronger material. In some embodiments, the choice of scaffold material can result in a weaker material. In some embodiments, as described in Example 13, the compositions provided herein can include a strength similar (e.g., in tear) to natural (e.g., bovine) hide while being thinner than natural hide.

[0081] In some embodiments, the scaffold may comprise natural substances such as collagen (e.g., collagen matrix), natural adhesives (e.g., fibrin glue, cold glue, animal glue, blood protein glue, casein glue, or plant glue such as starch and dextrin glue). In some embodiments, the scaffold may comprise polylactic acid, polyacetolactone, polycaprolactone, hydrogel, or any combination thereof. In some embodiments, the scaffold may include silk. In some embodiments, the scaffold may be made of silk. In some embodiments, the scaffold may comprise fibroin, cellulose, cotton, acetate fiber, acrylic fiber, latex fiber, linen, nylon, rayon, velvet, modified acrylic fiber, olefin polyester, saran, vinylon, wool, jute, hemp, bamboo, flax, or any combination thereof. In some embodiments, the scaffold may comprise fibers. In some embodiments, the fibers may be silk, cotton, wool, wood, especially cellulose extracted from wood, plants, algae, polyamide, modified cellulose, poly(p-phenylene terephthalamide), acrylic fibers such as fibers of polymethyl methacrylate or 2-hydroxyethyl polymethacrylate, polyolefin fibers such as fibers of polyethylene or polypropylene, glass, silica, aramid, carbon in the form of graphite, polytetrafluoroethylene, insoluble collagen, polyester, polyvinyl chloride, polyvinylidene chloride, polyvinyl alcohol, polyacrylonitrile, chitosan, polyurethane, poly(urethane-urea), polyethylene glycol phthalate, and fibers formed from blends of polymers such as the above polymers, such as polyamide / polyester fibers, or any combination thereof. In some embodiments, the scaffold comprises cellulose or modified cellulose. In some embodiments, the modified cellulose may include rayon, viscose, acetate fiber, rayon acetate fiber, or any combination thereof.

[0082] In some embodiments, such as those described in Example 1, the scaffold is a bamboo / cotton blend, single-strand nylon, double-strand nylon, or single-strand nylon. In some embodiments, such as those described in Example 1, a high level of growth and spindle-shaped cells are achieved using a bamboo / cotton scaffold.

[0083] In some embodiments, the scaffold may comprise a polymer. In some embodiments, the polymer may comprise a biopolymer. In some embodiments, the biopolymer may include, but is not limited to, chitin, chitosan, elastin, collagen, keratin, or polyhydroxyalkanoates. In some embodiments, the polymer may be biodegradable, bio-stable, or a combination thereof. In some embodiments, the polymer in the scaffold may be a natural polymer. In some embodiments, exemplary natural polymers may include polysaccharides such as alginate, cellulose, dextran, amylopectin, polyhyaluronic acid, chitin, poly(3-hydroxyalkanoate), poly(3-hydroxyoctanoate), poly(3-hydroxy fatty acid), or any combination thereof. In some embodiments, the polymer may include polyethylene (PE), polypropylene (PP), polyethylene terephthalate (PET), polyamide 6,6 (PA 6,6), polyamide 11 (PA 11), polyvinylidene fluoride (PVDF), polyethylene furandicarboxylate (PEF), polyurethane (PU), polyhydroxyalkanoate (PHA), polyhydroxybutyrate (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), polylactic acid (PLA), polycaprolactone (PCL), polybutylene succinate (PBS), polyglycolic acid (PGA), poly(lactic acid-co-glycolic acid) (PLGA), polyvinyl alcohol (PVOH), alginate, polyethylene glycolated fibrin (P-fibrin), polyglycerol sebacate (PGS), poly(L-lactic acid) (PLLA), polylactic acid-glycolic acid (PLGA), poly(D,L-lactic acid) / polyethylene glycol / poly(D-L-lactic acid) (PDLLA-PEG), hyaluronic acid (HA), or any combination thereof. In some embodiments, the scaffold may further comprise chemical derivatives of natural polymers. In some embodiments, the chemical derivatives may include substitution and / or addition of chemical groups such as alkyl, alkylene, hydroxylation, oxidation, another chemical modification, or any combination thereof. In some embodiments, the natural polymer may also be selected from proteins such as collagen, zein, casein, gelatin, gluten, and serum proteins. In some embodiments, the polymer in the scaffold may be a biodegradable synthetic polymer, including polyalpha-hydroxy acids such as poly-L-lactic acid (PLA), polyglycolic acid (PGA), or their copolymers (e.g., poly(D,L-lactic acid-co-glycolic acid) (PLGA)) and hyaluronic acid.

[0084] In some embodiments, the scaffold may comprise polyvinyl alcohol (PVA), polyvinyl acetate (PVA), polycaprolactone (PCL), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polyethylene terephthalate (PET), nylon, polyethylene (PE), polyethylene furanoate (PEF), polypropylene (PP), polyvinyl alcohol (PVA), polyhydroxyalkanoate (PHA), polyhydroxybutyrate (PHB), polycaprolactone (PCL), alginate, oxidized alginate, cotton, bast fiber, viscose, modal, lyocell, plant-based protein fiber, bio-based material, viscose, cellulose, alginate fiber, thermoplastic starch, or any combination thereof. In some embodiments, the scaffold comprises polyhydroxyalkanoate (PHA). In some embodiments, the scaffold comprises polyhydroxybutyrate (PHB). In some embodiments, the scaffold comprises polycaprolactone (PCL). In some embodiments, the scaffold comprises oxidized alginate. In some embodiments, the scaffold comprises polyvinyl alcohol (PVOH). In some embodiments, the scaffold comprises polyvinyl acetate (PVA).

[0085] In some embodiments, such as those described in Example 11, the scaffold may comprise 3M's Type G Thinsulate material, which comprises 3 main layers, 2 spunbonded layers, and one airlaid nonwoven layer, which is a sandwich structure. In some embodiments, the scaffold may include a towel-like material made of PET ( Figure 41 A-B). In some embodiments, the scaffold may include two fabrics, separated by fibers between the two fabrics such that the two outer fabrics are spaced a certain distance apart. In some embodiments, the scaffold may comprise a single fiber flock of lyocell fibers (e.g., "Fibertex"). In some embodiments, the scaffold may include a needlepunched nonwoven material that is pressed on one side and laminated to the other side by suture layers. In the above materials, the directions of the two needlepunched nonwoven materials may be such that the pressed side of each of the two materials can face outward or inward. In some cases, the needlepunched nonwoven material may be autoclaved before assembly. In some embodiments, the scaffold may include one or more vicryl meshes connected together. In some cases, as described in Example 11, the different scaffolds described herein may be used to achieve different tissue characteristics.

[0086] In some embodiments, the scaffold can be bioabsorbable. In some embodiments, a bioabsorbable scaffold can be a non-cytotoxic structure or material that is capable of containing or supporting living cells and maintaining them in a desired configuration for a period of time. In some embodiments, the term "bioabsorbable" can refer to any material that can be broken down into non-toxic by-products that can be excreted from or metabolized within the body. In some embodiments, exemplary bioabsorbable materials for scaffolds can include poly(lactic acid), poly(glycolic acid), poly(trimethylene carbonate), poly(dimethyltrimethylene carbonate), poly(amino acids), tyrosine-derived poly(carbonates), poly(carbonates), poly(caprolactone), poly(p-dioxanone), poly(esters), poly(ester-amides), poly(anhydrides), poly(orthoesters), collagen, gelatin, albumin, proteins, polysaccharides, mucopolysaccharides, saccharides, glycosaminoglycans, poly(ethylene glycol), poly(propylene glycol), poly(acrylates), poly(methacrylates), poly(vinyl alcohol), hyaluronic acid, chondroitin sulfate, heparin, dermatan sulfate, versican, copolymers, polymer blends, polymer mixtures, oligomers containing bioabsorbable bonds, or any combination thereof.

[0087] In some embodiments, the scaffold can be reticular. In some embodiments, the reticulum can be a network of materials (such as wires, ropes, strands, fibers, or any combination thereof) that can be woven or otherwise connected. In some embodiments, the network can include materials that can be artificial, biological, or any combination thereof. In some embodiments, the network can have pores, and the pore diameter can be regular or irregular, the shape can be regular or irregular, the pattern can be regular or irregular, or any combination thereof. In some embodiments, the network can be two-dimensional or three-dimensional. In some embodiments, the network can have pores with a diameter, spacing, or any combination thereof of about 10 nm - 10 cm. In some embodiments, the pore diameter can generally be about: 10 nm, 20 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 12 mm, 15 mm, 2 cm, 2.5 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, or 10 cm. In some embodiments, the pore diameter can be at least 10 nm (such as 50 nm, 100 nm, 500 nm, 1 mm). In some embodiments, the pore diameter can be at most 1 cm (such as 5 cm, 10 mm, 5 mm, 1 mm). The network diameter can be about 20 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm,.6 mm,.7 mm,.8 mm,.9 mm, 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 12 mm, 15 mm, 2 cm, 2.5 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, or 10 cm. In some embodiments, the network diameter can be at least 20 nm (such as, 50 nm, 100 nm, 500 nm, 1 mm). In some embodiments, the network diameter can be at most 3 cm (such as, 1 cm, 500 mm, 100 mm, 10 mm, 1 mm). In some embodiments, the diameter of the wires (such as fibers, wires, nets, etc.) or materials forming the network can be a diameter of about 50 nm - about 10 mm. In some embodiments, the network can not be a scaffold.

[0088] In some embodiments, the scaffold can be a support structure for cell proliferation. In some embodiments, the scaffold can be permeable to fluids, nutrients, such that cell culture medium can contact the surface of the cell layer.

[0089] In some embodiments, the scaffold can comprise a textile composed of entangled fibers. In some embodiments, the fibers can be entangled by weaving, knitting, or nonwoven fabric manufacturing techniques or some combination thereof. In some embodiments, the scaffold can be porous, biocompatible, sterilizable, mechanically and chemically stable, uniform, and any combination thereof. In some embodiments, the scaffold can comprise a three-dimensional structure. In some embodiments, cells can be seeded within the scaffold. In some embodiments, the scaffold can have different thicknesses. In some embodiments, the scaffold can have a thickness suitable for forming a cell layer. In some embodiments, the scaffold can have a thickness of about 0.1 mm - about 10 mm, such as about 0.1 mm - about 5 mm, about 0.1 mm - about 4 mm, about 0.1 mm - about 3 mm, about 0.1 mm - about 2 mm, about 0.1 mm - about 1 mm, about 0.2 mm - about 1 mm, about 0.3 mm - about 1 mm, about 0.4 mm - about 1 mm, about 0.5 mm - about 1 mm, 0.3 mm - about 1.5 mm, about 0.4 mm - about 1.2 mm, about 0.6 mm - about 1.2 mm, or about 0.7 mm - about 1.5 mm. In some embodiments, the scaffold can have a thickness of about 0.5 mm - 1 mm. In some embodiments, the scaffold can be at least 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.8 mm, 1 mm, 2 mm, 3 mm, 4 mm, or 5 mm in thickness. In some embodiments, the scaffold can be at most 0.5 mm, 0.8 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm in thickness. In some embodiments, the scaffold can have the length and / or width of the cell layer to be placed and / or grown on the scaffold. In some embodiments, the scaffold can have the length and / or width of the cell layer described herein. In some embodiments, the scaffold can comprise pore sizes less than 1 nm. In some embodiments, the scaffold can comprise pore sizes greater than 1 nm. In some embodiments, the scaffold can comprise pore sizes of 10 μm - 900 μm.

[0090] In some cases, as described in Example 3, the pore size may affect tissue ingrowth. In certain cases, the larger the pore size, the better the tissue growth ( Figure 15 A - F). In certain cases, the smaller the pore size, the more weight the fibers contribute, creating the illusion of a heavier, stronger hide ( Figure 16 A - F).

[0091] In some embodiments, the scaffold may comprise a three-dimensional woven material. In some embodiments, the three-dimensional woven material may comprise a spacer fabric. In some embodiments, the spacer fabric may comprise a front side in contact with a filler and a back side in contact with the filler, wherein the filler separates the front side from the back side. In some embodiments, the front side is separated from the back side by the filler to create a gap to allow entry of nutrients, removal of waste, cell attachment, cell growth, or any combination thereof. In some embodiments, the three-dimensional woven material may comprise a pile fabric. In some embodiments, the three-dimensional woven material may comprise a terry cotton fabric, a napped tweed, a velvet, a corduroy, a velveteen, or any combination thereof. In some embodiments, the scaffold may comprise a multi-layer scaffold material. In some embodiments, the multi-layer scaffold material may comprise a combination of different shape factors. In some embodiments, the different shape factors may comprise a non-woven material, a woven material, a needle-punched material, a three-dimensional structure, or any combination thereof. In some embodiments, the scaffold may comprise a three-layer composite material, wherein the three-layer composite material may comprise two outer layers and one inner layer. In some embodiments, the two outer layers may comprise surface layer characteristics, and the inner layer may comprise bulk properties. In some embodiments, the multi-layer may comprise multi-layer thin materials. In some embodiments, the multi-layers may be fused together, held together by entanglement, laminated together, stitched together, adhered together, woven together, printed together, or any combination thereof.

[0092] In some embodiments, the non-woven needle-punched material may comprise a first porosity. In some embodiments, the scaffold may comprise a non-woven configuration that can create a first porosity. In some embodiments, the first porosity may comprise an average pore size of about 30 - 70 μm. In some embodiments, the first porosity may comprise an average pore size of about 80 - 120 μm. In some embodiments, the first porosity may comprise an average pore size of about 50 μm or about 100 μm. In some embodiments, the scaffold may comprise a second porosity created by needle punching.

[0093] In some embodiments, the scaffold may comprise a terry cloth. In some embodiments, the terry cloth may comprise a one-piece textile including a high-porosity portion (fiber loops) and a stable backbone (woven mesh base), which constitute two main structural requirements of the scaffold: a high-porosity environment for tissue growth and mechanical support for the mechanics of end-product use.

[0094] In some embodiments, the scaffold may comprise a spacer fabric. In some embodiments, the spacer fabric may comprise a stable backbone and a high-porosity environment. In some embodiments, the center may be high-porosity and high-volume to enable growth of large tissues. In some embodiments, the two separated surfaces may be different from each other, such that further specified functions may be available. In some embodiments, the upper surface may be highly porous and dissolvable to have a smooth fiber-free upper surface, while the lower surface may be made of a stable material to provide mechanical support.

[0095] In some embodiments, the cell layer is not formed on the scaffold. In some embodiments, the dermis layer may not be formed on the scaffold (e.g., collagen matrix). In some embodiments, the cruelty-free leather does not contain a scaffold. In some embodiments, cells may be formed on the scaffold and then substantially removed from the scaffold before tanning. In some embodiments, the scaffold may include a dissolvable scaffold, where the scaffold may be contacted with a solvent of the dissolvable scaffold.

[0096] In some embodiments, the solvent provided herein can be any suitable solvent to dissolve or otherwise contain a degrading agent required to degrade or otherwise dissolve the scaffold. In some embodiments, the solvent includes water. In some embodiments, the solvent includes an organic solvent. In some embodiments, the solvent includes acetone, benzylamine, ethyl acetate, or a combination thereof. In some embodiments, the solvent includes acetone. In some embodiments, the solvent includes ethyl acetate. In other embodiments, the solvent contains benzylamine. In some embodiments, the solvent includes benzene, acetonitrile, ethanol, ether, dichloromethane, or tetrahydrofuran. In some embodiments, the temperature of the solvent is below the boiling point of the solvent. In some embodiments, the solvent can be at a temperature of at least 30 °C (e.g., at least 40 °C, at least 50 °C, at least 60 °C, at least 70 °C, at least 80 °C, at least 90 °C, at least 100 °C, or at least 110 °C). In some embodiments, the solvent can be at a temperature of at most 200 °C (e.g., at most 180 °C, at most 160 °C, at most 140 °C, at most 120 °C, at most 100 °C). In some embodiments, the solvent can be at a temperature of about 40 °C - about 50 °C. In some embodiments, the solvent can be at a temperature of 25 °C - about 125 °C. In some embodiments, the solvent can be at a temperature of about 25 °C - about 100 °C.

[0097] In some embodiments, the scaffold may comprise a thermoplastic polymer. In some embodiments, the thermoplastic polymer comprises polyvinyl acetate (PVA), polyvinyl alcohol (PVOH), or polylactic acid (PLA). In some embodiments, the thermoplastic polymer comprises polyvinyl acetate (PVA). In some embodiments, the thermoplastic polymer comprises polyvinyl alcohol (PVOH). In some embodiments, the thermoplastic polymer comprises polylactic acid (PLA). In some embodiments, the scaffold comprises polyhydroxyalkanoates (PHA), polyhydroxybutyrate (PHB), polycaprolactone (PCL), or oxidized alginate. In some embodiments, the thermoplastic polymer comprises polyhydroxyalkanoates (PHA). In some embodiments, the thermoplastic polymer comprises polyhydroxybutyrate (PHB). In some embodiments, the thermoplastic polymer comprises polycaprolactone (PCL). In some embodiments, the thermoplastic polymer comprises oxidized alginate.

[0098] In some embodiments, the thermoplastic polymer comprises polyvinyl alcohol (PVA). In some embodiments, the solvent comprises water. In some embodiments, the thermoplastic polymer may comprise polyvinyl alcohol (PVA), and wherein the solvent may comprise water.

[0099] In some embodiments, the scaffold comprises polylactic acid (PLA). In some embodiments, the solvent comprises benzyl, ethyl, haloalkane, or combinations thereof. In some embodiments, the scaffold may comprise polylactic acid (PLA), and wherein the solvent may comprise benzyl, ethyl, haloalkane, or combinations thereof. In some embodiments, the solvent may comprise benzylamine.

[0100] In some embodiments, the scaffold may comprise a bio-based material, such as the bio-based materials described elsewhere herein. In some embodiments, the solvents provided herein comprise cellulase (e.g., an enzyme that degrades bio-based materials). In some embodiments, the scaffold may comprise a bio-based material, and the solvent may comprise an enzyme that degrades bio-based materials. In some embodiments, the scaffold may comprise cellulose and the solvent may comprise cellulase.

[0101] In some embodiments, removing the scaffold material from the cell and scaffold composition may include using a dissolution method. In some embodiments, dissolution may include contacting the scaffold with a solvent. In some embodiments, the solvent may include benzylamine, hot water, an enzyme, or any combination thereof. In other embodiments, the solvent comprises an organic solvent. In some embodiments, the organic solvent includes acetone, benzylamine, or ethyl acetate. In some embodiments, the solvent may be applied to the scaffold at certain stages of growth or post-treatment to remove the scaffold. In some embodiments, the temperature of the solvent varies during dissolution. In some embodiments, the temperature of the solvent is below the boiling point of the solvent. In some embodiments, the temperature of the solvent does not exceed 150 °C (e.g., does not exceed 140 °C, does not exceed 130 °C, does not exceed 120 °C, does not exceed 110 °C, does not exceed 100 °C). In some embodiments, the temperature of the solvent is at least 30 °C (e.g., at least 40 °C, at least 50 °C, at least 60 °C, at least 70 °C, at least 80 °C, at least 90 °C, at least 100 °C, or at least 110 °C). In some embodiments, the temperature of the solvent is about 25 °C - about 100 °C. In some embodiments, the temperature of the solvent is about 40 °C - about 50 °C.

[0102] In some embodiments, removing the scaffold material from the cell and scaffold composition may include using a degradation method. In some embodiments, the scaffold degrades over time by hydrolytic degradation. In some embodiments, the scaffold degrades over time by hydrolytic degradation in a neutral aqueous solution. In some embodiments, the scaffold comprises polyhydroxyalkanoate (PHA), polyhydroxybutyrate (PHB), polycaprolactone (PCL), polylactic acid (PLA), or oxidized alginate. In some embodiments, the scaffold comprises polyhydroxyalkanoate (PHA). In some embodiments, the scaffold comprises polyhydroxybutyrate (PHB). In some embodiments, the scaffold comprises polycaprolactone (PCL). In some embodiments, the scaffold comprises polylactic acid (PLA). In some embodiments, the scaffold comprises oxidized alginate.

[0103] In some embodiments, solubilization may include contacting the scaffold with a solubilizing agent. In some embodiments, the solvent may comprise a solubilizing agent. In some embodiments, the solubilizing agent may comprise an enzyme. In some embodiments, the solubilizing agent comprises cellulase. In some embodiments, the enzyme may include cellulase. In some embodiments, the enzyme may comprise cellulase to digest cellulose. In some embodiments, the solubilizing agent comprises lipase. In some embodiments, the solubilizing agent comprises alginate lyase. In some embodiments, the scaffold comprises calcium alginate and the solubilizing agent comprises alginate lyase. In other embodiments, the scaffold comprises an ester-containing polymer and the solubilizing agent comprises an esterase. In some embodiments, the scaffold comprises an ester-containing polymer and the solubilizing agent comprises lipase.

[0104] In some embodiments, the solubilizing agent comprises a chelating agent. In some embodiments, the solubilizing agent comprises ethylenediaminetetraacetic acid (EDTA). In some embodiments, the scaffold comprises alginate and the solubilizing agent comprises ethylenediaminetetraacetic acid (EDTA).

[0105] In some embodiments, the solvent includes a corrosive agent. In some embodiments, the solvent comprises a strong acid. In some embodiments, the strong acid includes hydrochloric acid, nitric acid, hydroiodic acid, perchloric acid, chloric acid, or a combination thereof. In some embodiments, the strong acid includes hydrochloric acid. In some embodiments, the strong acid includes nitric acid. In some embodiments, the strong acid includes perchloric acid. In some embodiments, the strong acid includes hydroiodic acid. In some embodiments, the strong acid includes chloric acid. In other embodiments, the solvent comprises a strong base. In some embodiments, the strong base includes lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, strontium hydroxide, barium hydroxide, or a combination thereof. In some embodiments, the strong base includes sodium hydroxide. In some embodiments, the strong base includes lithium hydroxide. In some embodiments, the strong base includes potassium hydroxide. In some embodiments, the strong base includes calcium hydroxide. In some embodiments, the strong base includes strontium hydroxide. In some embodiments, the strong base includes barium hydroxide. In some embodiments, the solvent may comprise a chemical, such as sodium hydroxide, to degrade PLA. In some embodiments, the solvent comprises an oxidizing agent, such as to partially or completely degrade the material. In some embodiments, the oxidizing agent is sodium periodate. In some embodiments, the reagent can directly break down the scaffold material into smaller pieces, which can then be more easily solvated and removed from the cell and scaffold composition. In some embodiments, the scaffold removal process can be carried out at any stage of the manufacturing process. In some embodiments, the scaffold removal process can be carried out after tissue growth has started, during tissue growth, after tissue growth has been completed but before the liming and tanning processes, during the liming and tanning processes, or after the liming and tanning processes to convert the tissue into leather. In some embodiments, a composition is disclosed herein that comprises a dissolvable scaffold in contact with an extracellular matrix comprising collagen, wherein the scaffold is in contact with a solvent for the dissolvable scaffold.

[0106] In some embodiments, the scaffold may further comprise polyglycolic acid (PGA), polybutylene succinate (PBS), a bioabsorbable synthetic polymer, cellulose, cellulose acetate, acrylic, fiber, linen, rayon, velvet, modified acrylic, olefin polyester, saran, vinylon, wool, jute, hemp, bamboo, flax, or any combination thereof. In some embodiments, the scaffold may further comprise bio-based nylon, bio-based PET, bio-based PEF, bio-based polylactic acid (PLA), or any combination thereof. In some embodiments, the scaffold may comprise nylon 1,6, nylon 4,6, nylon 510, nylon 5,6, nylon 5,12, nylon 6, nylon 6,6, nylon 11, nylon 10.10, nylon 12, or any combination thereof. In some embodiments, the scaffold may include bast fibers, wherein the bast fibers may include flax, hemp, linen, jute, ramie, kenaf, sisal, or any combination thereof.

[0107] In some embodiments, any one of the scaffolds provided herein has a thickness of about 0.1 mm to about 4 mm. In some embodiments, the scaffold may have a thickness of about 1 mm to about 3 mm. In some embodiments, the scaffold may have a thickness of about 1 mm. In some embodiments, the scaffold may have a thickness of about 2 mm. In some embodiments, the scaffold may have a thickness of about 3 mm.

[0108] In some embodiments, any one of the scaffolds provided herein may comprise fibers having a decitex of about 0.1 decitex to about 50 decitex. In some embodiments, the scaffold comprises fibers having a decitex of at least 0.1 decitex (e.g., at least 1 decitex, at least 2 decitex, at least 5 decitex, at least 10 decitex, at least 20 decitex, at least 30 decitex, at least 40 decitex). In some embodiments, the scaffold comprises fibers having a decitex of at most 50 decitex (e.g., at most 40 decitex, at most 30 decitex, at most 20 decitex, at most 15 decitex, at most 10 decitex). In some embodiments, the scaffold comprises fibers having a decitex of about 0.1 decitex to about 30 decitex. In some embodiments, the scaffold comprises fibers having a decitex of about 1 decitex to about 10 decitex. In some embodiments, the scaffold may comprise fibers having a decitex of about 6.7 decitex. In some embodiments, the scaffold comprises fibers having a decitex of about 1.7 decitex. In some embodiments, the scaffold comprises fibers having a decitex of about 3.3 decitex.

[0109] In some embodiments, any one of the scaffolds provided herein may comprise fibers having a diameter of about 1 μm to about 100 μm. In some embodiments, the scaffold comprises fibers having a diameter of at least 1 μm (e.g., at least 2 μm, at least 5 μm, at least 10 μm, at least 20 μm, at least 30 μm, at least 40 μm, at least 50 μm). In some embodiments, the scaffold comprises fibers having a diameter of at most 100 μm (e.g., at most 90 μm, at most 80 μm, at most 70 μm, at most 60 μm, at most 50 μm). In some embodiments, the scaffold comprises fibers having a diameter of about 1 μm to about 70 μm. In some embodiments, the scaffold comprises fibers having a diameter of about 10 μm to about 50 μm. In some embodiments, the scaffold comprises fibers having a diameter of about 10 μm to about 100 μm.

[0110] In some embodiments, compositions are disclosed herein that comprise a scaffold at least partially coated with a scaffold in contact with a collagen-containing extracellular matrix, wherein the scaffold is at least partially coated with a coating comprising: Matrigel, vitronectin, fibronectin, a protein extracted from soybeans, a protein extracted from peas, a protein extracted from corn, a synthetically produced peptide, an RNA-binding glycine-rich (RBG) protein, polylysine, a synthetic protein, an RGD peptide, polylysine, polyarginine, polyornithine, a recombinant protein, an oligomer, a polymer, glycidyltrimethylammonium chloride (GTMAC), a carbohydrate-binding module, a cellulose-binding domain, a starch-binding domain, or a combination thereof. In some embodiments, as described in Example 5, at least partially coating the scaffold with wheat protein or pea protein increased the percentage of cells on the scaffold compared to an untreated control ( Figure 5 ). In some embodiments, conjugation of pea (e.g., sweet pea) protein is more effective than wheat flour protein ( Figure 5 , Figure 6 A-F).

[0111] In some embodiments, the scaffold is at least partially coated with a carbohydrate-binding module. In some cases, the carbohydrate-binding module is a protein domain present in a carbohydrate-active enzyme such as a glycoside hydrolase. In some instances, the carbohydrate-binding module has carbohydrate-binding activity. In some embodiments, the carbohydrate-binding module is selected from CBM3, CBM4, CBM6, CBM9, CBM17, CBM20, CBM21, CBM25, CBM28, CBM32, and CBM49. In certain cases, the carbohydrate-binding module is a cellulose-binding domain. In some embodiments, the scaffold is at least partially coated with a cellulose-binding domain. In some embodiments, the scaffold comprises cellulose. In some embodiments, the carbohydrate-binding module is a starch-binding module. In some embodiments, the scaffold comprises starch. In some embodiments, the carbohydrate-binding module is associated with an enzyme. In some cases, the enzyme does not hydrolyze the scaffold.

[0112] In some embodiments, the scaffold can be modified to contain functional groups. In certain cases, the functional groups are natural to the scaffold. In other cases, the functional groups are the result of modification. In some embodiments, the scaffold comprises amine hydroxyl, thiol, tyrosyl, amine, sulfate, aldehyde, hydrazide, aziridine, aryl azide, acrylate, epoxide, or carboxylic acid groups. In some embodiments, the scaffold comprises amine hydroxyl. In some embodiments, the scaffold comprises thiol. In some embodiments, the scaffold comprises a tyrosyl group. In some embodiments, the scaffold comprises a carboxylic acid group. In some embodiments, the scaffold comprises an amine. In some embodiments, the scaffold comprises sulfate. In some embodiments, the scaffold comprises aldehyde. In some embodiments, the scaffold comprises hydrazide. In some embodiments, the scaffold comprises aziridine. In some embodiments, the scaffold comprises aryl azide. In some embodiments, the scaffold comprises acrylate. In some embodiments, the scaffold comprises epoxide. In some embodiments, the scaffold is at least partially coated with GTMAC. In some embodiments, GTMAC provides primary amines on the surface of the scaffold.

[0113] In some embodiments, the coating can comprise a modification. In some embodiments, the modification can include reduction modification, addition modification, or a combination thereof. In some embodiments, the modification can include reduction modification, addition modification, or a combination thereof. In some embodiments, reduction modification can include using a base to cleave a point of the molecule and generate a more reactive chemical to increase attachment or generate some surface roughness. In some embodiments, addition modification can include adding functional groups with glycidyltrimethylammonium chloride (GTMAC) to increase the positive charge of the surface and increase attachment or graft polymer brushes to modulate the charge, density, hardness, or a combination thereof of the scaffold surface. In some embodiments, the scaffold is at least partially coated with a coating comprising GTMAC. In some embodiments, the coating comprises primary amines, such as provided by at least a partial coating of GTMAC. In some embodiments, at least partially coating with a coating comprising GTMAC increases the surface charge of the scaffold.

[0114] In some embodiments, the modification comprises hydrolysis. In some embodiments, the hydrolysis is to expose chemically reactive groups such as hydroxyl, carboxylic acid, ketone, etc. In some embodiments, the modification exposes hydroxyl. In some embodiments, the modification exposes carboxylic acid groups. In some embodiments, the modification exposes ketone groups. In some embodiments, the hydrolysis is completed under high pressure. In some embodiments, the hydrolysis is completed under autoclaving conditions. In some embodiments, the hydrolysis is completed by an enzymatic reaction. In some embodiments, the hydrolysis is completed under acidic conditions. In some embodiments, the hydrolysis is completed under basic conditions.

[0115] In some embodiments, the modification includes oxidation. In some embodiments, the modification includes oxidation with sodium periodate or any other suitable oxidizing agent.

[0116] In some embodiments, the modification includes a Maillard reaction, such as that described in Example 5. In some cases, the scaffold is immersed in a protein-rich solution under autoclaving conditions to treat the surface. In certain cases, compared to an unmodified scaffold ( Figure 20 B), the Maillard reaction helps the pea protein to bind to lyocell ( Figure 20 C), and cell spreading is improved. In certain cases, the scaffolds herein are oxidized with sodium periodate, as described in Example 5. In certain cases, a combined modification method is used, such as a combination of the Maillard reaction and oxidation, which can result in cell spreading ( Figure 20 F).

[0117] In some embodiments, in any composition provided herein, the collagen associates with the scaffold. In some embodiments, the association includes bonding. In some embodiments, the bonding includes covalent or non-covalent bonds. In some embodiments, in any composition provided herein, the collagen binds to the scaffold. In some embodiments, the association of the collagen with the scaffold includes crosslinking of the collagen and the scaffold.

[0118] In some embodiments, the collagen associates with the scaffold by non-specific adsorption, such as bonding. In some cases, non-specific adsorption includes van der Waals interactions, hydrogen bonds, depletion interactions, electrostatic interactions, or a combination thereof. In some embodiments, the collagen associates with the scaffold by van der Waals interactions. In some embodiments, the collagen associates with the scaffold by hydrogen bonds. In some embodiments, the collagen associates with the scaffold by depletion interactions. In some embodiments, the collagen associates with the scaffold by electrostatic interactions. In some cases, coating the surface at least partially with a coating described in other parts of this document can increase the strength of non-specific adsorption. In other embodiments, modification of the scaffold, such as the modification described in other parts of this document, increases the strength of non-specific adsorption of collagen to the scaffold.

[0119] In some embodiments, the collagen associates with the scaffold by covalent interactions. In some embodiments, the collagen associates with the scaffold by covalent bonds. In some cases, modification, such as the modification described in other parts of this document, can enable the collagen to interact covalently with the scaffold, such as covalent bonding.

[0120] In some embodiments, the scaffold comprises a carbodiimide. In some embodiments, the scaffold comprises an N-hydroxysuccinimide ester (NHS ester). In some embodiments, the collagen comprises a carbodiimide. In some embodiments, the collagen comprises an N-hydroxysuccinimide ester (NHS ester). In some embodiments, the carbodiimide is N,N′-dicyclohexylcarbodiimide (DCC). In some embodiments, the carbodiimide is 1-ethyl-3-(3-(dimethylaminopropyl)carbodiimide (EDC). In some embodiments, the collagen associates with the scaffold by EDC / NHS coupling (such as the coupling of an NHS ester and a carbodiimide), for example, forming a covalent bond with the scaffold. In some embodiments, fibroblasts and / or fibroblast-like cells comprise a carbodiimide. In some embodiments, fibroblasts and / or fibroblast-like cells comprise an N-hydroxysuccinimide ester (NHS ester). In some embodiments, fibroblasts and / or fibroblast-like cells associate with the scaffold by EDC / NHS coupling, such as forming a covalent bond with the scaffold.

[0121] In some embodiments, the scaffold comprises a click chemistry module. In some embodiments, the scaffold comprises an azide. In some embodiments, the scaffold comprises an alkyne. In some embodiments, the collagen comprises a click chemistry module. In some embodiments, the collagen comprises an azide. In some embodiments, the scaffold comprises an alkyne. In some embodiments, the collagen associates with the scaffold by a click chemical reaction (such as the reaction of an azide and an alkyne), such as forming a covalent bond with the scaffold. In some embodiments, fibroblasts and / or fibroblast-like cells comprise a click chemistry module. In some embodiments, fibroblasts and / or fibroblast-like cells comprise an azide. In some embodiments, fibroblasts and / or fibroblast-like cells comprise an alkyne. In some embodiments, fibroblasts and / or fibroblast-like cells associate with the scaffold by a click chemical reaction (such as the reaction of an azide and an alkyne), such as forming a covalent bond with the scaffold.

[0122] In some embodiments, the scaffold comprises a Michael donor. In some embodiments, the scaffold comprises a Michael acceptor. In some embodiments, the collagen comprises a Michael donor. In some embodiments, the scaffold comprises a Michael acceptor. In some embodiments, the collagen associates with the scaffold by coupling of a Michael donor and a Michael acceptor. In some embodiments, fibroblasts and / or fibroblast-like cells comprise a Michael donor. In some embodiments, fibroblasts and / or fibroblast-like cells comprise a Michael acceptor. In some embodiments, fibroblasts and / or fibroblast-like cells associate with the scaffold by coupling of a Michael donor and a Michael acceptor. In some embodiments, the Michael donor comprises an enolate. In some embodiments, the Michael acceptor comprises an α,β-unsaturated carbonyl.

[0123] In some embodiments, the scaffold comprises a thiol. In some embodiments, the scaffold comprises a maleimide. In some embodiments, the collagen comprises a thiol. In some embodiments, the collagen comprises a maleimide. In some embodiments, the collagen associates with the scaffold by coupling of a thiol and a maleimide, such as forming a covalent bond with the scaffold. In certain cases, the collagen associates with the scaffold in the presence of (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (sulfo-SMCC), where both the scaffold and the collagen comprise thiols. In some embodiments, fibroblasts and / or fibroblast-like cells comprise a thiol. In some embodiments, fibroblasts and / or fibroblast-like cells comprise a maleimide. In some embodiments, fibroblasts and / or fibroblast-like cells associate with the scaffold by coupling of a thiol and a maleimide, such as forming a covalent bond with the scaffold. In certain cases, fibroblasts and / or fibroblast-like cells associate with the scaffold in the presence of (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (sulfo-SMCC), where the scaffold and the fibroblasts and / or fibroblast-like cells both comprise thiols.

[0124] In some embodiments, the collagen associates with the scaffold by the Maillard reaction (such as a reaction between a reducing sugar and a protein), such as forming a covalent bond with the scaffold. In some embodiments, fibroblasts and / or fibroblast-like cells associate with the scaffold by the Maillard reaction (such as a reaction between a reducing sugar and a protein), such as forming a covalent bond with the scaffold.

[0125] In some embodiments, the scaffold may comprise a thermoplastic polymer, which may subsequently be substantially removed from the extracellular matrix prior to tanning. In some embodiments, the thermoplastic polymer may comprise polyvinyl alcohol (PVA), and wherein the method may include substantially removing the PVA by contacting the PVA with water at a temperature of about 18 °C to about 90 °C prior to tanning. In some embodiments, the thermoplastic polymer may comprise polylactic acid (PLA), and wherein the method may include substantially removing the PLA by contacting the PLA with a solvent to remove the PLA. In some embodiments, the solvent may comprise benzyl, ethyl, haloalkane, benzylamine, or any combination thereof. In some embodiments, the scaffold may include a dissolvable scaffold, which may be substantially removed by contacting the scaffold with a solvent. In some embodiments, the method of fabricating the scaffold may include needling the scaffold using a loom to wind fibers into a nonwoven scaffold material. In some embodiments, the loom may include barbed knitting needles. In some embodiments, needling may create pores in the scaffold material. In some embodiments, the method of preparing the scaffold may include at least partially coating the scaffold with a coating comprising: matrix glue, vitronectin, fibronectin, proteins extracted from soybeans, proteins extracted from peas, proteins extracted from corn, synthetically produced peptides, RNA-binding glycine-rich (RBG) proteins, polylysine, synthetic proteins, RGD peptides, polylysine, polyarginine, polyornithine, recombinant proteins, oligomers, polymers, or any combination thereof.

[0126] In some embodiments, compositions are disclosed herein that comprise a scaffold in contact with an extracellular matrix. In some embodiments, the extracellular matrix may comprise collagen. In some embodiments, the scaffold and cell composition may have at least one component of natural skin, such as melanocytes, hair follicles, sweat glands, and nerve endings. In some cases, cruelty-free leather may be distinguished from normal natural skin by the absence of at least one of these components. In some embodiments, cells, scaffolds, and cell composition leather that exhibit an abnormal phenotype or have at least one genotype alteration may include all of these components.

[0127] In some embodiments, compositions are disclosed herein that comprise isolated animal cells in contact with a scaffold. In some embodiments, the isolated animal cells can be isolated animal fibroblasts or fibroblast-like cells. In some embodiments, the extracellular matrix can be produced by the isolated animal fibroblasts or fibroblast-like cells. In some embodiments, the isolated animal cells can be immortalized isolated animal cells. In some embodiments, the immortalized isolated animal cells can grow beyond the Hayflick limit. In some embodiments, the immortalized isolated animal cells can grow through about 40 cell divisions, about 50 cell divisions, or about 60 cell divisions. In some embodiments, the isolated animal cells can be bovine or porcine cells. In some embodiments, the isolated animal cells can include human cells. In some embodiments, the composition can be at least partially decellularized. In some embodiments, at least partial decellularization can comprise a substantially absence of intact cells in the composition.

[0128] In some embodiments, other components can be added to the scaffold and cell composition. These other components can include myoepithelial cells, duct cells, secretory cells, alveolar cells, Langerhans cells, Merkel cells, adhesions, mammary glands, or any mixture thereof. In some embodiments, the cruelty-free leather can comprise one or more of the following: nerve cells, connective tissue (including bone, cartilage, cells that differentiate into osteoblasts and chondrocytes, and lymphoid tissue), epithelial cells (including endothelial cells that line cavities and blood vessels or channels, exocrine secretory epithelial cells, epithelial absorptive cells, keratinized epithelial cells, and extracellular matrix-secreting cells), and undifferentiated cells (such as embryonic cells, stem cells, and other precursor cells).

[0129] In some embodiments, the scaffold and cell composition can comprise hair follicles. The hair follicles can comprise one or more structures, including papillae, matrix, root sheath, bulge, infundibulum, arrector pili muscle, sebaceous gland, apocrine sweat gland, or any combination thereof. The hair follicles can comprise one or more hair follicle cells, including dermal papilla cells, outer root sheath cells, or any combination thereof. In some embodiments, the hair follicles can be in the epidermal layer. In some embodiments, the hair follicles can be in the dermal layer. In some embodiments, the hair follicle cells can differentiate from progenitor cells, such as stem cells. In some embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% of the hair follicle cells can differentiate from induced pluripotent stem cells.

[0130] In some embodiments, the scaffold and cell composition can be free of hair, blood vessels, sebaceous glands, hair follicles, oil glands, nerves, or any combination thereof, before or after tanning.

[0131] Cell type

[0132] In some embodiments, one or more cells in at least a portion of the cruelty-free leather can differentiate from progenitor cells, such as stem cells. In some embodiments, the cruelty-free leather can be produced from the engineered cells disclosed herein or tissues containing the engineered cells. In some embodiments, fibroblasts in the cruelty-free leather can differentiate from stem cells. In some embodiments, keratinocytes in the cruelty-free leather can differentiate from stem cells. In some embodiments, melanocytes in the cruelty-free leather can differentiate from stem cells.

[0133] In some embodiments, the stem cells can include embryonic stem cells (ESCs), adult stem cells, somatic stem cells, tissue-specific stem cells, mesenchymal stem cells, induced pluripotent stem cells (iPSCs), or any combination thereof. In some embodiments, the stem cells can be totipotent, pluripotent, or multipotent. In some embodiments, the stem cells can include adult stem cells, umbilical cord blood stem cells, or a combination thereof. Embryonic stem cells can be derived from a fertilized embryo that may be less than 1 week old. Induced pluripotent stem cells can be obtained by inducing the expression of one or more selected from the following in any somatic cell: Oct3, Oct4, Sox2, Klf4, TERT, Bmi1, CcnD1, Cdk4, SV40 large T antigen, c-Myc, or a fragment of any of these genes. In some embodiments, the somatic cells can include adult somatic cells. In some embodiments, the somatic cells can include fibroblasts. In some embodiments, the exogenous vector can contain or encode genes that induce pluripotency. In some embodiments, the exogenous vector can contain a plasmid. In some embodiments, induced pluripotent stem cells can be obtained by an active protein product or a bioactive fragment thereof. In some embodiments, one or more other genes can also be induced to reprogram somatic cells into induced pluripotent stem cells. In some embodiments, the genes used to induce pluripotency can include NANOG, UTF1, LIN28, SALL4, NR5A2, TBX3, ESSRB, DPPA4, SV40LT, REM2, MDM2, and cyclin D1. In some embodiments, the genes can be from humans. In some embodiments, the genes can be from mammals, birds, reptiles, amphibians, fish, invertebrates, or any combination thereof.

[0134] In some embodiments, various delivery methods can be used to regulate gene expression to reprogram somatic cells into iPSCs. In some embodiments, exemplary delivery methods can include naked DNA delivery, adenoviral vectors, electroporation, chemical delivery, mechanical delivery, polymer-based systems, microinjection, retroviral vectors (e.g., MMLV-derived retroviruses), lentiviral vectors (e.g., excisable lentiviruses), or any combination thereof. In some embodiments, somatic cells can include adult somatic cells. In some embodiments, somatic cells can be transfected with a vector for delivery of genes that induce pluripotency. In some embodiments, the vector can include a viral vector. In some embodiments, the vector can include a retroviral vector. In some embodiments, the genes that induce pluripotency can include Oct3, Oct4, Sox2, Klf4, TERT, Bmi1, CcnD1, Cdk4, SV40 large T antigen, c-Myc, fragments of any of these, or any combination thereof. In some embodiments, Sendai virus can be used as a delivery system. In some embodiments, somatic cells can include adult somatic cells. In some embodiments, somatic cells can be transfected with an episomal vector. In some embodiments, the episomal vector can include a plasmid. In some embodiments, the episomal vector can deliver Oct3, Oct4, Sox2, Klf4, TERT, Bmi1, CcnD1, Cdk4, SV40 large T antigen, c-Myc, fragments of any of these, or any combination thereof.

[0135] In some embodiments, compositions comprising isolated cells are disclosed herein, and the isolated cells can be used to produce cruelty-free leather, cruelty-free leather, isolated epidermal layers, isolated dermal layers, stratified structures, products produced therefrom, methods of producing the same, or any combination thereof. In some embodiments, methods and compositions comprising isolated cells are disclosed herein. In some embodiments, the isolated cells can include cells derived from animals. In some embodiments, the cells can be derived from mammals, birds, reptiles, amphibians, fish, invertebrates, or any combination thereof. In some embodiments, the cells can be derived from antelopes, bears, beavers, bison, boars, camels, caribou, cats, cows, deer, dogs, elephants, elk, foxes, giraffes, goats, hares, humans, horses, ibex, kangaroos, lions, llamas, lynxes, minks, moose, oxen, peccaries, pigs, rabbits, rhinos, seals, sheep, lambs, squirrels, tigers, whales, wolves, yaks, or zebras. In some embodiments, the animals can be primates, cows, sheep, pigs, horses, dogs, cats, rodents, rabbits, fish, birds, or reptiles. In some embodiments, the cells can be derived from birds. In some embodiments, the birds can include chickens, ducks, emus, geese, grouse, ostriches, pheasants, pigeons, quails, or turkeys. In some embodiments, the cells can be derived from reptiles, such as turtles, snakes, lizards, amphibians, crocodiles, or alligators. In some embodiments, the cells can be derived from amphibians. In some embodiments, the amphibians can include frogs, toads, fire salamanders, or newts. In some embodiments, the cells can be derived from fish. In some embodiments, the fish can include anchovies, bass, catfish, carp, cod, eels, flounders, puffers, groupers, haddocks, halibuts, herrings, mackerels, dolphinfishes, manta rays, marlins, orange roughies, perches, pickerels, pollocks, salmons, sardines, sharks, snappers, sole, stingrays, sailfishes, tilapias, trouts, tunas, or pollacks. In some embodiments, the isolated cells can be obtained from a biopsy. In some embodiments, the isolated cells can include isolated animal cells. In some embodiments, the isolated animal cells can include primate cells, bovine cells, ovine cells, porcine cells, equine cells, canine cells, feline cells, rodent cells, avian cells, marsupials, reptile cells, or rabbit cells. In some embodiments, the isolated animal cells can be genetically engineered cells. The cells can include a cell line having multiple cells. In some embodiments, the isolated animal cells can be immortalized cells. In some embodiments, the cells can include human cells, fibroblasts, stem cells, or any combination thereof.In some embodiments, the cells can be adipose tissue-derived cells (e.g., adipocytes), chondrocytes, osteocytes, osteoblasts, myofibroblasts, satellite cells, myoblasts, muscle cells, keratinocytes, keratinocytes, melanocytes, Langerhans cells, basal cells, smooth muscle cells, umbilical cord cells, pluripotent stem cells, mesenchymal stem cells, embryonic stem cells, or any combination thereof. In some embodiments, the cells can produce extracellular matrix (ECM). In some embodiments, the ECM proteins can include collagen, type I collagen, type III collagen, elastin, fibronectin, laminin, or any combination thereof. In some embodiments, the engineered tissue can comprise the cells described herein. In some embodiments, the isolated animal cells can comprise engineered isolated animal cells. In some embodiments, the engineered isolated animal cells can comprise genetically engineered cells. In some embodiments, the genetically engineered cells can comprise exogenous polynucleotides. In some embodiments, the genetically engineered cells can comprise genes for collagen production. In some embodiments, the collagen genes can be P4HA, P4HB, COL1A1, COL1A2, COL2A1, COL3A1, or any combination thereof. In some embodiments, the collagen genes can have an altered promoter that can alter the expression of the collagen genes, e.g., increase or decrease. In some embodiments, the collagen genes can be from humans. In some embodiments, the collagen genes can be from animals, mammals, birds, reptiles, amphibians, fish, invertebrates, or any combination thereof. In some embodiments, one or more exogenous genes can cause immortalization. In some embodiments, the exogenous genes can comprise mutants of hTERT, TERT, Bmi1, CcnD1, Cdk4, Cdk4, TAg (SV40 large T), SV40, c-myc, H-ras, Ela, c-mMycER. TAM, E6, E7, HER-2, SRC, EGFR, Abl, Atk02, Aml1, Axl, Bcl, Dbl, EGFR, ERBB, Ets-1, Fms, Fos, Fps, Gli, Gsp, Her2, Hox11, Hst, Il-3, Int-2, Jun, Kit, KS3, K-SAM, Lbc, Lck, L-myc, Lyl-1, Lyt-10, Mas, MDM-2, Mll, Mos, Myb, Neu, N-Myc, Ost, Pax-5, Pim-1, PRAD-1, Ras-K, Ras-N, Ret, Ros, Ski, Sis, Set, Src, Tal1, Tan1, Tiam1, Tsc2, Trk, or any combination thereof. In some embodiments, the isolated animal cell may comprise an immortalized cell, a tissue developed therefrom, or any combination thereof. In some embodiments, the exogenous polynucleotide may encode: (i) a polypeptide that interacts with a tumor suppressor protein or a fragment thereof and can alter the activity of the tumor suppressor protein or a fragment thereof, (ii) a polynucleotide that can encode a polypeptide that interacts with a tumor suppressor protein or a fragment thereof, or (iii) a combination of (i) and (ii). In some embodiments, the activity of the tumor suppressor protein or a fragment thereof can be determined by an in vitro assay. In some embodiments, the immortalized cell may have random mutations or multiple mutations. In some embodiments, the mutations can be generated by UV mutagenesis, chemical mutagenesis, or any combination thereof. In certain cases, the immortalized cell may have a targeted mutation, for example, a targeted mutation can be generated by the CRISPR system. In some embodiments, the mutations can be in cell cycle genes, oncogenes, metabolic genes, or any combination thereof. In some embodiments, the immortalized cell may have mutations in genes, promoter regions, intragenic regions, intergenic regions, or any combination thereof. In some embodiments, the genes may include oncogenes, cell cycle genes, or a combination thereof. In some embodiments, the immortalized cell may have increased or decreased expression of oncogenes or genes involved in the regulation of cell proliferation.In some embodiments, the immortalized cells can grow through about 30 cell divisions, about 40 cell divisions, about 50 cell divisions, about 60 cell divisions, about 70 cell divisions, about 80 cell divisions, about 90 cell divisions, about 100 cell divisions, about 150 cell divisions, about 200 cell divisions, about 250 cell divisions, about 300 cell divisions, about 350 cell divisions, about 400 cell divisions, about 450 cell divisions, about 500 cell divisions, about 550 cell divisions, about 600 cell divisions, about 650 cell divisions, about 700 cell divisions, about 750 cell divisions, about 800 cell divisions, about 850 cell divisions, about 900 cell divisions, about 950 cell divisions, about 1,000 cell divisions, about 5,000 cell divisions, about 10,000 cell divisions, about 50,000 cell divisions, or about 100,000 cell divisions. In some embodiments, the Hayflick limit or Hayflick number can include the limited number of cell doublings that primary cells can grow to. In some embodiments, the immortalized cells can grow beyond the Hayflick limit. In some embodiments, the immortalized cell line can grow to produce at least 1 million square feet of cruelty-free leather per year.

[0136] In some embodiments, in any of the compositions provided herein, the scaffold can be seeded with any suitable collagen-producing cells. In some embodiments, the scaffold can be seeded with primary cells. In some embodiments, the scaffolds described herein can be seeded with collagen-producing cells, including fibroblasts, keratinocytes, melanocytes, epithelial cells, keratin cells, Langerhans cells, basal cells, or combinations thereof. In some embodiments, the scaffolds provided herein can be seeded with fibroblasts. In some embodiments, the scaffolds provided herein can be seeded with keratinocytes. In some embodiments, the scaffolds provided herein can be seeded with melanocytes. In some embodiments, the scaffolds provided herein can be seeded with epithelial cells. In some embodiments, the scaffolds provided herein can be seeded with keratin cells. In some embodiments, the scaffolds provided herein can be seeded with Langerhans cells. In some embodiments, the scaffolds provided herein can be seeded with basal cells.

[0137] Method for manufacturing cruelty-free leather

[0138] In some embodiments, methods of making the compositions disclosed herein are disclosed, which include inoculating isolated animal fibroblasts or fibroblast-like cells onto a scaffold to form a composition. In some embodiments, a cell layer can be formed by preparing a plurality of multicellular bodies comprising one or more types of isolated cells and arranging such multicellular bodies to form a cell layer. In some embodiments, the cell layer can be formed by arranging a plurality of multicellular bodies adjacent to one another, wherein the plurality of multicellular bodies can fuse to form a planar layer. In some embodiments, the cells can grow three-dimensionally. In some embodiments, the cells can grow in suspension. In some embodiments, forming the cell layer can include using a scaffold. In some embodiments, the cell layer can be formed by arranging a plurality of isolated cells, multicellular bodies, or a combination thereof on a scaffold. In some embodiments, the forming step can include arranging or placing multicellular bodies or inoculating a plurality of isolated animal cells on a support substrate that allows the multicellular bodies, the plurality of isolated animal cells, or a combination thereof to fuse to form a layer (e.g., a substantially planar layer). In some embodiments, the multicellular bodies or layers can be arranged adjacent to one another horizontally and / or vertically. In some embodiments, the cell layer can be formed without a scaffold. In some embodiments, the cell layer can be formed on a scaffold and the scaffold can subsequently be at least partially removed. In some embodiments, the scaffold can include a support substrate. In some embodiments, the support substrate can be permeable to fluids, gases, and nutrients and allow cell culture medium to contact all surfaces of the multicellular bodies and / or the layer during arrangement and subsequent fusion. In some embodiments, the support substrate can be made of natural biomaterials such as collagen, fibronectin, laminin, and other extracellular matrices. In some embodiments, the support substrate can be made of synthetic biomaterials such as hydroxyapatite, alginate, agarose, polyglycolic acid, polylactic acid, and copolymers thereof. In some embodiments, the support substrate can be a solid, semi-solid, or a combination of solid and semi-solid support elements. In some embodiments, the support substrate can be planar to facilitate the production of a planar layer. In some embodiments, the support substrate can be elevated or raised above a non-permeable surface, such as a portion of a cell culture environment (e.g., a culture dish, a cell culture flask, etc.) or a bioreactor. In some embodiments, the permeable, elevated support substrate can help prevent premature cell death, can help enhance cell growth, and can promote multicellular body fusion to form a layer. In some embodiments, the cell layer can include a dermal layer. In some embodiments, cruelty-free leather can include a dermal layer or at least a partially decellularized portion. In some embodiments, the dermal layer can be an engineered dermal equivalent, such as an artificial dermal layer formed in vitro. In some embodiments, the dermal layer can include connective tissue cells. In some embodiments, the dermal layer can include fibroblasts or fibroblast-like cells.In some embodiments, fibroblasts or fibroblast-like cells in the dermis may express one or more markers, including but not limited to cluster of differentiation 10 (CD10), cluster of differentiation 73 (CD73), cluster of differentiation 44 (CD44), cluster of differentiation 90 (CD90), cluster of differentiation 105 (CD105), type I collagen, type III collagen, prolyl-4-hydroxylase β fibroblast, or a combination thereof. In some embodiments, the dermis may contain other types of cells, such as immune cells, macrophages, adipocytes, or a combination thereof. In some embodiments, the dermis may contain engineered cells. In some embodiments, the cell layer may contain immortalized cells, bovine cells, fibroblasts, or any combination thereof. In some embodiments, the cell layer may contain immortalized bovine fibroblasts. In some embodiments, the dermis may contain matrix components other than cells. In some embodiments, the matrix components may include collagen, elastin, fibrous extracellular matrix, extracellular gel-like substances mainly composed of glycosaminoglycans, proteoglycans, glycoproteins, or any combination thereof. In some embodiments, the extracellular gel-like substance mainly composed of glycosaminoglycans may contain hyaluronic acid. In some embodiments, the dermis may contain a matrix support. In some embodiments, the matrix support may be a scaffold. In some embodiments, the matrix support may contain a contracted collagen gel. In some embodiments, the pure collagen matrix may be a polyglycolic acid mesh or a collagen and glycosaminoglycan matrix covered with a silicone rubber membrane (C-GAG), a biopolymer, or any combination thereof. In some embodiments, the biopolymer may contain chitosan. In some embodiments, the matrix may be inoculated with fibroblasts. In some embodiments, inoculation with fibroblasts may produce an organotypic model. In some embodiments, the cell layer may include dermis of natural origin, keratinocytes, or a combination thereof. In some embodiments, the dermis of natural origin may be obtained from allogeneic cadaver skin. In some embodiments, keratinocytes may form a keratinocyte sheet. In some embodiments, the cell layer may contain freeze-dried inactivated dermis from cadaver skin to support the keratinocyte sheet.

[0139] In some embodiments, the methods provided herein may further include cell growth under shaking conditions. In some embodiments, as described in Example 1, shaking conditions result in the consumption of more glucose. In some embodiments, the methods provided herein may further include cell growth under static conditions.

[0140] In some embodiments, the methods provided herein further include inoculating cells at any suitable seeding density determined by those skilled in the art. In some embodiments, at least 30k cells / cm 2 (e.g., at least 50k cells / cm 2 , at least 62.5k cells / cm 2, at least 100k cells / cm 2 , at least 125k cells / cm 2 , at least 200k cells / cm 2 , at least 400k cells / cm 2 , at least 600k cells / cm 2 , at least 800k cells / cm 2 , at least 1M cells / cm 2 ) are seeded. In some embodiments, the cells are seeded at a density of up to 2M cells / cm 2 (e.g., up to 1.5M cells / cm 2 , up to 1.25M cells / cm 2 , 1M cells / cm 2 , up to 800k cells / cm 2 , up to 600k cells / cm 2 , up to 400k cells / cm 2 , up to 200k cells / cm 2 , up to 100k cells / cm 2 ) are seeded. In some embodiments, the cells are seeded at a density of no more than 200k cells / cm 2 . In some embodiments, the cells are seeded at a density of about 30k cells / cm 2 - about 2M cells / cm 2 . In some embodiments, the cells are seeded at a density of about 62.5k cells / cm 2 - about 1M cells / cm 2 . In some embodiments, the cells are seeded at a density of about 62.5k cells / cm 2 - about 200k cells / cm 2 . In some cases, a lower seeding density (e.g., 30k cells / cm 2 ) will produce more collagen than a high seeding density( Figure 24 ).

[0141] In some embodiments, the methods provided herein further include incubating for any suitable time. In some embodiments, the method includes incubating for at least 1 week (e.g., at least 1.5 weeks, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 8 weeks). In some embodiments, the method includes incubating for at most 12 weeks (e.g., at most 10 weeks, at most 8 weeks, at most 6 weeks, at most 4 weeks, at most 2 weeks, at most 1 week). In some embodiments, the method includes incubating for about 1 week - about 12 weeks. In some embodiments, the method includes incubating for about 4 weeks - about 8 weeks. In some embodiments, the method includes incubating for about 4 weeks. In some embodiments, the method includes incubating for about 6.5 weeks. In some embodiments, the method includes incubating for about 8 weeks.

[0142] In some embodiments, the methods provided herein further include soaking the scaffold in a protein-containing solution (e.g., serum) before culturing. In some embodiments, the soaking includes soaking the scaffold in fetal bovine serum. In some embodiments, soaking may be important for cell attachment and proliferation. In some cases, soaking causes sticky proteins to adsorb to the surface, thereby increasing cell attachment.

[0143] In some embodiments, the thickness of the dermis layer can be designed to suit the function or use of the cruelty-free leather. In some embodiments, the dermis layer can have a thickness of about 0.01 mm - about 50 mm. In some embodiments, the dermis layer can have a thickness of about 0.01 mm - about 10 mm, about 0.01 mm - about 8 mm, about 0.01 - about 5 mm, about 0.02 - about 5 mm, about 0.05 - about 5 mm, about 0.1 - about 5 mm, about 0.1 - about 2 mm, about 0.1 - about 1 mm, about 0.1 - about 0.8 mm, or about 0.1 - about 0.5 mm. In some embodiments, the dermis layer can have a thickness of about 0.02 mm - 5 mm. For example, the dermis layer can have a thickness of about 0.1 mm - 0.5 mm. In some embodiments, the dermis layer can have a thickness of about 0.2 mm - 0.5 mm. In some embodiments, the thickness of the dermis layer can be at least 0.001 mm, 0.01 mm, 0.02 mm, 0.04 mm, 0.08 mm, 0.1 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1 mm, 2 mm, 4 mm, 8 mm or 10 mm. In some embodiments, the thickness of the dermis layer can be at most 50 mm, 40 mm, 20 mm, 10 mm, 8 mm, 4 mm, 2 mm, 1 mm, 0.8 mm, 0.4 mm, 0.2 mm, 0.1 mm, 0.08 mm, 0.04 mm, 0.02 mm or 0.01 mm. In some embodiments, the dermis layer can have a thickness of at least about 50 mm.

[0144] In some embodiments, the length of the dermis layer can be designed to suit the function or use of the cruelty-free leather. In some embodiments, the dermis layer can have a length of from about 0.01 mm to about 50 m. In some embodiments, the dermis layer can have a length of from about 0.01 mm to about 10 mm, from about 0.01 mm to about 8 mm, from about 0.01 to about 5 mm, from about 0.02 to about 5 mm, from about 0.05 to about 5 mm, from about 0.1 to about 5 mm, from about 0.1 to about 2 mm, from about 0.1 to about 1 mm, from about 0.1 to about 0.8 mm, or from about 0.1 to about 0.5 mm. In some embodiments, the dermis layer can have a length of from about 0.02 mm to 5 mm. For example, the dermis layer can have a length of from about 0.1 mm to 0.5 mm. In some embodiments, the dermis layer can have a length of from about 0.2 mm to 0.5 mm. In some embodiments, the length of the dermis layer can be at least 0.001 mm, 0.01 mm, 0.02 mm, 0.04 mm, 0.08 mm, 0.1 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1 mm, 2 mm, 4 mm, 8 mm, or 10 mm. In some embodiments, the length of the dermis layer can be at most 50 mm, 40 mm, 20 mm, 10 mm, 8 mm, 4 mm, 2 mm, 1 mm, 0.8 mm, 0.4 mm, 0.2 mm, 0.1 mm, 0.08 mm, 0.04 mm, 0.02 mm, or 0.01 mm. In some embodiments, the dermis layer can have a length of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 700, 1000 mm. In some embodiments, the dermis layer can have a length of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400 m. In some embodiments, the dermis layer can have a length of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400 cm.

[0145] In some embodiments, the width of the dermal layer can be designed to suit the function or use of the cruelty-free leather. In some embodiments, the dermal layer can have a width of from about 0.01 mm to about 50 μm. In some embodiments, the dermal layer can have a width of from about 0.01 mm to about 10 mm, from about 0.01 mm to about 8 mm, from about 0.01 to about 5 mm, from about 0.02 to about 5 mm, from about 0.05 to about 5 mm, from about 0.1 to about 5 mm, from about 0.1 to about 2 mm, from about 0.1 to about 1 mm, from about 0.1 to about 0.8 mm, or from about 0.1 to about 0.5 mm. In some embodiments, the dermal layer can have a width of from about 0.02 mm to 5 mm. In some embodiments, the dermal layer can have a width of from about 0.1 mm to 0.5 mm. In some embodiments, the dermal layer can have a width of from about 0.2 mm to 0.5 mm. In some embodiments, the width of the dermal layer can be at least 0.001 mm, 0.01 mm, 0.02 mm, 0.04 mm, 0.08 mm, 0.1 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1 mm, 2 mm, 4 mm, 8 mm, or 10 mm. In some embodiments, the width of the dermal layer can be at most 50 mm, 40 mm, 20 mm, 10 mm, 8 mm, 4 mm, 2 mm, 1 mm, 0.8 mm, 0.4 mm, 0.2 mm, 0.1 mm, 0.08 mm, 0.04 mm, 0.02 mm, or 0.01 mm. In some embodiments, the dermal layer can have a width of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 700, 1000 mm. In some embodiments, the dermal layer can have a width of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700 cm. In some embodiments, the dermal layer can have a width of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400 m.

[0146] In some embodiments, the cruelty-free leather can comprise one or more dermal layers. In some embodiments, the cruelty-free leather can have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 40, 60, 80, or 100 dermal layers. In some embodiments, when the cruelty-free leather can comprise more than one dermal layer, the dermal layers can be placed on top of another dermal layer. In some embodiments, the cruelty-free leather can comprise two dermal layers, such as a first dermal layer and a second dermal layer. In some embodiments, the first dermal layer can be placed on top of the second dermal layer.

[0147] In some embodiments, the dermis or at least a partially decellularized portion thereof can be stratified, e.g., having multiple sublayers. In some embodiments, the sublayers can have different compositions, e.g., different fiber concentrations. In some embodiments, the sublayers of the dermis or at least a partially decellularized portion thereof can have different thicknesses, densities, or combinations thereof. In some embodiments, the dermis or at least a partially decellularized portion thereof can have a papillary dermis, a reticular dermis, any at least partially decellularized portion thereof, or any combination thereof. In some embodiments, the papillary dermis or at least a partially decellularized portion thereof can comprise loose areolar connective tissue, loosely arranged fibers, at least a partially decellularized portion of these, or any combination thereof. In some embodiments, the loosely arranged fibers can include collagen fibers. In some embodiments, the reticular dermis can comprise dense irregular connective tissue, including collagen fibers and dermal elastic fibers.

[0148] In some embodiments, the dermis layer or at least a partially decellularized portion thereof may comprise a free collagen matrix or lattice that can contract and homogenize in all directions. In some embodiments, fibroblasts (e.g., immortalized bovine fibroblasts), and other suitable types of dermal cells, may be distributed in a continuous collagen gel. In some embodiments, the dermal equivalent may comprise at least one type I collagen matrix in which fibroblasts may be distributed. In some embodiments, the dermal equivalent may also contain other extracellular matrix components. In some embodiments, the extracellular matrix components may include collagen, such as collagen IV, laminin, nidogen, fibronectin, proteoglycan, glycosaminoglycan, or hyaluronic acid. In some embodiments, the dermis layer may contain type IV collagen and laminin, nidogen, or a combination thereof. In some embodiments, the concentrations of these different components may be adjusted. For example, in some embodiments, the concentration of laminin may be about 1% - about 15% of the final volume. In some embodiments, the concentration of collagen IV may be about 0.3% - about 4.5% of the final volume. In some embodiments, the concentration of nidogen may be about 0.05% - about 1% of the final volume. In some embodiments, the collagen may be bovine-derived collagen, rat-derived collagen, fish-derived collagen, natural collagen from any other source, or collagen produced by genetic engineering that can contract in the presence of fibroblasts, or any combination thereof. In some embodiments, the collagen may be from a non-natural source. In some embodiments, the matrix may be a non-tensed collagen gel obtained by horizontal and vertical contraction that does not cause preferential tissue effects on fibroblasts. In some embodiments, the matrix (also referred to as "free") may not adhere to a support, and its volume may change without limitation, imparting varying thicknesses and diameters. In some embodiments, the thickness of the dermal equivalent may be at least 0.05 cm, and in some embodiments, about 0.05 - 2 cm. In some embodiments, the thickness may be increased without compromising the beneficial properties of the skin equivalent or cruelty-free leather. In some embodiments, the thickness may be about 3 mm - about 20 cm or more. In some embodiments, the cruelty-free leather may comprise only the dermis layer.

[0149] In some embodiments, the cell and scaffold composition may comprise an epidermis layer (e.g., an artificial epidermis layer). In some embodiments, the epidermis layer may be an engineered epidermal equivalent, such as an artificial epidermis layer formed in vitro.

[0150] In some embodiments, the epidermal layer may comprise one or more types of cells, including keratinocytes, melanocytes, Langerhans cells, Merkel cells, and inflammatory cells. In some embodiments, the epidermal layer may comprise keratinocytes. In some embodiments, the keratinocytes in the epidermal layer may include epithelial keratinocytes, basal keratinocytes, proliferative basal keratinocytes, differentiated suprabasal keratinocytes, or any combination thereof.

[0151] In some embodiments, the epidermal layer may comprise engineered cells. In some embodiments, the epidermal layer may comprise immortalized cells. In some embodiments, the epidermal layer may comprise at least basal keratinocytes, such as keratinocytes that may be undifferentiated. In some embodiments, the epidermal layer may further comprise partially differentiated keratinocytes as well as fully differentiated keratinocytes. In some embodiments, one or more epidermal layers in the cruelty-free leather may transition from undifferentiated basal keratinocytes to fully differentiated keratinocytes as progression may occur from the dermal-epidermal junction where basal keratinocytes may be located.

[0152] In some embodiments, basal keratinocytes may express hemidesmosomes, which may be used to help anchor the epidermal layer and the dermal layer together. In some embodiments, basal keratinocytes may also be used for skin regeneration. In some embodiments, the epidermal layer in the cruelty-free leather herein may have basal keratinocytes that provide these functions. In some embodiments, the cruelty-free leather comprising such basal keratinocytes is capable of regeneration. In some embodiments, in one or more epidermal layers in the cruelty-free leather, the difference between basal keratinocytes and differentiated keratinocytes may be that both E- and P-cadherins may be present in epidermal keratinocytes along the basement membrane zone (BMZ), but keratinocytes that are differentiated and located away from the BMZ may express only E-cadherin.

[0153] In some embodiments, the basal keratinocytes of the epidermal layer may be arranged in a layer in direct contact with the dermal layer, serving as a boundary between differentiated keratinocytes and fibroblasts. In another scenario, there may be a gap between the basal keratinocytes and the dermal layer. Additionally, there may be a gap between basal keratinocytes and other basal keratinocytes, and a gap between differentiated keratinocytes and the dermal layer. In the latter case, there may be a gap between basal or differentiated keratinocytes and the dermal layer, and the dermal layer and the epidermal layer may not contact each other evenly but may be adjacent to each other. In some embodiments, the dermal layer and the epidermal layer may be adjacent because there may typically be fluid between the dermal layer and the epidermal layer, but substantially no other intermediate materials, such as cell layers, collagen layers, matrix layers, or other supports.

[0154] In some embodiments, keratinocytes in the epidermal layer may express one or more markers. In some embodiments, the markers may include, but are not limited to, keratin 14 (KRT14), tumor protein p63 (p63), desmoglein 3 (DSG3), integrin, beta 4 (ITGB4), laminin, alpha 5 (LAMA5), keratin 5 (KRT5), isoforms of tumor protein p63 (such as TAp63), laminin, beta 3 (LAMB3), and keratin 18 (KRT18).

[0155] In some embodiments, the thickness of the epidermal layer can be designed to suit the function or use of the cruelty-free leather. In some embodiments, the epidermal layer can have a thickness of about 0.001 mm to about 10 mm. In some embodiments, the epidermal layer can have a thickness of about 0.005 mm to about 10 mm, about 0.005 mm to about 5 mm, about 0.005 mm to about 2 mm, about 0.01 mm to about 10 mm, about 0.01 mm to about 5 mm, about 0.01 mm to about 2 mm, about 0.01 mm to about 1, about 0.01 mm to about 0.8 mm, about 0.01 mm to about 0.4 mm, about 0.01 mm to about 0.2 mm, about 0.01 mm to about 0.1 mm, about 0.05 mm to about 0.4 mm, about 0.05 mm to about 0.2 mm, about 0.05 mm to about 0.1 mm, about 0.1 mm to about 0.4 mm, about 0.1 mm to about 0.2 mm, about 0.08 mm to about 1 mm, or about 0.05 mm to about 1.5 mm. In some embodiments, the epidermal layer can have a thickness of about 0.01 mm to about 2 mm. In some embodiments, the epidermal layer can have a thickness of about 0.1 mm to about 0.22 mm. In some embodiments, the thickness of the epidermal layer can be at least 0.001 mm, 0.01 mm, 0.02 mm, 0.04 mm, 0.08 mm, 0.1 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1 mm, 2 mm, 4 mm, 8 mm, or 10 mm. In some embodiments, the thickness of the dermal layer can be at most 50 mm, 40 mm, 20 mm, 10 mm, 8 mm, 4 mm, 2 mm, 1 mm, 0.8 mm, 0.4 mm, 0.2 mm, 0.1 mm, 0.08 mm, 0.04 mm, 0.02 mm, or 0.01 mm. In some embodiments, the thickness values described herein can be the thickness of the epidermal layer and the basement membrane substitute.

[0156] In some embodiments, the length of the epidermal layer can be designed to suit the function or use of the cruelty-free leather. In some embodiments, the epidermal layer can have a length of from about 0.01 mm to about 50 m. In some embodiments, the epidermal layer can have a length of from about 0.01 mm to about 10 mm, from about 0.01 mm to about 8 mm, from about 0.01 to about 5 mm, from about 0.02 to about 5 mm, from about 0.05 to about 5 mm, from about 0.1 to about 5 mm, from about 0.1 to about 2 mm, from about 0.1 to about 1 mm, from about 0.1 to about 0.8 mm, or from about 0.1 to about 0.5 mm. In some embodiments, the epidermal layer can have a length of from about 0.02 mm to 5 mm. In some embodiments, the epidermal layer can have a length of from about 0.1 mm to 0.5 mm. In some embodiments, the epidermal layer can have a length of from about 0.2 mm to 0.5 mm. In some embodiments, the length of the epidermal layer can be at least 0.001 mm, 0.01 mm, 0.02 mm, 0.04 mm, 0.08 mm, 0.1 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1 mm, 2 mm, 4 mm, 8 mm, or 10 mm. In some embodiments, the length of the epidermal layer can be at most 50 mm, 40 mm, 20 mm, 10 mm, 8 mm, 4 mm, 2 mm, 1 mm, 0.8 mm, 0.4 mm, 0.2 mm, 0.1 mm, 0.08 mm, 0.04 mm, 0.02 mm, or 0.01 mm. In some embodiments, the epidermal layer can have a length of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 700, 1000 mm. In some embodiments, the epidermal layer can have a length of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400 μm.

[0157] In some embodiments, the width of the epidermal layer can be designed to suit the function or use of cruelty-free leather. In some embodiments, the epidermal layer can have a width of from about 0.01 mm to about 50 m. In some embodiments, the epidermal layer can have a width of from about 0.01 mm to about 10 mm, from about 0.01 mm to about 8 mm, from about 0.01 to about 5 mm, from about 0.02 to about 5 mm, from about 0.05 to about 5 mm, from about 0.1 to about 5 mm, from about 0.1 to about 2 mm, from about 0.1 to about 1 mm, from about 0.1 to about 0.8 mm, or from about 0.1 to about 0.5 mm. In some embodiments, the epidermal layer can have a width of from about 0.02 mm to 5 mm. In some embodiments, the epidermal layer can have a width of from about 0.1 mm to 0.5 mm. In some embodiments, the epidermal layer can have a width of from about 0.2 mm to 0.5 mm. In some embodiments, the width of the epidermal layer can be at least 0.001 mm, 0.01 mm, 0.02 mm, 0.04 mm, 0.08 mm, 0.1 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1 mm, 2 mm, 4 mm, 8 mm, or 10 mm. In some embodiments, the width of the epidermal layer can be at most 50 mm, 40 mm, 20 mm, 10 mm, 8 mm, 4 mm, 2 mm, 1 mm, 0.8 mm, 0.4 mm, 0.2 mm, 0.1 mm, 0.08 mm, 0.04 mm, 0.02 mm, or 0.01 mm. In some embodiments, the epidermal layer can have a width of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 700, 1000 mm. In some embodiments, the epidermal layer can have a width of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400 cm. In some embodiments, the epidermal layer can have a width of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400 m.

[0158] In some embodiments, the epidermal layer can be stratified, e.g., having multiple sub-layers. In some embodiments, the sub-layers can have different cell compositions, e.g., different types of keratinocytes. In some embodiments, the sub-layers can contain engineered cells. In some embodiments, the sub-layers of the epidermal layer can have different thicknesses and / or densities. In some embodiments, the epidermal layer can have one or more cornified layers (stratum corneum), clear / translucent layer (stratum lucidum), granular layer (stratum granulosum), spinous layer (stratum spinosum), basal layer / germinative layer (stratum basale / germinativum), or any combination thereof. In some embodiments, the epidermal layer can contain a functional epidermal permeability barrier (e.g., organized lipid bilayer in the stratum corneum). In some embodiments, the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, or stratum basale / germinativum can have a thickness of from about 0.0001 mm to about 5 mm. In some embodiments, the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, or stratum basale / germinativum can have a thickness of at least about 0.001 mm, 0.01 mm, 0.02 mm, 0.04 mm, 0.08 mm, 0.1 mm, 0.15 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1 mm, 2 mm, 4 mm, 8 mm, or 10 mm. In some embodiments, the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, or stratum basale / germinativum can have a thickness of at most about 50 mm, 40 mm, 20 mm, 10 mm, 8 mm, 4 mm, 2 mm, 1 mm, 0.8 mm, 0.4 mm, 0.2 mm, 0.15 mm, 0.1 mm, 0.08 mm, 0.04 mm, 0.02 mm, or 0.01 mm.

[0159] In some embodiments, the epidermal layer can further contain cells that produce pigments (e.g., melanin). In some embodiments, such pigment-producing cells can be melanocytes. In some embodiments, the melanocytes in the epidermal layer can express one or more markers. In some embodiments, such markers can include, but are not limited to, sex determining region Y-box 10 (Sox-10), microphthalmia-associated transcription factor (MITF-M), premelanosome protein (gp-100), dopachrome tautomerase (DCT), tyrosinase (TYR), and Melan-A (MLANA). In some embodiments, cruelty-free leather can not contain the epidermal layer.

[0160] Provided herein are methods of making any of the compositions provided herein. In some embodiments, the method comprises at least partially coating a scaffold with a coating comprising: Matrigel, vitronectin, fibronectin, proteins extracted from soy, proteins extracted from peas, proteins extracted from corn, synthetically produced peptides, RNA-binding glycine-rich (RBG) proteins, polylysine, synthetic proteins, RGD peptides, polylysine, polyarginine, polyornithine, recombinant proteins, oligomers, polymers, GTAMC, carbohydrate-binding modules, cellulose-binding domains, starch-binding domains, or combinations thereof. In some embodiments, the method further comprises inoculating the scaffold with isolated animal fibroblasts or fibroblast-like cells to form the composition. In some embodiments, the collagen, fibroblasts, and / or fibroblast-like cells associate with the scaffold by any of the means described elsewhere herein, such as non-specific adsorption, covalent interaction, or specific adsorption.

[0161] In some embodiments, a method of making cruelty-free leather may include forming and tanning a cell and scaffold composition. In some embodiments, the method may include further processing the cell and scaffold composition, e.g., to achieve the thickness and texture of natural leather. In some embodiments, tanning may be performed after at least partial decellularization of the cell layer. In some embodiments, tanning may produce cruelty-free leather similar to natural leather, which may be a durable and flexible material produced by tanning animal hides and skins (usually cowhide). Tanning herein may refer to the process of treating animal skins to produce leather or treating the compositions disclosed herein to produce cruelty-free leather. Tanning may be performed in various ways, including vegetable tanning (e.g., using tannic acid), chrome tanning (chromium salts, including chromium sulfate), aldehyde tanning (using glutaraldehyde or oxazolidine compounds), synthetic tanning agents (synthetic tannins, using aromatic polymers), bacterial staining, etc. In some embodiments, tanning may be metal-free. In some embodiments, tanning may be an environmentally friendly process. In some embodiments, tanning may be performed to convert the proteins in the hides, furs, or compositions disclosed herein into a stable material that does not spoil, while keeping the material flexible. In some embodiments, chromium may be used as a tanning material. In some embodiments, tanning may include chromium, aluminum, zirconium, titanium, iron, sodium aluminosilicate, formaldehyde, glutaraldehyde, oxazolidine, isocyanate, carbodiimide, polycarbamoyl sulfate, tetrahydroxyphosphonium sulfate, sodium p-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]benzenesulfonate, pyrogallol, catechol, synthetic tanning agents, or any combination thereof. In some embodiments, tanning may be performed on engineered cells of a scaffold. In some embodiments, the tissue that can be tanned may include fibers or a plurality of fibers (e.g., polyester fibers, synthetic fibers, natural fibers). In some embodiments, the pH of the cell layer or layered structure may be adjusted (e.g., decreased; e.g., to a pH of about 2.8 - 3.2)) to enhance tanning. In some embodiments, after tanning, the pH may be increased ("basified" to a slightly higher level, e.g., a pH of about 3.8 - 4.2). In some embodiments, the pH described herein may be at least 1. In some embodiments, the pH described herein may be 14 or less. In some embodiments, tanning may be performed on a cell layer, e.g., a dermal layer, an epidermal layer, engineered cells, an immortalized cell layer, laminin, fibronectin, collagen, or any combination thereof. In some embodiments, a tissue (e.g., a tissue from engineered cells) may be tanned. In some embodiments, tanning may also be performed on a layered structure, e.g., a layered structure comprising at least a dermal layer. In certain cases, tanning may also be performed on synthetic leather. In some embodiments, tanning may be performed after forming a cell layer (e.g., a dermal layer or an epidermal layer). In some embodiments, tanning may be performed after forming a layered structure.

[0162] In some embodiments, the methods provided herein may further include processing. The processing may occur on one or more cell layers, such as one or more cell layers of a scaffold. The (further) processing may be performed on a hide (such as a tanned hide). In some embodiments, the processing may be selected from the group consisting of: preservation, soaking, softening, pickling, de-acidification, dilution, retanning, lubrication, crusting, wetting, squeezing, trimming, re-chroming, neutralization, dyeing, fatliquoring, filling, stripping, fatliquoring, whitening, fixing, shaping, drying, conditioning, grinding, staking, buffing, finishing, oiling, brushing, padding, impregnating, spraying, roll coating, curtain coating, polishing, electroplating, embossing, ironing, glazing, tumbling, and any combination thereof.

[0163] In some embodiments, the cruelty-free leather may comprise collagen and extracellular matrix components produced by cells in the dermis layer and / or epidermis layer disclosed herein. In some embodiments, the cruelty-free leather may comprise at least partially decellularized dermis layer and / or epidermis layer as disclosed herein. In some embodiments, the cruelty-free leather does not comprise an epidermis layer. In some embodiments, the cruelty-free leather may further comprise at least a portion of hair follicle cells, endothelial cells, smooth muscle cells, dermal papilla cells, immune system cells (such as lymphocytes, dendritic cells, mast cells, macrophages, or Langerhans cells), adipocytes, nerve cells, Schwann cells, and mixtures thereof. In some embodiments, the cruelty-free leather may comprise at least a portion of engineered cells (e.g., cells comprising molecular switches). The cruelty-free leather may comprise immortalized cells. In some embodiments, the cruelty-free leather may comprise isolated cells. In some embodiments, the cruelty-free leather may comprise a cell line.

[0164] In some embodiments, the cruelty-free leather may comprise hair. In some embodiments, the cruelty-free leather may comprise hair in one or more layered structures. In some embodiments, the cruelty-free leather may include fur. In some embodiments, the hair (such as fur) may be natural, synthetic, or a combination thereof. In some embodiments, the hair (e.g., fur) may be grown by cells in the cruelty-free leather or added exogenously to the cruelty-free leather. In some embodiments, the cruelty-free leather may not have any hair.

[0165] The cruelty-free leather may comprise at least a portion of prokaryotic cells, eukaryotic cells, or a combination thereof. In some embodiments, the cruelty-free leather may comprise at least a portion of bacterial cells, such as Escherichia coli. In some embodiments, the cruelty-free leather may comprise at least a portion of eukaryotic cells (such as bovine cells, porcine cells, human cells, Saccharomyces cerevisiae).

[0166] In some embodiments, one or more cells in at least a portion of the cruelty-free leather can be genetically engineered cells. The term "genetically engineered" can refer to an artificial alteration of the nucleic acid content of a cell. Thus, genetically engineered cells can include cells in which one or more nucleotides have been inserted, deleted, and / or substituted in the cell genome, as well as cells in which there are alterations including the introduction of self-replicating extrachromosomal nucleic acid inserted into the cell. Genetically engineered cells also include cells in which the transcription of one or more genes has been altered, such as increased or decreased.

[0167] In some embodiments, the thickness of the leather unit can be measured in millimeters, ounces, or irons. In some embodiments, one ounce can be equal to 1 / 64 inch or 0.0156 inches or 0.396 mm. In some embodiments, one iron can be equal to 1 / 48 inch or 0.0208 inches or 0.53 mm.

[0168] Method of Use

[0169] In some embodiments, disclosed herein is a method comprising transplanting a composition disclosed herein onto a patient in need of a skin graft for treating skin loss or damage. In some embodiments, the patient may have a laceration, a contusion, an injury, a sore, a burn, a wound, a surgical wound, surgically excised skin, necrotic skin, or any combination thereof. In some embodiments, disclosed herein is a method comprising tanning a composition disclosed herein to produce cruelty-free leather. In some embodiments, disclosed herein is a method of using the cruelty-free leather disclosed herein as a substitute for traditional leather in leather products. Disclosed herein is a leather product comprising the cruelty-free leather disclosed herein. In some embodiments, leather products may include watch straps, belts, straps, packaging, shoes, boots, footwear, gloves, clothes, bags, handbags, wallets, coin purses, wallets, key bags, credit card cases, pen bags, backpacks, boxes, wallets, saddles, harnesses, whips, suitcases, travel goods, rucksacks, briefcases, document bags, briefcases, briefcases, pet supplies, holsters, collars, hunting and fishing supplies, gun boxes, cutlery boxes, holsters, stationery, writing pads, book covers, camera boxes, glasses cases, cigarette cases, cigar boxes, jewelry boxes, mobile phone cases, sporting goods, balls, basketballs, footballs, rugby or any combination thereof. In some embodiments, clothes may include tops, bottoms, outerwear or any combination thereof. In some embodiments, bags may include handbags with or without shoulder straps. In some embodiments, luggage may include large travel cases, suitcases, travel bags, cosmetic cases, wash bags or any combination thereof. In some embodiments, disclosed herein is a method for treating a disease or illness using a composition disclosed herein. Disclosed herein, in some embodiments, are kits comprising a composition as disclosed herein or a leather product as disclosed herein. DETAILED DESCRIPTION

[0170] Example 1. Cell attachment on nylon and bamboo / cotton scaffolds

[0171] Bovine dermal fibroblasts were attached to single or double strand nylon scaffolds or 40:60 bamboo / cotton scaffolds for 17 days under static and shaking growth conditions to evaluate cell attachment to various scaffold materials. Cell growth was assessed by measuring the residual glucose level before each feed. Generally, shaking conditions resulted in more glucose consumption. The bamboo / cotton blend consumed the most glucose under static and shaking conditions, while glucose consumption in the double strand nylon under shaking increased steadily over time. Figure 1 A. Figure 1 B. Figure 1 C. Figure 1 E. Figure 1 F. Figure 1 G. Figure 1 I and Figure 1As seen in , microscopic examination of cells stained with calcein-AM and Hoescht dye showed that cell attachment on the surface of single-strand nylon was lower than that on the surface of double-strand nylon, and a clear extracellular matrix was visibly formed on the scaffold on the double-strand nylon surface. As Figure 1 D, Figure 1 H, and Figure 1 the microscopic examination seen in showed optimal growth of cells attached to bamboo / cotton scaffolds and spindle-shaped cells under both shaking and static conditions. These data shown by the nylon data indicate that woven materials may not provide sufficient surface area or curvature for good cell attachment. Alternatively, non-woven materials (such as non-woven materials of bamboo / cotton mixtures) exhibited significantly improved cell attachment, while non-woven nylon (such as needle-punched nylon) scaffolds showed increased cell attachment compared to simple meshes, further highlighting the advantages of non-woven materials.

[0172] Example 2. Scaffold Dissolution Conditions

[0173] Poly(vinyl alcohol) (PVOH) and poly(lactic acid) (PLA) were tested as soluble scaffolds for fibroblast attachment to prevent fiber protrusion and improve tissue quality. The conditions required to dissolve the PVOH scaffold were deionized water or DMEM for 1 minute at 95 °C. The conditions required to dissolve PLA needed the use of organic solvents overnight, such as benzylamine or ethyl acetate. The success of dissolving the scaffold was determined by observing visual changes to evaluate scaffold removal and potential raw hide damage, as well as the weight of the scaffold before and after dissolution, as shown in Table 1. PLA was dissolved overnight in benzylamine, resulting in a 72% reduction in scaffold weight, while dissolution in ethyl acetate under similar conditions resulted in a 93% weight loss of the scaffold. The effects of the dissolution conditions on raw hides were evaluated on both raw salted hides (before tanning) and tanned but un-lipidized hides (after tanning).

[0174] Exposing pre-tanned hides on PVOH scaffolds to high temperature (95 °C) would damage the tissue. However, exposing tanned hides on PVOH scaffolds to high temperature (95 °C) did not result in complete destruction but did result in a reduction in raw hide quality while causing approximately 100% removal of the scaffold. Lower dissolution temperatures (90 °C) for longer periods of time in water (including 45 minutes or 1.5 hours) resulted in 27.3% and 46.7% scaffold removal, respectively, but would cause damage to the raw hides because the denaturation temperature of tanned hides is approximately 70 °C. Due to the lower denaturation temperature, a lower dissolution temperature of 40 - 50 °C is preferred to avoid raw hide damage. However, exposure to benzylamine and ethyl acetate did not cause significant changes to pre-tanned and tanned hides on PLA scaffolds. This data emphasizes the ability to dissolve PLA and PVOH scaffolds under various conditions to help prevent fiber protrusion through tissues and improve tissue quality.

[0175] Table 1

[0176] Conditions Weight before dissolution (mg) Weight after dissolution (mg) Reduction % Benzylamine, overnight 85.1 23.7 72％ Ethyl acetate, overnight 82.6 5.7 93％ Deionized water, 95°C, 1 min Not measured Not measured ～100％ DMEM, 95°C, 1 min Not measured Not measured ～100％ 90°C, 45 minutes 51.9 37.7 27.3％ 90°C, 1.5 hours 59.7 31.8 46.7％

[0177] Example 3. Influence of Scaffold Pore Size on Tissue Quality

[0178] A series of PLA scaffold materials with different pore sizes were tested, including 55 μm, light punch 55 μm (55LP), 80 μm, and 100 μm, and the tissue quality was evaluated as a function of pore size in the form of a cardholder. The scaffolds were assembled onto a frame and placed in a bioreactor. The scaffolds were inoculated with an inoculation solution (2.75×10 6 cells / mL) and kept stationary, while shaking conditions were initiated after 1 week. There was no feeding for 2 days after inoculation, and thereafter the cells were cultured with a complete medium exchange every 7 days for 27 days of tissue growth, and the medium was sampled daily. Tissue biopsies were performed using a sterile 4 mm biopsy punch. No significant differences were observed in sulfated glycosaminoglycan (sGAG) after 24 or 96 hours (n = 2 biopsies), and there were also no any significant differences in collagen and total protein concentrations between scaffolds with different pore sizes (n = 3 biopsies)( Figure 31 ). The 55LP scaffold exhibited the highest flexural stiffness of the raw material and the rawhide. This indicates that the increased thickness and moment of inertia translate into an increase in material stiffness. Scaffolds with smaller pore sizes were heavier before and after culture, making the tanned rawhide feel firmer, as Figure 13 shown. However, the net weight of the tissue content calculated on all scaffolds was similar and independent of the pore size, as Figure 13 shown. Histological examinations were completed on cross-sections of the tissue stained with trichrome. Compared with Figure 16 A-F with an average pore size of 50 μm, larger pore sizes generally had better tissue ingrowth, as Figure 15 A-F (where the average pore size was 100 μm) shown. It was observed that the 55LP scaffold with the smallest pore size and the largest starting thickness had poor tissue ingrowth, as Figure 14 A-F shown. Generally, materials with large pore sizes showed more inconsistencies and light spots during scanning, as Figure 2 A, Figure 2 B, Figure 2 C, and Figure 2 D shown, but generally, the PLA scaffolds increased the consistency with the pressed PET scaffolds. The tissue weights calculated under all conditions were similar and independent of the pore size. Histological examinations were completed on cross-sections of the tissue stained with trichrome. Compared with Figure 3 B with a porosity of 50 μm, larger pore sizes generally resulted in better tissue ingrowth, as Figure 3As shown in A. Scaffolds with large pores provide better tissue ingrowth, while small pore diameters result in an increase in the weight of the fibers, creating the illusion of a heavier and stronger leather, demonstrating the importance of the porosity of the scaffold material.

[0179] Example 4. Effect of soaking in PLA

[0180] PLA scaffolds were tested to determine if soaking in fetal bovine serum (FBS) was required to fully achieve cell attachment and proliferation. Soaking in FBS allows for the adsorption of adhesive proteins to the surface, thus permitting increased cell attachment. Two boxes of 5 square scaffolds made of 1.7 dtex PLA and 60 μm pore size were prepared, and one control box contained PET. One set of 5 scaffolds was filled with pure FBS to cover the channels and soaked overnight at 37 °C, while the second set and the control (PET scaffolds) were not (see Figure 4A ). After aspirating the FBS, all channels were seeded with fibroblasts. These scaffolds were harvested at different times. The scaffolds were biopsied 1 time on day 1, 2 times on day 2, and 2 times on day 4 using a sterile biopsy punch, and the media cells were saved for analysis. The biopsies on day 2 and day 4 were used for further analysis. Three of these biopsies were used for DNA determination, and one was used for calcein imaging. The DNA determination showed that FBS-soaked PLA scaffolds had the highest initial cell attachment compared to unsoaked PLA scaffolds ( Figure 4B ), so soaking the channels and the adsorption of proteins to the surface had a beneficial effect.

[0181] Example 5. Protein-scaffold conjugation for cell attachment and proliferation

[0182] Poor cell attachment to biomaterials after seeding can lead to cell death, reduced viability, and poor collagen production, which can be addressed by increasing the cell compatibility of the biomaterial surface. As observed in Example 2, traditional cell culture proteins can be applied to increase cell attachment. Low-cost, cruelty-free options were also evaluated. Proteins extracted from sweet pea and wheat flour were conjugated to PLA and lyocell and compared to their respective unmodified scaffold materials to determine if functionalization of the scaffold surface could enhance cell attachment and proliferation. Using carbodiimide crosslinking chemistry (EDC / NHS), the protein was conjugated to PLA via a bond between the activated carboxylic acid of PLA and the amine of the protein. Attempts to enhance the conjugation of the protein to PLA included adding carboxyl groups to PLA prior to conjugation with the protein. The protein was conjugated to lyocell via the Maillard reaction in an autoclave. Autoclaving was completed one, two, or four times. The cell culture conditions were as follows: 500 k / cm in tissue culture 2, the 48-well plates were treated with DMEM HG containing 10% FBS. Cell attachment and proliferation were measured by MTT and DNA assays, and the results showed that conjugation of the protein to lyocell, rather than PLA, significantly enhanced cell attachment and proliferation compared to its unmodified counterpart. Figure 5 The percentages of cells on pea protein, wheat protein, and untreated (NT) scaffolds after 1 and 4 days are shown, highlighting the increase exhibited after surface modification of lyocell. As Figure 6 shown in Figure 6 D, the fluorescence images after 1 day indicated that cells aggregated in unmodified lyocell, while after conjugation with pea protein (as Figure 6 shown in Figure 6 A, Figure 6 B, and Figure 5 C), spindle-shaped growth occurred, highlighting the increased cell compatibility after modification. Conjugation of pea or wheat protein to PLA resulted in little change in spindle-shaped cell growth, as Figure 6 shown in Figure 6 A, Figure 6 B, Figure 6 C,

[0183] E, and Figure 25 F. These examples demonstrate that the cell compatibility of the scaffold surface can be increased by treating the scaffold surface with groups that increase biocompatibility, such as these or other groups including polypeptides. In addition, chemical processes for modulating surface properties have the potential to increase cell affinity or tissue effects, which can be achieved by a variety of synthetic methods, such as processing with sodium hydroxide or glycidyltrimethylammonium chloride (GTMAC). Figure 17 A-C) or cell activity (as Figure 18 shown in Figure 18 ). However, as Figure 17 shown in

[0184] Test other conjugation methods to see if the utilization of these low-cost proteins can be improved, including Maillard reaction, surface activation, and EDC / NHS conjugation. The Maillard reaction was simulated by autoclaving the scaffolds while soaking them in a protein-rich solution. In some cases, the scaffold surface was activated by oxidation with sodium periodate and then the scaffold was inoculated as such, or then conjugated with proteins via the Maillard reaction. The protein was conjugated to PLA using carbodiimide crosslinker chemistry (EDC / NHS) via a bond between the activated carboxylic acid of PLA and the amine of the protein. It was found that the Maillard reaction contributed to the conjugation of pea protein to lyocell, as Figure 20 shown in Figure 20 C, and cell spreading was improved compared to the unmodified scaffolds, as Figure 21 shown in Figure 18 B. It was also found that covalently binding pea protein to PLA using EDC / NHS chemistry increased cell viability by more than 50% compared to unmodified PLA, as Figure 21 shown, where it was found that previous cold coating did not change cell viability, as Figure 20 shown. Oxidation of lyocell itself did not result in long-lived cell attachment. Although a significant increase in cell viability was seen on the first day after inoculation, however, as Figure 20 shown in Figure 21 , this viability decreased significantly on day 4, and as

[0185] shown in Figure 19 E, this decrease in cell viability could be confirmed by the lack of cell spreading in the calcein images taken on day 4. However, the combination of oxidation and the Maillard reaction with pea protein on lyocell made the cells appear to be very well distributed, as

[0186] Figure 20 F, however, this increase in cell distribution was not reflected in the Figure 21 cell viability numbers shown.

[0185] The Maillard reaction was further investigated by adjusting the autoclave cycle number. One, two, or four drying cycles on the autoclave were completed and compared to soaking only in a cold solution without any autoclave cycles. As Figure 19 shown, compared to the untreated conditions, one and two autoclave cycles produced the highest cell viability on day 4 after inoculation, with the viability more than tripled. It was found that cold soaking greatly increased cell viability on day 1, however, this viability decreased significantly on day 4. This decrease in viability may be due to the separation of protein groups from the scaffold, thus reducing the viability. The initial increase in viability compared to one, two, or four autoclave cycles may be due to a higher initial quality of the protein, which underwent fewer denaturing autoclave cycles.

[0186] These examples of activating the scaffold surface and conjugating protein sources demonstrate that the cell compatibility of the scaffold surface can be improved by treating the scaffold surface. In addition, the chemical processes for regulating surface properties have the potential to increase cell affinity or tissue effects. Although not shown here, other methods that can be used to activate the fiber surface include treatment with sodium hydroxide or glycidyltrimethylammonium chloride (GTMAC).

[0187] Example 6. Effect of seeding density on tissue effect

[0188] The effect of cell seeding density on tissue effect was evaluated to optimize the seeding concentration of bovine dermal fibroblasts. Optimization was completed on small-scale PET scaffolds. After incubation for 6.5 weeks (as short as 4 weeks or as long as 8 weeks), the seeding densities of 125k / cm 2 , 250k / cm 2 , 500k / cm 2 and 1M / cm 2 (as low as 30k / cm 2 and as high as 1M / cm 2 ) were examined, and the samples were preserved for collagen analysis and scanning electron microscopy (SEM) imaging. After digestion for 24 or 96 hours after biopsy with an 8mm biopsy punch, the collagen concentration was evaluated. After both time periods, there was no statistically significant difference in collagen concentration between any of the seeding densities. When the scale was enlarged to an inoculation area of 220cm 2 , seeding densities of 500k / cm 2 , 125k / cm 2 and 62.5k / cm 2 were examined, and the same effect was detected. These seeded scaffolds were incubated for 8 weeks and processed similarly to evaluate collagen production. After digestion for 24 or 96 hours, there was no statistically significant difference in the amount of collagen produced between the inoculation areas with seeding densities of 65k / cm 2 and 125k / cm 2 ( Figure 22 and Figure 23 ), but there was a difference between the areas with seeding densities of 125k / cm 2 -500k / cm 2 , where collagen increased with increasing seeding density. Figure 24 It was shown that the soluble collagen readings were significantly higher when using a seeding density of 30k / cm 2 .

[0189] Example 7. Tissue and leather growth process

[0190] The cell source (immortalized / isolated cells) was thawed and grown to reach the required number of cells. The scaffold material was prepared on the frame and sterilized together with the bioreactor container. The cells were seeded (500k cells / cm2 to the scaffold material in the bioreactor, add tissue growth medium, and then culture, changing regularly. To transform the grown tissue into leather, remove the engineered animal hide from the growth medium and the frame and optionally clean it. Optionally, the hide can be salted, desalted, limed, or softened. Then, tan the hide to prepare leather, which can optionally be subsequently processed (i.e., add oil emulsion, add dye, add retanning agent).

[0191] Example 8. Effect of Fiber Diameter and Sterilization Parameters on Fiber Strength

[0192] Fiber diameter can have a significant impact on the tactile feel of the resulting leather, especially its roughness. In addition, the roughness of the material and fiber diameter can affect cell performance. A series of PLA fiber diameters ranging from 1 - 40 dtex have been examined. As shown in Table 2, increasing the fiber thickness from the control (100 gsm) to 6.7 dtex results in lower areal density, collagen, and DNA content.

[0193] Table 2

[0194] Tests Control (100 gsm) PLA 6.7 dtex PLA <![CDATA[Glutamine consumption [μM / cm 2 *]]> 73.6 69.3 Thickness [mm] 1.44 1.36 <![CDATA[Areal density [g / cm 2 > 0.159 0.157 Average pixel value [pixel value]* 155 151 Collagen [mg / biopsy] 2.19 1.89 DNA [μg / biopsy] 27.2 31.2 Dry weight [mg / biopsy] 6.16 10.5** Histological ingrowth [yes / no] No No Grade after tanning [A, B, C, D] A D

[0195] Example 9. Histological Images of Cross - Sections of Cell and Scaffold Combinations

[0196] Figure 7 A and Figure 7 B show histological images of 5 - μm cross - sections of tissue stained with trichrome on the scaffold, where the markers highlight dense tissue, fibers, cell nuclei, and low - density tissue. Figure 8 A shows a histological image of a 5 - μm cross - section of natural cowhide stained with trichrome, where the markers indicate the location of the texture and the dermis. Figure 8 B shows a histological image of a 5 - μm cross - section of the top layer (granules) of cowhide stained with trichrome. Figure 9 A shows a fluorescence micrograph of fibroblasts at 4 - fold magnification after adding calcein AM. Live cells show green fluorescence, and the arrows highlight cell spreading, indicating affinity for the scaffold. Figure 9 B shows a fluorescence micrograph of fibroblasts at 10 - fold magnification after adding calcein AM. Live cells show green fluorescence, and the arrows highlight cell spreading, indicating affinity for the scaffold. The second arrow highlights the fibers illuminated by white light. Figure 10 A shows a fluorescence micrograph of fibroblasts at 10 - fold magnification after adding calcein AM, such that live cells show green fluorescence. Cell spreading and attachment highlight good affinity for the scaffold. Figure 10B shows a fluorescence microscopic image of fibroblasts magnified 10 times after adding calcein AM, making the live cells show green fluorescence. The rounding and aggregation of cells indicate low affinity for the scaffold. Figure 11 Showing a histological image of a 5-μm cross-section of the tissue stained by trichrome staining, where the markers indicate the panoramic view of the slide, the scale bar, the cursor position, and the tissue thickness.

[0197] Example 10. Influence of the basic fiber material on tissue effect

[0198] Cell attachment and tissue growth have been attempted on a variety of different materials. In this example, cell attachment of various fiber types was first tested, and then tissue growth was tested. A variety of sustainable bio-based materials and less sustainable but commercial materials were tested. All materials tested here are needle-punched nonwoven materials. PET materials and some nylon materials were used as controls. Two different PLA materials were tested, one simply called PLA and the other called 50-50. The 50-50 material contains two types of PLA fibers, each containing 50% by mass. Half is the normal high melting temperature PLA used in the first sample type. The other half of the fibers are special core-sheath fibers, where the core is a high melting temperature polymer and the outer layer or sheath is a lower melting temperature polymer. This special fiber structure allows the needle-punched nonwoven fabric to be more easily formed into the desired shape because any heat treatment will easily melt or soften the sheath material while maintaining the integrity of the core material. Two viscose materials with different cross-sections, circular and trilobal, are respectively designated as regular and trilobal viscose. Viscose is a cellulose fiber made by dissolving wood or other cellulose fibers in a caustic sulfide solution and then reconstructing the dissolved pulp into fibers. Finally, Lyocell materials were tested. Different from viscose, Lyocell is dissolved by a solvent process, which can significantly reduce toxic waste compared with viscose.

[0199] The initial attachment test shows that in the case of PET, nylon, PET, and 50-50 scaffolds, the glucose consumption was high after 7 days of culture, as Figure 26 shown. Glucose consumption, similar to MTT, can be used as an indirect measurement of the affinity of cells for the substrate because only with sufficient affinity of cells for their substrate can significant glucose consumption and cell activity be achieved. The cellulose-based materials showed less glucose consumption than the aforementioned materials. Among the cellulose-based materials, cotton showed the highest consumption, but this was behind the other materials mentioned above, as Figure 26 shown. Under these culture conditions, it was found that the cells did not have a high affinity for the surface of the cellulose-based materials. However, when the culture medium was changed, it was found that the cells attached to the Lyocell-based materials.

[0200] In subsequent tissue culture experiments, other materials were tested. Scaffolds composed of polyvinyl alcohol (also known as PVOH or PVA) were tested, as well as three different Lyocell scaffolds. These three new Lyocell scaffolds used fibers of different weights and diameters, including 1.7, 3.3, and 6.7 dtex fibers. In addition, scaffolds composed of alginate were tested. Another scaffold consisted of Vicryl mesh, which was woven from PLGA fibers into a mesh. The total collagen content and the collagen content normalized by the total protein content were evaluated for each grown tissue. The highest collagen in each biopsy was achieved under PET hPL conditions, showing the influence of both media conditions and scaffold conditions on the deposited tissue, as Figure 27 shown. Tissues grown on PLA and PVOH had the second highest amount of collagen, followed by PET in FBS, and then the Vicryl mesh. The scores of different Lyocell materials with different fiber weights varied greatly from each other. The 3.3 dtex scaffold seemed to produce the most collagen among the three, while the 1.7 dtex and 6.7 dtex seemed to have very little tissue deposition. At least, the alginate scaffold produced almost no collagen at all. Then, small-scale square tissues were tanned, marked, and made distinguishable from each other, as Figure 28 shown. While hPL PET did produce the most collagen and seemed to be stained the darkest.

[0201] Example 11. Influence of Scaffold Shape Factor on Tissue Effect

[0202] Many biomaterial scaffold shape factors were tested herein, and their influence on tissue formation was evaluated. The control used in this experiment was a simple needle-punched nonwoven material that had been thermally coated on one side to a given thickness such that one face was smooth; this situation was referred to as the "pressed control". The 3M Thinsulate G-type material was tested here, which consisted of three main layers, two spunbond and one airlaid nonwoven layer, which formed a sandwich structure, so it was a spunbond layer, then airlaid, and then spunbond again. The material was polyester and might be coated with various materials to achieve maximum insulation properties. The material was also held together at certain intervals by sutures, and for this experiment, sutures were applied to produce a more regular thickness throughout the material. This situation will be referred to as "Thinsulate". Next was a towel-like material made of PET material, which was called the "Secant Towel". This material could be Figure 41It can be seen in more detail in A-B. Here, a pattern of looped fibers originating from the cross-knit surface can be seen, which is in the form of a terry cotton fabric. Next is a thicker material, which consists of two fabrics separated by fibers between the two fabrics, separating the two outer fabrics by a set distance. These types of fibers are commonly known as spacer fabrics and are referred to here as "secant spacers". Next is "Fibertex", which is another needle-punched nonwoven fabric. However, this fabric (unlike the "pressed control") is not pressed; unlike the control consisting of a group of PET fibers, this fabric consists only of a single fiber population of lyocell fibers. Next is "double-layer pressing", where two "pressed control" fabrics are stacked one on top of the other, with the rough surfaces facing inwards towards each other and the smooth sides facing outwards. These two fabrics are laminated together by stitching. Next is the "smooth surface laminate", where a vicryl mesh is laminated on top of the "pressed control" fabric by stitches, such that the vicryl mesh is adjacent to the smooth surface of the "pressed control" fabric. Next is the "rough surface laminate", which is the same as the "smooth surface laminate", except for the arrangement of the mesh and the "pressed control" fabric, as the rough surface of the "pressed control" fabric is now adjacent to the mesh. Next is the "autoclaved laminate", which is a replica of the rough surface laminate, except that the scaffolds have been laminated together and then autoclaved, where the others are autoclaved before assembly. Finally, there is the "only absorbable" one, where two vicryl meshes are laminated together such that the scaffold is entirely composed of knitted absorbable material.

[0203] After the complete incubation period on the cardholder-scale material, the biochemical content and physical properties of the dermis were analyzed. When normalized by the volume of the biopsies taken, the "only absorbable" condition produced the highest concentration of collagen at the 96-hour digestion time point, as Figure 32 shown, but this result may be discounted as the thickness and volume of the biopsies were less than any other condition, as Figure 33 shown. As Figure 32 shown, the collagen density of Thinsulate, Fibertex, and the rough surface laminate ranked second highest among the tested shape factors. When observing other physical and biochemical characteristics, it can be seen that the secant towel had the highest DNA content, while the secant spacer had the highest thickness. Compared to other conditions, the control double-layer pressing had high DNA, thickness, and weight contents. In Figure 34 it can be seen directly that Fibertex had very similar thickness, weight, hydroxyproline, and DNA contents compared to the pressed control condition. This is significant as the shape factors of the scaffold samples were the most similar. In Figure 35Among them, the collagen values of the compression control and Fibertex were compared after 4 weeks and 8 weeks of tissue growth. Different from the hydroxyproline measurement conducted at 8 weeks, it was found that the soluble collagen in Fibertex was slightly lower at 8 weeks but higher at 4 weeks. Since the internal structure of Fibertex was slightly different from that of the compression control, differences in growth kinetics were expected.

[0204] In addition to biochemical changes, these different shape factors showed an impact on the way tissues form in three dimensions. By using histology, it was possible to observe how different scaffolds formed tissues differently. In the compression control, it could be seen that the tissue was relatively evenly distributed throughout the thickness of the scaffold, and the tissue deposition on one surface was slightly denser. On the contrary, the bilayer compression scaffold had a very distinct tissue deposition gradient, with a high density at the edges and a very small density in the center, and some completely empty areas, as Figure 36 shown. The bilayer compression scaffold was much thicker than the compression control, weakening the influence of material thickness. The secant towel material, similar to the compression control, produced relatively continuous tissue deposition, as Figure 37 shown. Different from the compression control, the secant towel seemed to have a unique surface pattern on the side with a wavy surface. There were high-density fibers near the peaks, while in the valleys, there were few fibers (if any). The local collagen distribution of the compression control and the secant towel could be compared. In the compression control, as Figure 38 shown in A - B, the collagen deposited on the surface was naturally very flat, while in the secant towel, the collagen deposition was more random, and in comparison, this arrangement looked more round and isotropic, as Figure 39 shown in A - B. The smooth surface laminate and the rough surface laminate could be distinguished by the nature of the two outer surfaces produced in tissue deposition. In the smooth surface laminate, where the rough surface of the compression control faced outward, the tissue cross-section showed a smooth surface and a rough surface, as Figure 40 shown. In contrast, in the rough surface laminate, the rough surface of the compression control faced inward, and the two smooth outer surfaces in the histological cross-section were as Figure 42 shown. In the autoclaved laminate, the two smooth surfaces were similar to the rough surface laminate in Figure 43 , however, a denser collagen layer was seen on one surface, which might be due to differences in the Vicryl properties after autoclaving, which might cause a certain amount of hydrolytic degradation before inoculation. In Fibertex, a slightly different tissue deposition compared to the compression control was observed. Where there were torn voids in the histological cross-section, some discontinuous tissue formation could be seen, however, relatively continuous tissue deposition existed outside these tears, as Figure 44 In Fibertex, a slightly different tissue deposition compared to the compression control was observed. Where there were torn voids in the histological cross-section, some discontinuous tissue formation could be seen, however, relatively continuous tissue deposition existed outside these tears, as Figure 45As shown. In the sandwich structure from Thinsulate, a slightly thicker tissue cross-section can be seen, but tissue deposition is relatively continuous throughout the thickness. Although the fiber webs on the upper and lower surfaces of the spunbond layer are denser, good tissue penetration through the surface can be seen, and there does not seem to be an impermeable layer in Figure 46 it. Finally, very dense tissue formation was observed in the absorbable tissue, but not in any other tissue, as Figure 47 shown. Different from other materials, this material starts out very thin, has very high cell affinity, and dissolves at the end of the culture. All these factors may contribute to the high tissue density observed through histological and biochemical data. For help with interpretation, see Figure 11 to better understand the anatomy of the histological images.

[0205] Through this experiment, various shape factors were tested, including needled nonwovens, 3-layer sandwiches, mesh and nonwoven laminates, terry cotton fabrics, spacer fabrics, and plain knitted meshes. Other shape factors have been tested, such as the woven mesh shown in Example 1. Different effects of these shape factors on tissue formation can be seen. Various effects, such as double-layer stacked hollow tissue, and incredibly dense tissue in the case of only absorbable, and tissue continuity in the towel-like fabric despite the lack of fibers, can be used to achieve the desired tissue properties. The data indicate that the shape factors may have an impact on the tissue effect.

[0206] Example 12. Influence of Scaffold Attachment Factors on Leather Effect

[0207] The modification of the surface of poly(lactic acid) (PLA) and PLA scaffolds was tested to evaluate the efficacy of covalent and non-covalent attachment factors on the surface. The poly(lactic acid) (PLA) scaffolds were coated with glycidyltrimethylammonium chloride (GTAMC), which enabled the introduction of amines (PLA-NH2) on the polymer surface, which can non-specifically adsorb to collagen. The PLA scaffolds were coated with pea protein and hydrolyzed under acidic conditions to expose hydroxyl, carboxylic acid, and ketone (PLA-hyd), which can then non-specifically adsorb to collagen. The PLA scaffolds were coated with pea protein and also oxidized using sodium periodate (PLA-ox) to increase the non-specific adsorption of collagen. For each of the three scaffolds and the PLA control, fibroblasts were inoculated and allowed to grow to produce an extracellular matrix, which includes collagen. The scaffolds were optionally removed by dissolving in benzylamine, ethyl acetate, or acetone. For example, the resulting materials were examined histologically and microscopically to determine the density and distribution of collagen resulting from the above scaffold modifications. The resulting materials can be tanned to produce cruelty-free leather and the raw hide thickness and strength can be compared.

[0208] In another embodiment, a polylactic acid (PLA) scaffold is coated with a chemically modified polymeric material. In one embodiment, the polymeric material coating the PLA is further modified by adding a carbodiimide linker EDC (PLA-EDC). Fibroblasts are seeded onto the PLA-EDC and allowed to grow such that an extracellular matrix containing collagen is formed. The collagen is modified with N-hydroxysuccinimide. The collagen and the carbodiimide linker of the PLA-EDC are chemically crosslinked to produce a covalently bound scaffold-polymeric material. In another embodiment, the polymeric material coating the PLA is further modified by the addition of azide (-N3) (PLA-N3). Fibroblasts are seeded onto the PLA-N3 and allowed to grow, producing an extracellular matrix containing collagen. Then, the collagen is modified with an alkyne (which crosslinks with the azide in a click chemical reaction) to produce a covalently crosslinked scaffold-collagen material. The PLA-N3-collagen and PLA-EDC-collagen materials are then optionally decellularized prior to examination and compared to the PLA control by histology and microscopy to examine collagen density and distribution. The resulting material can also optionally be tanned to produce cruelty-free leather, and the raw hide thickness and strength can be compared to the PLA control.

[0209] Example 13. Leather Strength and Thickness

[0210] The average double tear strength and thickness of raw hides prepared with different scaffolds were measured and compared to the average double tear strength and thickness of a typical cow hide crust. The average double tear strength was measured using the ISO 3377-2 method. The measurement results are shown in Table 3.

[0211] Table 3

[0212] Average double tear strength (N) Thickness (mm) Tanned raw hide w / PLA scaffold 3 0.4-0.6 Tanned raw hide w / rayon scaffold 19 0.4-0.6 Tanned raw hide w / PET scaffold 31 0.4-0.6 Typical cowhide outer skin 20-25 1.0-1.2

[0213] The resulting double tear strength and thickness can indicate that the compositions provided herein comprise a scaffold in contact with an extracellular matrix, and that the methods provided herein can produce scaffolds whose strength can be adjusted by simply modifying the scaffold material. Additionally, the data indicate that when compared to cow hide crust, the compositions provided herein can comprise a strength similar to natural leather, but the material is thinner.

[0214] While the preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Many variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. The following claims are intended to define the scope of the invention and cover the methods and structures within the scope of these claims and their equivalents.

Claims

1. A composition comprising a scaffold in contact with an extracellular matrix comprising collagen, wherein the scaffold comprises a non-woven needled material.

2. A composition comprising a scaffold in contact with an extracellular matrix comprising collagen, wherein the scaffold comprises a three-dimensional woven material.

3. A composition comprising a scaffold in contact with an extracellular matrix comprising collagen, wherein the scaffold comprises polycaprolactone (PCL), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polyethylene terephthalate (PET), nylon, polyethylene (PE), polyethylene furanoate (PEF), polypropylene (PP), polyvinyl alcohol (PVA), cotton, bast fiber, viscose, modal, lyocell, plant-based protein fiber, bio-based material, viscose, cellulose, alginate fiber, thermoplastic starch, or any combination thereof.

4. A composition comprising a scaffold that is at least partially coated, the scaffold being in contact with an extracellular matrix comprising collagen, wherein the scaffold is at least partially coated with a coating comprising: Matrigel, vitronectin, fibronectin, a protein extracted from soybeans, a protein extracted from peas, a protein extracted from corn, a synthetically produced peptide, an RNA-binding glycine-rich (RBG) protein, polylysine, a synthetic protein, an RGD peptide, polylysine, polyarginine, polyornithine, a recombinant protein, an oligomer, a polymer, or any combination thereof.

5. A composition comprising a dissolvable scaffold in contact with an extracellular matrix comprising collagen, wherein the scaffold is in contact with a solvent that can dissolve the scaffold.

6. The composition according to any one of claims 1, 2, 4, or 5, wherein the scaffold comprises polycaprolactone (PCL), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polyethylene terephthalate (PET), nylon, polyethylene (PE), polyethylene furanoate (PEF), polypropylene (PP), polyvinyl alcohol (PVA), polyhydroxyalkanoate (PHA), polyhydroxybutyrate (PHB), oxidized alginate, cotton, bast fiber, viscose, modal, lyocell, plant-based protein fiber, bio-based material, viscose, cellulose, alginate fiber, thermoplastic starch, or any combination thereof.

7. The composition according to any one of claims 2-6, wherein the scaffold comprises a non-woven needled material.

8. The composition according to claim 1 or claim 7, wherein the non-woven needled material comprises a first porosity.

9. The composition according to claim 8, wherein the scaffold comprises a non-woven structure that produces the first porosity.

10. The composition according to claim 9, wherein the first porosity comprises an average pore size of about 30-70 μm.

11. The composition according to claim 9, wherein the first porosity comprises an average pore size of about 80-120 μm.

12. The composition according to claim 9, wherein the first porosity comprises an average pore size of about 50 μm or about 100 μm.

13. The composition according to any one of claims 8-12, wherein the scaffold comprises a second porosity produced by needling.

14. The composition according to any one of claims 1-3 or 5, wherein the scaffold is at least partially coated with a coating comprising: matrix glue, vitronectin, fibronectin, proteins extracted from soybeans, proteins extracted from peas, proteins extracted from corn, synthetically produced peptides, RNA-binding glycine-rich (RBG) proteins, polylysine, synthetic proteins, RGD peptides, polylysine, polyarginine, polyornithine, recombinant proteins, oligomers, polymers, glycidyl trimethyl ammonium chloride (GTMAC), carbohydrate-binding modules, cellulose-binding domains, starch-binding domains, or combinations thereof.

15. The composition according to claim 4 or 14, wherein the scaffold comprises amine hydroxyl groups, thiol groups, tyrosyl groups, or carboxylic acid groups.

16. The composition according to any one of claims 4, 14, or 15, wherein the scaffold is at least partially coated with the carbohydrate-binding module.

17. The composition according to any one of claims 14-16, wherein the carbohydrate-binding module is a cellulose-binding domain.

18. The composition according to claim 17, wherein the scaffold comprises cellulose.

19. The composition according to claim 16, wherein the carbohydrate-binding module is a starch-binding module.

20. The composition according to claim 19, wherein the scaffold comprises starch.

21. The composition according to any one of claims 16-20, wherein the carbohydrate-binding module is associated with an enzyme.

22. The composition according to claim 21, wherein the enzyme does not hydrolyze the scaffold.

23. The composition according to claim 4 or any one of claims 14-22, wherein the coating comprises a modification.

24. The composition according to claim 23, wherein the modification comprises a reduction modification, an addition modification, or a combination thereof.

25. The composition according to claim 23, wherein the modification of the scaffold increases the strength of non-specific adsorption of the collagen to the scaffold.

26. The composition according to claim 23, wherein the modification comprises hydrolysis.

27. The composition according to claim 26, wherein the modification exposes chemical reactive groups comprising: hydroxyl groups, carboxylic acids, ketones, or combinations thereof.

28. The composition according to claim 23, wherein the modification comprises oxidation.

29. The composition according to claim 28, wherein the oxidation is carried out with sodium periodate.

30. The composition according to any one of claims 23-29, wherein the scaffold comprises amines, carboxylic acids, sulfates, aldehydes, hydrazides, thiol groups, aziridines, aryl-azides, acrylates, or epoxides.

31. The composition according to any one of claims 14-30, wherein the scaffold is at least partially coated with a coating comprising GTMAC.

32. The composition according to claim 31, wherein the scaffold comprises primary amines.

33. The composition according to claim 31 or 32, wherein at least partially coating with a coating comprising GTMAC increases the surface charge of the scaffold.

34. The composition according to any one of claims 1-33, wherein the collagen is associated with the scaffold.

35. The composition according to any one of claims 1 - 34, wherein the collagen associates with the scaffold by non - specific adsorption.

36. The composition according to any one of claims 1 - 35, wherein the collagen associates with the scaffold by van der Waals interactions.

37. The composition according to any one of claims 1 - 36, wherein the collagen associates with the scaffold by hydrogen bonding.

38. The composition according to any one of claims 1 - 37, wherein the collagen associates with the scaffold by depletion interaction.

39. The composition according to any one of claims 1 - 38, wherein the collagen associates with the scaffold by electrostatic interaction.

40. The composition according to any one of claims 1 - 34, wherein the collagen associates with the scaffold by covalent interaction.

41. The composition according to any one of claims 1 - 40, wherein the scaffold comprises carbodiimide or N - hydroxysuccinimide ester (NHS ester).

42. The composition according to any one of claims 1 - 41, wherein the collagen comprises carbodiimide or N - hydroxysuccinimide ester (NHS ester).

43. The composition according to claim 41 or 42, wherein the carbodiimide is N,N′ - dicyclohexylcarbodiimide (DCC) or 1 - ethyl - 3 - (3 - dimethylaminopropyl)carbodiimide (EDC).

44. The composition according to any one of claims 41 - 43, wherein the collagen associates with the scaffold by EDC / NHS coupling.

45. The composition according to any one of claims 1 - 34, wherein the scaffold comprises azide or alkyne.

46. The composition according to any one of claims 1 - 34 or 45, wherein the collagen comprises azide or alkyne.

47. The composition according to any one of claims 45 or 46, wherein the collagen associates with the scaffold by click chemistry reaction.

48. The composition according to any one of claims 1 - 34, wherein the scaffold comprises a Michael donor or a Michael acceptor.

49. The composition according to any one of claims 1 - 34 or 48, wherein the collagen comprises a Michael donor or a Michael acceptor.

50. The composition according to any one of claims 48 or 49, wherein the collagen associates with the scaffold by the coupling of the Michael donor and the Michael acceptor.

51. The composition according to any one of claims 48 - 50, wherein the Michael donor includes enolate.

52. The composition according to any one of claims 48 - 51, wherein the Michael acceptor contains α,β - unsaturated carbonyl.

53. The composition according to any one of claims 1 - 34, wherein the scaffold comprises thiol or maleimide.

54. The composition according to any one of claims 1 - 34 or 53, wherein the collagen comprises thiol or maleimide.

55. The composition according to any one of claims 53 or 54, wherein the collagen associates with the scaffold by the coupling of thiol and maleimide.

56. The composition according to any one of claims 53 - 55, wherein the collagen associates with the scaffold in the presence of (sulfosuccinimidyl 4-(N - maleimidomethyl)cyclohexane - 1 - carboxylate) (sulfo - SMCC), and wherein the scaffold and the collagen contain thiols.

57. The composition according to any one of claims 1 - 34, wherein the collagen associates with the scaffold through the Maillard reaction.

58. The composition according to any one of claims 1 - 4 or 6 - 57, wherein the scaffold comprises a soluble scaffold, and wherein the scaffold is contacted with a solvent capable of dissolving the scaffold.

59. The composition according to claim 5 or 58, wherein the temperature of the solvent is lower than the boiling point of the solvent.

60. The composition according to claim 58 or 59, wherein the solvent is at a temperature of about 40 °C to about 50 °C.

61. The composition according to claim 58 or 59, wherein the scaffold comprises a thermoplastic polymer.

62. The composition according to claim 61, wherein the thermoplastic polymer comprises polyvinyl alcohol (PVA).

63. The composition according to claim 61, wherein the thermoplastic polymer comprises polyvinyl alcohol (PVA) or polyvinyl alcohol (PVOH).

64. The composition according to claim 61, wherein the thermoplastic polymer comprises polylactic acid (PLA).

65. The composition according to any one of claims 58 - 64, wherein the solvent comprises water.

66. The composition according to any one of claims 58 - 64, wherein the solvent comprises an organic solvent.

67. The composition according to claim 66, wherein the organic solvent comprises acetone, benzylamine or ethyl acetate.

68. The composition according to claim 67, wherein the solvent comprises benzylamine.

69. The composition according to claim 66, wherein the scaffold comprises polylactic acid (PLA), and wherein the solvent comprises benzyl, ethyl, haloalkane or a combination thereof.

70. The composition according to any one of claims 58 - 68, wherein the scaffold dissolves over time by hydrolysis degradation.

71. The composition according to any one of claims 58 - 70, wherein the scaffold degrades over time by hydrolysis degradation in a neutral aqueous solution.

72. The composition according to any one of claims 58 - 71, wherein the solvent comprises a solubilizing agent.

73. The composition according to claim 72, wherein the solubilizing agent comprises ethylenediaminetetraacetic acid (EDTA).

74. The composition according to claim 73, wherein the scaffold comprises alginate.

75. The composition according to claim 72, wherein the solubilizing agent comprises a strong acid.

76. The composition according to claim 75, wherein the strong acid comprises hydrochloric acid, nitric acid, hydroiodic acid, perchloric acid, chloric acid or a combination thereof.

77. The composition according to claim 72, wherein the solubilizing agent comprises a strong base.

78. The composition according to claim 77, wherein the strong base comprises lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, strontium hydroxide, barium hydroxide or a combination thereof.

79. The composition according to claim 72, wherein the solvent comprises an oxidizing agent.

80. The composition according to claim 79, wherein the oxidizing agent partially or completely degrades the scaffold.

81. The composition according to claim 79 or 80, wherein the oxidizing agent is sodium periodate.

82. The composition according to any one of claims 58 - 71, wherein the scaffold comprises a bio-based material, and the solvent comprises an enzyme that degrades the bio-based material.

83. The composition according to claim 82, wherein the solvent comprises cellulase.

84. The composition according to claim 82 or 83, wherein the scaffold comprises cellulose, and the solvent comprises cellulase.

85. The composition according to claim 82, wherein the scaffold comprises an ester-containing polymer.

86. The composition according to claim 82 or 85, wherein the solvent comprises esterase.

87. The composition according to any one of claims 82 - 86, wherein the solvent comprises lipase.

88. The composition according to claim 82, wherein the scaffold comprises calcium alginate.

89. The composition according to claim 82 or 88, wherein the solvent comprises alginate lyase.

90. The composition according to claim 1 or any one of claims 3 - 89, wherein the scaffold comprises a three-dimensional woven material.

91. The composition according to claim 2 or claim 90, wherein the three-dimensional woven material comprises spacer fabric.

92. The composition according to claim 91, wherein the spacer fabric includes a front surface in contact with the filler and a back surface in contact with the filler, wherein the filler separates the front surface from the back surface.

93. The composition according to claim 92, wherein a gap is created by separating the front surface from the back surface by the filler to allow entry of nutrients, removal of waste, cell attachment, cell growth, or any combination thereof.

94. The composition according to claim 2 or any one of claims 90 - 93, wherein the three-dimensional woven material comprises a pile fabric.

95. The composition according to claim 2 or any one of claims 90 - 94, wherein the three-dimensional woven material includes terry cotton fabric, napped tweed, velvet, corduroy, velveteen, or any combination thereof.

96. The composition according to any one of claims 1 - 95, wherein the scaffold comprises a multi-layer scaffold material.

97. The composition according to claim 96, wherein the multi-layer scaffold material comprises a combination of different shape factors.

98. The composition according to claim 97, wherein the different shape factors comprise non-woven material, woven material, needled material, three-dimensional structure, or any combination thereof.

99. The composition according to any one of claims 96 - 98, wherein the scaffold comprises a three-layer composite material, wherein the three-layer composite material comprises two outer layers and one inner layer.

100. The composition according to claim 99, wherein the two outer layers comprise surface layer characteristics, and the inner layer comprises bulk properties.

101. The composition according to any one of claims 96 - 100, wherein the multi-layer comprises multi-layer thin materials.

102. The composition according to any one of claims 96 - 101, wherein the layers are fused together, held together by entanglement, laminated together, stitched together, adhered together, woven together, printed together, or any combination thereof.

103. The composition according to any one of claims 1 - 102, wherein the composition further comprises isolated animal cells in contact with the scaffold.

104. The composition according to claim 103, wherein the isolated animal cells are isolated animal fibroblasts or fibroblast - like cells.

105. The composition according to any one of claims 103 - 104, wherein the isolated animal cells are immortalized isolated animal cells.

106. The composition according to claim 105, wherein the immortalized isolated animal cells can grow beyond the Hayflick limit.

107. The composition according to claim 105 or claim 106, wherein the immortalized isolated animal cells can grow through about 40 cell divisions, about 50 cell divisions, or about 60 cell divisions.

108. The composition according to any one of claims 103 - 107, wherein the isolated animal cells are bovine or porcine cells.

109. The composition according to any one of claims 103 - 107, wherein the isolated animal cells are human cells.

110. The composition according to any one of claims 1 - 109, wherein the extracellular matrix is produced by isolated animal fibroblasts or fibroblast - like cells.

111. The composition according to any one of claims 1 - 110, wherein the composition is at least partially decellularized.

112. The composition according to claim 111, wherein at least partial decellularization comprises substantially no intact cells in the composition.

113. The composition according to any one of claims 1 - 112, wherein the scaffold further comprises polyglycolic acid (PGA), polybutylene succinate (PBS), a bioabsorbable synthetic polymer, cellulose, cellulose acetate, acrylic, fiber, linen, rayon, velvet, modified acrylic, olefin polyester, saran, vinylon, wool, jute, hemp, bamboo, linen fabric, or any combination thereof.

114. The composition according to any one of claims 1 - 113, wherein the scaffold further comprises bio - based nylon, bio - based PET, bio - based PEF, bio - based polylactic acid (PLA), or any combination thereof.

115. The composition according to any one of claims 1 - 114, wherein the scaffold comprises nylon 1,6, nylon 4,6, nylon 510, nylon 5,6, nylon 5,12, nylon 6, nylon 6,6, nylon 11, nylon 10.10, nylon 12, or any combination thereof.

116. The composition according to any one of claims 1 - 115, wherein the scaffold comprises bast fibers, and the bast fibers comprise flax, hemp, linen fabric, jute, ramie, kenaf, sisal, or any combination thereof.

117. The composition according to any one of claims 1-116, wherein the scaffold has a thickness of about 0.1 mm to about 4 mm.

118. The composition according to any one of claims 1-116, wherein the scaffold has a thickness of about 1 mm to about 3 mm.

119. The composition according to any one of claims 1-116, wherein the scaffold has a thickness of about 1 mm.

120. The composition according to any one of claims 1-116, wherein the scaffold has a thickness of about 2 mm.

121. The composition according to any one of claims 1-116, wherein the scaffold has a thickness of about 3 mm.

122. The composition according to any one of claims 1-121, wherein the scaffold comprises fibers of about 6.7 dtex.

123. The composition according to any one of claims 1-122, wherein the scaffold comprises fibers having a diameter of about 1 μm to about 100 μm.

124. A method for preparing the composition according to any one of claims 1-123, which comprises inoculating isolated animal fibroblasts or fibroblast-like cells onto the scaffold to form the composition.

125. A method for preparing the composition according to any one of claims 1-123, wherein the scaffold comprises a thermoplastic polymer, and the thermoplastic polymer is subsequently substantially removed from the extracellular matrix before tanning.

126. The method according to claim 125, wherein the thermoplastic polymer comprises polyvinyl alcohol (PVA), and the method comprises substantially removing the PVA by contacting the PVA with water at a temperature of about 18 °C to about 90 °C before tanning.

127. The method according to claim 125, wherein the thermoplastic polymer comprises polylactic acid (PLA), and the method comprises removing the PLA by contacting the PLA with a solvent before tanning, thereby substantially removing the PLA.

128. A method for preparing the composition according to any one of claims 1-123, wherein the scaffold comprises a dissolvable scaffold, and the dissolvable scaffold is removed by contacting the scaffold with a solvent.

129. A method for preparing the composition according to any one of claims 1-123, which comprises needling the scaffold using a knitting machine to entangle the fibers in the nonwoven scaffold material.

130. The method according to claim 129, wherein the knitting machine comprises knitting needles with barbs.

131. The method according to claim 129, wherein the needling produces pores in the scaffold material.

132. A method for preparing the composition according to any one of claims 1-123, which comprises at least partially coating the scaffold with a coating comprising a matrix glue, vitronectin, fibronectin, a protein extracted from soybeans, a protein extracted from peas, a protein extracted from corn, a synthetically produced peptide, an RNA-binding glycine-rich (RBG) protein, a synthetic protein, an RGD peptide, polylysine, polyarginine, polyornithine, a recombinant protein, an oligomer, a polymer, GTMAC, a carbohydrate-binding module, a cellulose-binding domain, a starch-binding domain, or a combination thereof.

133. The method according to claim 132, which further comprises inoculating isolated animal fibroblasts or fibroblast-like cells onto the scaffold to form the composition.

134. A method which comprises transplanting the composition according to any one of claims 1-123 into a patient in need of a skin graft for treating skin loss or injury.

135. The method according to claim 134, wherein the patient has a laceration, a contusion, an injury, a sore, a burn, a wound, a surgical wound, surgically removed skin, necrotic skin, or any combination thereof.

136. A method which comprises tanning the composition according to any one of claims 1-123 to produce cruelty-free leather.

137. Cruelty-free leather produced by tanning the composition according to any one of claims 1-123.

138. A method which comprises: a) inoculating isolated animal fibroblasts or fibroblast-like cells onto a scaffold to form a cell layer on the scaffold, wherein the scaffold comprises polycaprolactone (PCL), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polyethylene terephthalate (PET), nylon, polyethylene (PE), polyethylene furanoate (PEF), polypropylene (PP), polyvinyl alcohol (PVA), cotton, bast fiber, viscose, modal, lyocell, plant-based protein fiber, alginate fiber, thermoplastic starch, or any combination thereof; b) growing the cells to produce a composition comprising an extracellular matrix; and c) tanning the composition comprising the extracellular matrix to form cruelty-free leather.

139. A method which uses the cruelty-free leather according to any one of claims 136-137 as a substitute for traditional leather in leather products.

140. The method according to claim 139, wherein the leather product comprises a watchband, a belt, a harness, a packaging, shoes, boots, footwear, gloves, clothes, bags, handbags, wallets, coin purses, billfolds, key cases, credit card holders, pen cases, backpacks, boxes, money clips, saddles, harnesses, whips, luggage, travel supplies, canvas backpacks, briefcases, document bags, attache cases, business cases, pet supplies, leather cases, collars, hunting and fishing supplies, gun cases, cutlery cases, holsters, stationery, writing pads, book covers, camera cases, glasses cases, cigarette cases, cigar cases, jewelry boxes, phone cases, sports supplies, balls, basketballs, footballs, rugby balls, or any combination thereof.

141. A leather product which comprises the cruelty-free leather according to any one of claims 136-137.

142. The leather product according to claim 141, which is included in the following: watch strap, belt, shoulder strap, packaging, shoes, boots, footwear, gloves, clothing, bag, handbag, wallet, coin purse, billfold, key case, credit card holder, pen case, backpack, box, money clip, saddle, harness, whip, trunk, travel supplies, canvas backpack, briefcase, document case, attache case, business case, pet supplies, leather case, collar, hunting and fishing supplies, gun case, cutlery case, holster, stationery, writing pad, book cover, camera case, glasses case, cigarette case, cigar case, jewelry box, mobile phone case, sports supplies, ball, basketball, football, rugby or any combination thereof cutlery case.

143. The method according to claim 140 or the leather product according to claim 142, wherein the clothing includes upper garments, lower garments, outer garments or any combination thereof.

144. The method according to claim 140 or the leather product according to claim 142, wherein the bag includes a handbag with or without a shoulder strap.

145. The method according to claim 140 or the leather product according to claim 142, wherein the trunk includes a large suitcase, carry-on suitcase, travel bag, cosmetic case, toiletry bag or any combination thereof.

146. A method of treating a disease or disorder using the composition according to any one of claims 1-123.

147. A kit comprising the composition according to any one of claims 1-123 or the leather product according to any one of claims 139-145.

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