Scaffold material for artificial dermis layer

Abstract

Disclosed herein are compositions for use as scaffolds for artificial dermal layers. Disclosed herein are methods for the manufacture and processing of scaffolds for artificial dermal layers. Disclosed herein are scaffolds for use in supporting immortalized fibroblasts.

Description

Detailed Description of the Invention

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 400,704, filed August 24, 2022, and U.S. Provisional Patent Application No. 63 / 515,982, filed July 27, 2023, each of which is incorporated by reference in its entirety.

[0002] [overview] Disclosed herein in some embodiments are compositions comprising a scaffold in contact with an extracellular matrix comprising collagen, wherein the scaffold can comprise a needle-punched nonwoven material. Disclosed herein in some embodiments are compositions comprising a scaffold in contact with an extracellular matrix comprising collagen, wherein the scaffold can comprise a three-dimensional woven material. Disclosed herein in some embodiments are compositions comprising a scaffold in contact with an extracellular matrix comprising collagen, wherein the scaffold can comprise polycaprolactone (PCL), polylactic acid (PLA), polylactic and polyglycolic acid (PLGA), polyethylene terephthalate (PET), nylon, polyethylene (PE), polyethylene furanoate (PEF), polypropylene (PP), polyvinyl alcohol (PVA), cotton, bast fiber, viscose, modal, lyocell, plant protein fiber, bio-based material, viscose, cellulose, alginate fiber, thermoplastic starch, or any combination thereof. In some embodiments, disclosed herein are compositions comprising an at least partially coated scaffold in contact with an extracellular matrix comprising collagen, wherein the scaffold is at least partially coated with a coating comprising Matrigel, vitronectin, fibronectin, soy protein extract, pea protein extract, corn protein extract, synthetically produced peptide, RNA-binding glycine-rich (RBG) protein, polylysine, synthetic protein, RGD peptide, polylysine, polyarginine, polyornithine, recombinant protein, oligomer, polymer, or any combination thereof. In some embodiments, disclosed herein are compositions comprising a dissolvable scaffold in contact with an extracellular matrix comprising collagen, wherein the scaffold is in contact with a solvent capable of dissolving the scaffold.In some embodiments, the scaffold may comprise polycaprolactone (PCL), polylactic acid (PLA), polylactic-polyglycolic acid (PLGA), polyethylene terephthalate (PET), nylon, polyethylene (PE), polyethylene furanoate (PEF), polypropylene (PP), polyvinyl alcohol (PVA), cotton, bast fiber, viscose, modal, lyocell, plant protein fiber, bio-based material, viscose, cellulose, alginate fiber, thermoplastic starch, or any combination thereof. In some embodiments, the scaffold may comprise a nonwoven fabric capable of producing a first plurality of pores. In some embodiments, the first plurality of pores may comprise an average pore size of about 30-70 μm. In some embodiments, the first plurality of pores may comprise an average pore size of about 80-120 μm. In some embodiments, the first plurality of pores may comprise an average pore size of about 50 μm or about 100 μm. In some embodiments, the scaffold may include a second plurality of pores created by needle punching. In some embodiments, the scaffold may be at least partially coated with a coating comprising Matrigel, vitronectin, fibronectin, soy protein extract, pea protein extract, corn protein extract, synthetically produced peptide, RNA-binding glycine-rich (RBG) protein, polylysine, synthetic protein, RGD peptide, polylysine, polyarginine, polyornithine, recombinant protein, oligomer, polymer, or any combination thereof. In some embodiments, the scaffold is at least partially coated with a coating comprising Matrigel, vitronectin, fibronectin, soy protein extract, pea protein extract, corn protein extract, synthetically produced peptide, RNA-binding glycine-rich (RBG) protein, polylysine, synthetic protein, RGD peptide, polylysine, polyarginine, polyornithine, recombinant protein, oligomer, polymer, GTMAC, carbohydrate-binding module, cellulose-binding domain, starch-binding domain, or combinations 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 can comprise a modification. In some embodiments, the modification can comprise a reductive modification, an additive modification, or a combination thereof. In some embodiments, the modification comprises hydrolysis. In some embodiments, the modification exposes chemically active groups including hydroxyls, carboxylic acids, ketones, or combinations thereof. In some embodiments, the modification comprises oxidation. In some embodiments, the oxidation occurs with sodium periodate. In some embodiments, the scaffold comprises amines, carboxylic acids, sulfates, aldehydes, hydrazides, sulfhydryls, diazirines, aryl azides, acrylates, and epoxides. 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 via non-specific adsorption. In some embodiments, the collagen is associated with the scaffold via van der Waals interactions. In some embodiments, the collagen is associated with the scaffold via hydrogen bonding. In some embodiments, the collagen is associated with the scaffold via depletion interactions. In some embodiments, the collagen is associated with the scaffold via electrostatic interactions.In some embodiments, modifying the scaffold increases the strength of nonspecific adsorption of collagen to the scaffold. In some embodiments, the collagen is coupled to the scaffold via a covalent interaction. In some embodiments, the scaffold comprises a carbodiimide or an N-hydroxysuccinimide ester (NHS ester). In some embodiments, the collagen comprises a carbodiimide or an 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 is coupled to the scaffold via 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 is coupled to the scaffold via a 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 is associated with the scaffold via 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 a thiol or maleimide. In some embodiments, the collagen comprises a thiol or maleimide. In some embodiments, the collagen is associated with the scaffold via coupling of the thiol and maleimide. In some embodiments, the collagen is associated with the scaffold in the presence of (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (sulfo-SMCC), wherein the scaffold and collagen comprise a thiol. In some embodiments, the collagen is associated with the scaffold via a Maillard reaction.

[0003] In some embodiments, the compositions provided herein comprise a scaffold. In some embodiments, the scaffold disclosed herein may be a dissolvable scaffold, wherein the scaffold may be contacted with a solvent capable of dissolving the scaffold. In some embodiments, the temperature of the solvent is below the boiling point of the solvent. In some embodiments, the solvent may be at a temperature of about 40°C to about 50°C. In some embodiments, the scaffold may comprise a thermoplastic polymer. In some embodiments, the thermoplastic polymer may comprise polyvinyl alcohol (PVA) and the solvent may 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 may comprise polylactic acid (PLA) and the solvent may comprise a benzyl group, an ethyl group, a haloalkane, or a combination thereof. In some embodiments, the solvent may comprise benzylamine. In some embodiments, the scaffold dissolves over time via hydrolysis. In some embodiments, the scaffold degrades over time via 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 dissolution agent. In some embodiments, the dissolution agent comprises ethylenediaminetetraacetic acid (EDTA). In some embodiments, the scaffold comprises alginate. In some embodiments, the dissolution agent comprises a strong acid. In some embodiments, the strong acid comprises hydrochloric acid, nitric acid, hydroiodic acid, perchloric acid, chloric acid, or a combination thereof. In some embodiments, the dissolution agent comprises a strong base. In some embodiments, the strong base comprises lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, strontium hydroxide, barium hydroxide, or a combination thereof.In some embodiments, the dissolution agent comprises an oxidizing agent, which partially or completely degrades the scaffold, in some embodiments, the oxidizing agent is sodium periodate.

[0004] In some embodiments, the compositions provided herein comprise a scaffold. In some embodiments, the scaffolds disclosed herein may comprise a bio-based material and the solvent may comprise an enzyme that degrades the bio-based material. In some embodiments, the scaffold may comprise cellulose and the solvent may 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 alginate lyase.

[0005] 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 include a front surface in contact with a filler and a back surface in contact with a filler, the filler separating the front surface from the back surface. In some embodiments, separation of the front surface from the back surface by the filler may create a gap that allows for nutrient ingress, waste removal, cell adhesion, 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 comprise terry, frieze, velvet, corduroy, velveteen, or any combination thereof. In some embodiments, the scaffold may comprise multiple layers of scaffold material. In some embodiments, the multiple layers of scaffold material may comprise a combination of different form factors. In some embodiments, the different form factors may include nonwoven materials, woven materials, needle-punched materials, three-dimensional structures, or any combination thereof. In some embodiments, the scaffold may include a three-layer composite material, where the three-layer composite material may include two outer layers and one inner layer. In some embodiments, the two outer layers may include surface layer properties and the inner layer may include bulk properties. In some embodiments, the multiple layers may include multiple layers of thin material. In some embodiments, the multiple layers may be fused, held together by intertwining, laminated together, sutured together, glued together, woven together, baked together, or any combination thereof. In some embodiments, the compositions disclosed herein may further include 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 isolated animal fibroblasts or fibroblast-like cells.In some embodiments, the isolated animal cells may be isolated immortalized animal cells. In some embodiments, the isolated immortalized animal cells may be propagated beyond the Hayflick limit. In some embodiments, the isolated immortalized animal cells may be propagated 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 comprise human cells. In some embodiments, the compositions disclosed herein may be at least partially decellularized. In some embodiments, being at least partially decellularized may comprise a substantial absence of intact cells in the composition. In some embodiments, the scaffold may further comprise polyglycolic acid (PGA), polybutylene succinate (PBS), a bioabsorbable synthetic polymer, cellulose, acetate, acrylic, fiber, linen, rayon, velvet, modacrylic, olefin polyester, saran, vinyon, 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 comprise bast fibers, which may comprise flax, hemp, linen, jute, ramie, kenaf, sisal, or any combination thereof. In some embodiments, the scaffold may comprise a thickness of about 0.1 mm to about 4 mm. In some embodiments, the scaffold may comprise a thickness of about 1 mm to about 3 mm. In some embodiments, the scaffold may comprise a thickness of about 1 mm. In some embodiments, the scaffold may comprise a thickness of about 2 mm. In some embodiments, the scaffold may comprise a thickness of about 3 mm. In some embodiments, the scaffold may comprise fibers comprising a dtex of about 6.7 dtex.In some embodiments herein, the scaffold may comprise fibers comprising a diameter of about 1 μm to about 100 μm. In some embodiments, a method of making a composition disclosed herein is disclosed, comprising seeding isolated animal fibroblasts or fibroblast-like cells onto a scaffold to form a composition. In some embodiments, the scaffold may comprise a thermoplastic polymer that can be subsequently 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 comprise substantially removing the PVA prior to tanning by contacting the PVA with water having a temperature of about 18°C ​​to about 90°C. In some embodiments, the thermoplastic polymer may comprise polylactic acid (PLA), and the methods disclosed herein may comprise substantially removing the PLA prior to tanning by contacting the PLA with a solvent to remove the PLA. In some embodiments, the solvent may comprise a benzyl group, an ethyl group, a haloalkane, a benzylamine, or any combination thereof. In some embodiments, the scaffold may comprise a dissolvable scaffold, which may be substantially removed by contacting the scaffold with a solvent. In some embodiments, the method of manufacturing the scaffold may include needle-punching the scaffold using a needle loom to entangle fibers into a nonwoven scaffold material. In some embodiments, the needle loom may include barbed needles. In some embodiments, the needle-punching may create pores in the scaffold material. In some embodiments, the method of manufacturing the scaffold may include at least partially coating the scaffold with a coating comprising Matrigel, vitronectin, fibronectin, a protein extract from soybean, a protein extract from pea, a protein extract 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 a combination thereof.

[0006] Disclosed herein in some embodiments is a method of making any of the compositions provided herein, comprising at least partially coating a scaffold with a coating comprising Matrigel, vitronectin, fibronectin, soy protein extract, pea protein extract, corn protein extract, synthetically produced peptide, RNA-binding glycine-rich (RBG) protein, synthetic protein, RGD peptide, polylysine, polyarginine, polyornithine, recombinant protein, oligomer, polymer, GTAMC, carbohydrate-binding module, cellulose-binding domain, starch-binding domain, or a combination thereof. In some embodiments, the scaffold is at least partially coated with GTAMC to provide primary amines on the scaffold. In some embodiments, the carbohydrate-binding module is coupled to 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 comprises the addition of amines, carboxylic acids, sulfates, aldehydes, hydrazides, sulfhydryls, diazirines, aryl azides, acrylates, and epoxides. In some embodiments, the modification comprises hydrolysis, oxidation, enzymatic degradation, acidification, or basification. In some embodiments, the modification exposes one or more active groups comprising hydroxyl, carboxyl, ketone, or a combination thereof. In some embodiments, the method further comprises seeding isolated animal fibroblasts or fibroblast-like cells onto the scaffold to form the 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 via specific adsorption. In some embodiments, specific adsorption comprises an interaction with the 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 via non-specific adsorption. In some embodiments, the collagen is associated with the scaffold via non-specific adsorption. In some embodiments, the isolated animal fibroblasts and / or fibroblast-like cells are associated with the scaffold via non-specific adsorption. In some embodiments, the non-specific adsorption comprises van der Waals interactions. In some embodiments, the non-specific adsorption comprises hydrogen bonding. In some embodiments, the non-specific adsorption comprises depletion interactions. In some embodiments, the non-specific adsorption comprises electrostatic interactions. In some embodiments, modifying the scaffold increases non-specific adsorption of the 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 via covalent interactions. In some embodiments, the collagen is associated with the scaffold via covalent interactions. In some embodiments, the isolated animal fibroblasts and / or fibroblast-like cells are associated with the scaffold via covalent interactions. In some embodiments, the scaffold comprises a carbodiimide or N-hydroxysuccinimide ester (NHS ester). In some embodiments, the collagen, isolated animal fibroblasts, and / or fibroblast-like cells comprise a carbodiimide or N-hydroxysuccinimide ester (NHS ester). In some embodiments, the collagen is associated with the scaffold via EDC / NHS coupling. In some embodiments, the fibroblasts and / or fibroblast-like cells are associated with the scaffold via EDC / NHS coupling. In some embodiments, the scaffold comprises an azide or an alkyne. In some embodiments, the collagen, isolated animal fibroblasts, and / or fibroblast-like cells comprise an azide or an alkyne.In some embodiments, the collagen is associated with the scaffold via a click chemistry reaction. In some embodiments, the collagen is associated with the scaffold via a click chemistry reaction. In some embodiments, the fibroblasts and / or fibroblast-like cells are associated with the scaffold via a 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 is associated with the scaffold via Michael donor and Michael acceptor coupling. In some embodiments, the fibroblasts and / or fibroblast-like cells are associated with the scaffold via Michael donor and Michael acceptor coupling. 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, fibroblasts, and / or fibroblast-like cells comprise a thiol or maleimide. In some embodiments, the collagen is associated with the scaffold via thiol and maleimide coupling. In some embodiments, the fibroblasts and / or fibroblast-like cells are associated with the scaffold via thiol and maleimide coupling. In some embodiments, the collagen is associated with the scaffold in the presence of sulfo-SMCC. In some embodiments, both the scaffold and collagen comprise a thiol. In some embodiments, the collagen, fibroblasts, and / or fibroblast-like cells are associated with the scaffold via the Maillard reaction. In some embodiments, the collagen is associated with the scaffold via the Maillard reaction.

[0007] Disclosed herein in some embodiments is a method comprising grafting a composition disclosed herein onto a patient in need of a skin graft for the treatment of lost or damaged skin. In some embodiments, the patient may have a laceration, bruise, lesion, sore, burn, wound, surgical wound, surgically removed skin, necrotic skin, or any combination thereof. Disclosed herein in some embodiments is a method comprising tanning a composition disclosed herein to create cruelty-free leather. Disclosed herein in some embodiments is a method of using the cruelty-free leather disclosed herein as a substitute for conventional leather in leather products. Disclosed herein is a leather product comprising the cruelty-free leather disclosed herein. In some embodiments, the leather goods may include watch straps, belts, suspenders, packaging, shoes, boots, footwear, gloves, clothing, bags, clutches, wallets, coin purses, billfolds, key pouches, credit card cases, pencil cases, backpacks, cases, purses, saddles, saddlery, whips, luggage, travel goods, rucksacks, portfolios, briefcases, briefcases, attaché cases, pet supplies, leashes, collars, hunting and fishing supplies, gun cases, cutlery cases, firearm holsters, stationery, writing implements, book covers, camera cases, eyeglass cases, cigarette cases, cigar cases, jewelry cases, cell phone holsters, sporting 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 a trunk, suitcase, travel bag, beauty case, toiletry, or any combination thereof. In some embodiments, disclosed herein are methods of using the compositions as disclosed herein for the treatment of a disease or disorder.Disclosed herein in some embodiments are kits comprising a composition as disclosed herein or a leather product as disclosed herein.

[0008] Disclosed herein are methods that include: The method includes: 1) seeding isolated animal fibroblasts or fibroblast-like cells onto a scaffold and allowing the cells to form on the scaffold, wherein the scaffold comprises polycaprolactone (PCL), polylactic acid (PLA), polylactic-polyglycolic acid (PLGA), polyethylene terephthalate (PET), nylon, polyethylene (PE), polyethylene furanoate (PEF), polypropylene (PP), polyvinyl alcohol (PVA), cotton, bast fiber, viscose, modal, lyocell, vegetable 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 an extracellular matrix to form a cruelty-free leather. In some embodiments, disclosed herein are methods of using the cruelty-free leather disclosed herein as a substitute for conventional leather in leather products. Disclosed herein are leather products comprising the cruelty-free leather disclosed herein. In some embodiments, the leather goods may include watch straps, belts, suspenders, packaging, shoes, boots, footwear, gloves, clothing, bags, clutches, wallets, coin purses, billfolds, key pouches, credit card cases, pencil cases, backpacks, cases, purses, saddles, saddlery, whips, luggage, travel goods, rucksacks, portfolios, briefcases, briefcases, attaché cases, pet supplies, leashes, collars, hunting and fishing supplies, gun cases, cutlery cases, firearm holsters, stationery, writing implements, book covers, camera cases, eyeglass cases, cigarette cases, cigar cases, jewelry cases, cell phone holsters, sporting 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 a trunk, suitcase, travel bag, beauty case, toiletry, or any combination thereof.Disclosed herein in some embodiments are methods of using a composition as disclosed herein for the treatment of a disease or disorder. Disclosed herein in some embodiments are kits comprising a composition as disclosed herein or a leather product as disclosed herein. [Incorporated by reference]

[0009] All publications, patents, and patent applications mentioned in this specification are herein incorporated 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. [Brief description of the drawing]

[0010] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0011] Figure 1A shows a microscopic image of unstained fibroblasts adhered to a 1-ply nylon scaffold under rocking conditions. Figure 1B shows a microscopic image of unstained fibroblasts adhered to a 2-ply nylon scaffold under rocking conditions. Figure 1C shows a microscopic image of unstained fibroblasts adhered to a 40:60 bamboo / cotton scaffold under rocking conditions. Figure 1D shows a microscopic image of fibroblasts adhered to a 1-ply cotton scaffold under rocking conditions. Figure 1E shows a microscopic image of calcein-AM stained fibroblasts adhered to a 1-ply nylon scaffold under rocking conditions. Figure 1F shows a microscopic image of calcein-AM stained fibroblasts adhered to a 2-ply nylon scaffold under rocking conditions. Figure 1G shows a microscopic image of calcein-AM stained fibroblasts adhered to a 40:60 bamboo / cotton scaffold under rocking conditions. Figure 1H shows a microscopic image of fibroblasts stained with calcein-AM adhered to a 1-ply cotton scaffold under rocking conditions. Figure 1I shows a microscopic image of fibroblasts stained with Hoechst stain adhered to a 1-ply nylon scaffold under rocking conditions. Figure 1J shows a microscopic image of fibroblasts stained with Hoechst stain adhered to a 2-ply nylon scaffold under rocking conditions. Figure 1K shows a microscopic image of fibroblasts stained with Hoechst stain adhered to a 40:60 bamboo / cotton blend scaffold under rocking conditions. Figure 1L shows a microscopic image of fibroblasts stained with Hoechst stain adhered to a 1-ply cotton scaffold under rocking conditions.

[0012] Figure 2A shows scan data for a skin grown on a lightly punched PLA scaffold with a 55 μm pore size. Figure 2B shows scan data for a skin grown on a PLA scaffold with a 55 μm pore size. Figure 2C shows scan data for a skin grown on a PLA scaffold with a 80 μm pore size. Figure 2D shows scan data for a skin grown on a PLA scaffold with a 100 μm pore size.

[0013] Figure 3A shows histological images of 5 μm cross sections of tissue stained with trichrome stain when the scaffold is PLA with an average pore size of approximately 100 μm, and Figure 3B shows histological images of 5 μm cross sections of tissue stained with trichrome stain when the scaffold is PLA with an average pore size of approximately 50 μm.

[0014] Figure 4A shows a schematic of the experiment performed to investigate the effect of FBS-soaked PLA scaffolds on cell adhesion and proliferation, while Figure 4B shows sample cells / biopsies grown on a non-serum-soaked PET control compared to serum-soaked and non-soaked PLA.

[0015] Figure 5 shows the percentage of cells on unmodified, pea protein-conjugated, and wheat protein-conjugated PLA and Lyocell scaffolds measured by MTT assay after 1 and 4 days, where NT is untreated, Pea is pea protein, and Wheat is wheat protein.

[0016] Figure 6A shows a fluorescence microscopy image of fibroblasts after 1 day growing on an unmodified PLA scaffold. Figure 6B shows a fluorescence microscopy image of fibroblasts after 1 day growing on a PLA scaffold conjugated with pea protein. Figure 6C shows a fluorescence microscopy image of fibroblasts after 1 day growing on a PLA scaffold conjugated with wheat protein. Figure 6D shows a fluorescence microscopy image of fibroblasts after 1 day growing on an unmodified lyocell scaffold. Figure 6E shows a fluorescence microscopy image of fibroblasts after 1 day growing on a lyocell scaffold conjugated with pea protein. Figure 6F shows a fluorescence microscopy image of fibroblasts after 1 day growing on a lyocell scaffold conjugated with wheat protein.

[0017] Figures 7A and 7B show histological images of 5 μm cross-sectional tissue stained with trichrome stain on the scaffold, with labels highlighting dense tissue, fibers, nuclei, and less dense tissue.

[0018] Figure 8A shows a histological image of a 5 μm cross section of natural bovine hide stained with trichrome stain, with the grain and corium locations labeled. Figure 8B shows a histological image of a 5 μm cross section of the superficial layer (grain) of bovine hide stained with trichrome stain.

[0019] Figure 9A shows a 4x zoom fluorescence microscopy image of fibroblasts after addition of calcein AM. Live cells fluoresce green, and the arrows highlight cell extensions, indicating affinity to the scaffold. Figure 9B shows a 10x zoom fluorescence microscopy image of fibroblasts after addition of calcein AM. Live cells fluoresce green, and the arrows highlight cell extensions, indicating affinity to the scaffold. The second arrow highlights fibers illuminated with white light.

[0020] Figure 10A shows a 10x zoom fluorescence microscope image of fibroblasts after addition of calcein AM, with live cells fluorescing green. Cell spreading and adhesion highlight high affinity to the scaffold. Figure 10B shows a 10x zoom fluorescence microscope image of fibroblasts after addition of calcein AM, with live cells fluorescing green. Cell rounding and clumping indicate low affinity to the scaffold.

[0021] Figure 11 shows a histological image of a 5 μm section of tissue stained with trichrome stain, with labels indicating the slide overview, scale bar, cursor position, and tissue thickness.

[0022] Figure 12 shows the residual glucose levels measured from the last four feeds from days 10 to 17 of tissue culture in fresh medium, 40:60 bamboo / cotton, 1-ply nylon, 2-ply nylon, and 1-ply cotton scaffolds under static and rocking conditions.

[0023] Figure 13 shows basic physical data for tissue on 100 gsm (blue), 50 gsm (red), 55 μm pore size light punch (55LP) (green), pressed PET (PPET) (purple), and VL1 on PPET scaffolds (blue). Physical data includes harvest thickness, crust thickness, harvest weight, raw material weight, crust weight, and calculated tissue-only weight.

[0024] Figures 14A and 14B show histological images of tissue grown on a light punch scaffold with a 55 μm pore size, with a 5 μm cross section stained with trichrome stain.

[0025] Figures 15A, 15B, 15C, 15D, 15E, and 15F show histological images of 5 μm cross sections of tissue grown on 50 gsm scaffolds stained with trichrome stain.

[0026] Figures 16A, 16B, 16C, 16D, 16E, and 16F show histological images of 5 μm cross sections of tissue grown on 100 gsm scaffolds stained with trichrome stain.

[0027] Figure 17A shows a fluorescence microscope image of fibroblasts grown on an unmodified PLA scaffold after 4 days. Figure 17B shows a fluorescence microscope image of fibroblasts grown on a PLA scaffold conjugated with pea protein after 4 days. Figure 17C shows a fluorescence microscope image of fibroblasts grown on a PLA scaffold conjugated with wheat protein after 4 days. Figure 17D shows fibroblasts grown on an unmodified lyocell scaffold after 4 days. Figure 17E shows fibroblasts grown on a lyocell scaffold conjugated with pea protein after 4 days. Figure 17F shows fibroblasts grown on a lyocell scaffold conjugated with wheat protein after 4 days.

[0028] FIG. 18 shows the cell viability at day 1 (blue) and day 4 (red) 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), normalized to the viability of fibroblasts grown on unmodified PLA (PLA NT).

[0029] FIG. 19 shows the relative cell viability after 1 day (blue) and 4 days (blue) of fibroblasts grown on unmodified lyocell (Lyo), lyocell conjugated with pea protein by cryoadsorption (Lyo pea cryo), lyocell conjugated with pea protein after 1 autoclave cycle (Lyo pea auto 1), lyocell conjugated with pea protein after 2 autoclave cycles (Lyo pea auto 2), and lyocell conjugated with pea protein after 4 autoclave cycles (Lyo pea auto 4) relative to fibroblasts grown on unmodified PLA (PLA NT).

[0030] Figure 20A shows a fluorescence microscopy image of fibroblasts grown on an unmodified PLA scaffold after 1 day. Figure 20B shows a fluorescence microscopy image of fibroblasts grown on an unmodified lyocell scaffold after 4 days. Figure 20C shows a fluorescence microscopy image of fibroblasts grown on a lyocell scaffold conjugated with pea protein, where the pea protein was conjugated via autoclaving after 4 days. Figure 20D shows a fluorescence microscopy image of fibroblasts grown on a PLA scaffold conjugated with pea protein after 4 days. Figure 20E shows a fluorescence microscopy image of fibroblasts grown on an oxidized lyocell scaffold after 4 days. Figure 20F shows a fluorescence microscopy image of fibroblasts grown on an oxidized lyocell scaffold functionalized with pea protein after 4 days.

[0031] Figure 21 shows the relative cell viability of fibroblasts grown on PLA conjugated with pea protein (PLA Pea), lyocell conjugated with pea protein (autoclaved Lyocell Pea), oxidized lyocell conjugated with pea protein (oxidized Lyocell Pea), oxidized lyocell, and unmodified lyocell scaffolds, relative to cells grown on unmodified PLA, after 1 day (blue) and 4 days (red).

[0032] Figure 22 shows a seeding density of 125k cells / cm 2 , 250k cells / cm 2 , 500k cells / cm 2 , and 1M cells / cm 2 The mean collagen concentrations obtained from tissue biopsies of 24 and 96 hours after digestion are shown.

[0033] Figure 23 shows a seeding density of 62.5k cells / cm 2 , 125k cells / cm 2 , 500k cells / cm 2 Figure 1 shows the collagen concentration (wet weight) of tissue obtained 24 hours (blue) or 96 hours (orange) after digestion.

[0034] Figure 24 shows a seeding density of 500k cells / cm 2 , 125k cells / cm 2 ,60k cells / cm 2 , and 30k cells / cm 2 Figure 1 shows the collagen concentration per unit wet weight in tissue obtained after 24 hours (orange) and 96 hours (blue) of digestion.

[0035] Figure 25A shows a fluorescence microscope image of fibroblasts that have successfully attached and spread onto the scaffold, and Figure 25B shows a fluorescence microscope image of fibroblasts that have failed to attach and clumped onto the scaffold.

[0036] Figure 26 shows the glucose concentration (g / L) after 1 day (blue), 3 days (red), and 7 days (green) for cell cultures growing on control scaffolds and nylon, PLA, 50:50 PLA:Bioc PLA, cotton, trilobal viscose, regular viscose, and lyocell scaffolds.

[0037] FIG. 27 shows the protein content (blue) and collagen content normalized to biopsy (red) of tissues grown on PET scaffolds in hPL medium and on 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 FBS medium.

[0038] Figure 28 shows tanned skins obtained from tissue growth on various scaffolds. The tissues in the top row, from left to right, are hPL PET, FBS PET, PVOH (not shown), PLA in the top row, and 1.7 dtex lyocell, 3.3 dtex lyocell, 6.7 dtex lyocell, alginate, and nylon mesh in the bottom row.

[0039] Figure 29 shows a control skin not exposed to high temperatures (left), a skin exposed to 95°C to remove the scaffold after tanning (center), and a skin exposed to 95°C to remove the scaffold before tanning (right).

[0040] Figure 30A shows pre-tanned skin (left) and post-tanned skin (right) after the skin was treated with benzylamine. Figure 30B shows pre-tanned skin (left) and post-tanned skin (right) after the skin was treated with ethyl acetate. Figure 30C shows skin that was not treated with benzylamine or ethyl acetate.

[0041] Figure 31 shows the collagen concentration (blue) and total protein concentration (red) of biopsy tissue grown on a 55 μm pore size scaffold (55-1), a 55 μm pore size Light Punch (55-LP-1) scaffold, an 80 μm pore size scaffold (80-1), and a 100 μm pore size scaffold (100-1).

[0042] Figure 32 shows collagen content normalized by volume after 24 hours (blue) or 96 hours (red) of digestion from tissue grown on pressed control (pressed PET), Thinsulate, secant towel, secant spacer, fibertex, pressed PET double stack, smooth facing laminate, rough facing laminate, autoclaved laminate, and absorbable-only scaffold materials.

[0043] Figure 33 shows potential normalization factors including DNA (μg), thickness (mm*5), and weight (mg*10) for tissue grown on pressed control (pressed PET), Thinsulate, Secant Towel, Secant Spacer, Fibertex, pressed PET double stack, smooth laminate, rough laminate, autoclaved laminate, and absorbable-only scaffold materials.

[0044] FIG. 34 shows the thickness, weight, hydroxyproline content, and DNA content from tissue grown on pressed control (PET) (blue) and Fibertex (red).

[0045] Figure 35 shows the collagen content of biopsies after 4 weeks of growth or 8 weeks of growth for tissue grown on pressed control (PET) (blue) and Fibeltex (red) after 24 or 96 hours of digestion.

[0046] FIG. 36 shows histological images of 5 μm cross sections of tissue grown on pressed PET scaffolds stained with trichrome stain.

[0047] FIG. 37 shows histological images of 5 μm cross sections of tissue grown on double layer pressed PET scaffolds stained with trichrome stain.

[0048] FIG. 38 shows a histological image of a 5 μm cross section of tissue grown on a knitted towel-like material stained with trichrome stain.

[0049] Figures 39A and 39B show histological images of 5 μm sections of tissue stained with trichrome stain showing the planar, aligned collagen arrangement.

[0050] Figures 40A and 40B show histological images of 5 μm cross sections of tissue grown on knitted towel-like material stained with trichrome stain.

[0051] 41A and 41B show images of a knitted towel-like material into which tissue can grow.

[0052] FIG. 42 shows histological images of 5 μm cross sections of tissue grown on Vicryl and pressed laminate scaffolds stained with trichrome stain.

[0053] FIG. 43 shows histological images of 5 μm cross sections of tissue grown on Vicryl and rough-pressed laminate scaffolds stained with trichrome stain.

[0054] FIG. 44 shows histological images of 5 μm cross sections of tissue grown on Vicryl and autoclaved pressed laminate scaffolds stained with trichrome stain.

[0055] Figure 45 shows a histological image of a 5 μm cross section of tissue grown on a Fibertex scaffold stained with trichrome stain.

[0056] Figure 46 shows a histological image of a 5 μm cross section of tissue grown on a Thinsulate scaffold stained with trichrome stain.

[0057] FIG. 47 shows histological images of 5 μm cross sections of tissue grown on Vicryl-only scaffolds stained with trichrome stain. [Detailed explanation]

[0058] Some aspects are described below with reference to illustrative applications. It should be understood that numerous specific details, relationships, and methods are described to provide a thorough understanding of the features described herein. The features described herein can be implemented without one or more of the specific details or in other ways. The features described herein are not limited by the illustrated order of acts or events, as some acts may occur in different orders and / or concurrently with other acts or events. Furthermore, not all illustrated acts or events may be required to implement a methodology in accordance with the features described herein.

[0059] The terms used herein are for descriptive purposes only and are not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that "including," "includes," "having," "has," "with," or variations thereof are used in either the detailed description and / or claims, such terms are intended to be inclusive in the same manner as the term "comprising."

[0060] In this disclosure, the terms "about" or "approximately" can mean a range of up to 10% of a given value. In this disclosure, the term "substantially" means something that can be made to a large extent or to a great extent.

[0061] The term "fibroblast" as used herein may include connective tissue cells found in the skin and tendons of the body. Fibroblasts can be obtained from skin or tendon biopsies. Fibroblasts are also ubiquitous in many tissues and organs other than skin and tendons. Fibroblasts may be a type of biological cell that synthesizes extracellular matrix and collagen. Fibroblasts may be a type of biological cell that generates the structural framework (stroma) of animal tissues and plays an important role in wound healing. Fibroblasts may be the most common cell in animal connective tissue. Fibroblasts may comprise the majority of tissue layers grown in culture, which may be tanned into leather.

[0062] As used herein, the term "fibroblast-like cells" can refer to fibroblasts expanded in culture, immortalized fibroblasts, or a combination thereof. Fibroblast-like cells can include differentiated cells that have a morphology, phenotype, or a combination thereof substantially similar to that of fibroblasts. Fibroblast-like cells can include genetic or phenotypic changes from fibroblasts while exhibiting substantially similar gene expression to fibroblasts. Fibroblast-like cells can have similar properties to fibroblasts, such as the ability to synthesize collagen, extracellular matrix, the structural matrix (stroma) of animal tissues, or a combination thereof. Fibroblast-like cells can produce substantially similar levels of collagen compared to fibroblasts.

[0063] As used herein, the term "pluripotent stem cell" may refer to any precursor cell that has the potential to form adult cells other than the placenta.

[0064] As used herein, the term "embryonic stem cell" or "ES cell" or "ESC" can refer to a precursor cell that has the potential to form any adult cell.

[0065] As used herein, the terms "induced pluripotent stem cell," "iPS cell," and "iPSC" may refer to a type of pluripotent stem cell artificially induced from a non-pluripotent cell (e.g., an adult somatic cell). Induced pluripotent stem cells may be similar to embryonic stem cells in their ability to form all adult cells, but they may not be derived from an embryo.

[0066] As used herein, the term "isolated" can refer to cells 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, a scaffold, a medium, or a combination thereof.

[0067] As used herein, the terms "decellularize" 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. "Decellularization" may refer to the process of removing cells to create a decellularized cell layer, and decellularization can be achieved through methods such as salting out or the use of detergents.

[0068] As used herein, the term "cruelty-free leather" may refer to a leather material described herein that can function as a leather material for any mammal or non-mammal. The disclosure herein can be practiced with humans and non-human mammals, such as non-human primates, as well as members of the families cattle, bovine, porcine, equine, canine, and feline, as well as rodents such as mice, rats, and guinea pigs, members of the ragmorph family including rabbits, fish including sharks and stingrays, birds including ostriches, and reptiles including lizards, snakes, and crocodiles. In some embodiments, cruelty-free leather may include artificial leather. In some embodiments, cruelty-free leather may include eco-friendly leather, plastic-free leather, or any combination thereof. In some embodiments, cruelty-free leather may include a tanned artificial cell layer, a tanned at least partially decellularized cell layer, or a combination thereof. In some embodiments, the cell layer or at least partially decellularized cell layer may comprise a dermis layer, an epidermis layer, an at least partially decellularized dermis layer, an at least partially decellularized epidermis layer, or any combination thereof. In some embodiments, the mammalian cruelty-free leather that may be formed may depend on the source of the cells, e.g., keratinocytes and fibroblasts, used in the inventions described herein; for example, if bovine keratinocytes and fibroblasts may be used to form a skin equivalent, a bovine cruelty-free leather may be formed. Cruelty-free leather composition

[0069] Disclosed herein in some embodiments are compositions comprising a scaffold for culturing cells. In some embodiments, the scaffold can provide a substrate or support for cells to attach to, grow on, generate extracellular matrix, or any combination thereof. In some embodiments, the scaffold and cell composition disclosed herein can be tanned to create cruelty-free leather. In some embodiments, the methods or compositions disclosed herein can be used to generate skin equivalents. In some embodiments, the compositions disclosed herein can be implanted into a patient in need thereof for the treatment of a disease or disorder. In some embodiments, cells, cell layers, layered structures, or cruelty-free leather can be seeded onto the scaffold. In some embodiments, the scaffold can include a matrix. In some embodiments, the scaffold can provide a certain stiffness (e.g., tear resistance), elasticity, or both. In some embodiments, the cruelty-free leather can include a portion of a scaffold or the entire scaffold. In some embodiments, the cruelty-free leather may not include a scaffold. In some embodiments, after assisting in layer formation in the cruelty-free leather, the scaffold can be removed from the final cruelty-free leather product. In some embodiments, the scaffold contained in the cruelty-free leather may degrade after a period of time. In some embodiments, the scaffold can be degradable, biodegradable, bioabsorbable, resorbable, or any combination thereof.

[0070] In some embodiments, the scaffold can comprise a three-dimensional woven material. In some embodiments, the three-dimensional woven material can comprise a spacer fabric. In some embodiments, the spacer fabric can include multiple sides. In some embodiments, the multiple sides can include a front side and a back side. In some embodiments, the sides can comprise a woven material. In some embodiments, a filler can separate the front side and the back side. In some embodiments, the filler can space the front side from the back side. In some embodiments, the filler can provide gaps to allow for nutrient entry, waste removal, cell growth, or any combination thereof.

[0071] In some embodiments, the scaffold may be made of natural materials, synthetic materials, or any combination thereof. In some embodiments, the scaffold may include a substrate. In some embodiments, the substrate may include a substrate for cell growth. In some embodiments, the scaffold may be formed using a net made of a bioabsorbable synthetic polymer. In some embodiments, the scaffold may be formed by attaching a nylon net to a silicone film. In some embodiments, the scaffold may include a two-layer structure of a collagen sponge and a silicone sheet. In some embodiments, the scaffold may be formed using an atelocollagen sponge. In some embodiments, the scaffold may be a sheet. In some embodiments, the scaffold may be formed by combining collagen sponges with different pore sizes. In some embodiments, the acellular dermal matrix (ADM) may be formed using at least partially decellularized fibrin glue, allogeneic skin, or a combination thereof. Scaffolding materials

[0072] Disclosed herein in some embodiments is a composition comprising a scaffold in contact with an extracellular matrix comprising collagen, wherein the scaffold may comprise a needle-punched nonwoven material. Disclosed herein in some embodiments is a composition comprising a scaffold in contact with an extracellular matrix comprising collagen, wherein the scaffold may comprise a three-dimensional woven material. Disclosed herein in some embodiments is a composition comprising a scaffold in contact with an extracellular matrix comprising collagen, wherein the scaffold may comprise polycaprolactone (PCL), polylactic acid (PLA), polylactic-polyglycolic acid (PLGA), polyethylene terephthalate (PET), nylon, polyethylene (PE), polyethylene furanoate (PEF), polypropylene (PP), polyvinyl alcohol (PVA), cotton, bast fiber, viscose, modal, lyocell, plant protein fiber, bio-based material, cellulose, alginate fiber, thermoplastic starch, or any combination thereof. In some embodiments, the scaffold may comprise polycaprolactone. In some embodiments, the scaffold may comprise polylactic acid. In some embodiments, the scaffold may comprise polylactic acid / polyglycolic acid. In some embodiments, the scaffold may comprise polyethylene terephthalate. In some embodiments, the scaffold may comprise nylon. In some embodiments, the scaffold may comprise polyethylene. In some embodiments, the scaffold may comprise polyethylene furanoate. In some embodiments, the scaffold may comprise polypropylene. In some embodiments, the scaffold may comprise polyvinyl alcohol. In some embodiments, the scaffold may comprise cotton. In some embodiments, the scaffold may comprise bast fibers. In some embodiments, the scaffold may comprise viscose. In some embodiments, the scaffold may comprise modal. In some embodiments, the scaffold may comprise lyocell. In some embodiments, the scaffold may comprise vegetable protein fibers. In some embodiments, the scaffold may comprise bio-based materials. In some embodiments, the scaffold may comprise cellulose. In some embodiments, the scaffold may comprise alginate fibers. In some embodiments, the scaffold may comprise thermoplastic starch.

[0073] The methods and compositions provided herein may offer design advantages over the use of natural leather. In some embodiments, the selection of a scaffold material, as described in Example 13, may affect the physical properties of the resulting material. In some embodiments, the selection of a scaffold material may affect the tear strength (e.g., average double tear strength) of the resulting material. In some embodiments, the selection of a scaffold material may result in a stronger material. In some embodiments, the selection of a scaffold material may result in a weaker material. In some embodiments, as described in Example 13, the compositions provided herein may be thinner than natural leather but comprise similar (e.g., tear) strength to natural (e.g., bovine) leather.

[0074] In some embodiments, the scaffold may comprise natural substances such as collagen (e.g., collagen matrix), natural adhesives (e.g., fibrin glue, cryogenic glue, animal glue, blood albumen glue, casein glue, or plant glues such as starch glue and dextrin glue). In some embodiments, the scaffold may comprise polylactide, polyglycolide, polycaprolactone, hydrogel, or any combination thereof. In some embodiments, the scaffold may comprise silk. In some embodiments, the scaffold may be made of silk. In some embodiments, the scaffold may comprise silk fibroin, cellulose, cotton, acetate, acrylic, latex fiber, linen, nylon, rayon, velvet, modacrylic, olefin polyester, saran, vinyon, wool, jute, hemp, bamboo, flax, or combinations thereof. In some embodiments, the scaffold may comprise fiber. In some embodiments, the fibers can be silk, cotton, wool, wood (e.g., cellulose extracted from wood, plants, or algae), polyamide, modified cellulose, poly-p-phenylene terephthalamide, acrylic fibers (e.g., polymethyl methacrylate or poly-2-hydroxyethyl methacrylate), polyolefin fibers (e.g., polyethylene or polypropylene fibers), glass, silica, aramid, carbon (e.g., graphite forms), poly(tetrafluoroethylene), insoluble collagen, polyester, polyvinyl chloride, polyvinylidene chloride, polyvinyl alcohol, polyacrylonitrile, chitosan, polyurethane, poly(urethane-urea), polyethylene phthalate, and fibers formed from blends of such polymers as those described above, 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 can comprise rayon, viscose, acetate, rayon acetate, or any combination thereof.

[0075] In some embodiments, the scaffold is a bamboo / cotton blend, 1-ply nylon, 2-ply nylon, or 1-ply nylon, such as that described in Example 1. In some embodiments, high levels of proliferation and spindle-like cells are achieved using a bamboo / cotton scaffold, such as that described in Example 1.

[0076] 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, biostable, 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, pullulan, polyhyaluronic acid, chitin, poly(3-hydroxyalkanoates), poly(3-hydroxyoctanoates), poly(3-hydroxy fatty acids), or any combination thereof. In some embodiments, the polymer is polyethylene (PE), polypropylene (PP), polyethylene terephthalate (PET), polyamide 6,6 (PA6,6), polyamide 11 (PA11), polyvinylidene fluoride (PVDF), polyethylene furanoate (PEF), polyurethane (PU), polyhydroxyalkanoate (PHA), polyhydroxybutyrate (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), polylactic acid (PLA), polycaprolactone (P The scaffold may comprise a polymer selected from the group consisting of poly(L-lactic acid), polybutylene succinate (PBS), polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA), polyvinyl alcohol (PVOH), alginate, copolymer PEGylated fibrin (P-fibrin), polyglycerol sebacate (PGS), poly(L-lactic acid) (PLLA), poly(lactic-coglycolic 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 also comprise a chemical derivative of a natural polymer. In some embodiments, the chemical derivative 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 albumen. In some embodiments, the polymer in the scaffold may be a biodegradable synthetic polymer, including poly alpha-hydroxy acids such as poly-L-lactic acid (PLA), polyglycolic acid (PGA) or copolymers thereof (e.g., poly-D,L-lactic-co-glycolic acid (PLGA)), and hyaluronic acid.

[0077] In some embodiments, the scaffold may comprise polyvinyl alcohol (PVA), polyvinyl acetate (PVA), polycaprolactone (PCL), polylactic acid (PLA), polylactic-polyglycolic 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, vegetable 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).

[0078] In some embodiments, the scaffold may include a sandwich of 3M Thinsulate material Type G, including three main layers, two spunbond and one airlaid nonwoven, as described in Example 11. In some embodiments, the scaffold may include a towel-like material made of PET (Figures 41A-B). In some embodiments, the scaffold may include two outer fabrics held apart by fibers running a distance between them. In some embodiments, the scaffold may include a single fiber mass of lyocell fibers (e.g., Fibertex). In some embodiments, the scaffold may include a needle-punched nonwoven material pressed on one side and laminated to the other side by a suture. In the above-mentioned materials, the orientation of the two needle-punched nonwoven materials may be such that the pressed sides of each of the two materials face outward or inward. In some examples, the needle-punched nonwoven material may be autoclaved before assembly. In some embodiments, the scaffold may comprise one or more vicryl meshes glued together. In some instances, different scaffolds described herein, such as those described in Example 11, may be used to achieve different tissue properties.

[0079] In some embodiments, the scaffold may be bioabsorbable. In some embodiments, the bioabsorbable scaffold may be a non-cytotoxic structure or material that may be capable of containing or supporting living cells and maintaining them in a desired configuration for a period of time. In some embodiments, the term "bioresorbable" may refer to any material that the body can break down into non-toxic by-products that can be excreted from or metabolized within the body. In some embodiments, exemplary bioabsorbable materials for the scaffold may 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(paradioxanone), poly(esters), poly(ester amides), poly(anhydrides), poly(orthoesters), collagen, gelatin, serum albumin, proteins, polysaccharides, mucopolysaccharides, carbohydrates, glycosaminoglycans, poly(ethylene glycol), poly(propylene glycol), poly(acrylates), poly(methacrylates), poly(vinyl alcohol), hyaluronic acid, chondroitin sulfate, heparin, dermatan sulfate, versican, copolymers, blends of polymers, mixtures of polymers, oligomers comprising bioabsorbable linkages, or combinations thereof.

[0080] In some embodiments, the scaffold can be a mesh. In some embodiments, the mesh can be a net of materials (e.g., threads, cords, strands, fibers, or any combination thereof) that can be woven or otherwise connected. In some embodiments, the mesh can comprise materials that can be artificial, biological, or any combination thereof. In some embodiments, the mesh can have pores that can be regular or irregular in size, regular or irregular in shape, regular or irregular in pattern, or any combination thereof. In some embodiments, the mesh can be two-dimensional or three-dimensional. In some embodiments, the mesh can have pores with diameters of about 10 nm to 10 cm, spacing, or a combination thereof. In some embodiments, the pore size can be generally 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 size can be at least 10 nm (e.g., 50 nm, 100 nm, 500 nm, 1 mm). In some embodiments, the pore size can be at most 1 cm (e.g., 5 cm, 10 mm, 5 mm, 1 mm). The mesh can have a diameter of about 20 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 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 mesh can have a diameter of at least 20 nm (e.g., 50 nm, 100 nm, 500 nm, 1 mm).In some embodiments, the mesh can have a diameter of up to 3 cm (e.g., 1 cm, 500 mm, 100 mm, 10 mm, 1 mm). In some embodiments, the strands (e.g., fibers, threads, webs, etc.) or material forming the mesh can have a diameter of about 50 nm to about 10 mm. In some embodiments, the mesh need not be a scaffold.

[0081] In some embodiments, the scaffold can provide a support structure for cell growth, hi some embodiments, the scaffold can be permeable to fluids, nutrients, etc., allowing cell culture medium to contact the surface of the cell layer.

[0082] In some embodiments, the scaffold may comprise a woven fabric comprising intertwined fibers. In some embodiments, the fibers may be intertwined by woven, knitted, or nonwoven fabric fabrication techniques, or some combination thereof. In some embodiments, the scaffold may be porous, biocompatible, sterilizable, mechanically and chemically stable, consistent, and any combination thereof. In some embodiments, the scaffold may comprise a three-dimensional structure. In some embodiments, cells may be seeded within the scaffold. In some embodiments, the scaffold may be of various thicknesses. In some embodiments, the scaffold may have a thickness that may be suitable for the formation of a cell layer. In some embodiments, the scaffold may have a thickness of about 0.1 mm to about 10 mm, e.g., about 0.1 mm to about 5 mm, about 0.1 mm to about 4 mm, about 0.1 mm to about 3 mm, about 0.1 mm to about 2 mm, about 0.1 mm to about 1 mm, about 0.2 mm to about 1 mm, about 0.3 mm to about 1 mm, about 0.4 mm to about 1 mm, about 0.5 mm to about 1 mm, about 0.3 mm to about 1.5 mm, about 0.4 mm to about 1.2 mm, about 0.6 mm to about 1.2 mm, or about 0.7 mm to about 1.5 mm. In some embodiments, the scaffold may have a thickness of about 0.5 mm to 1 mm. In some embodiments, the scaffold may have a thickness of 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 some embodiments, the scaffold may be up to 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 thick. In some embodiments, the scaffold may have the length and / or width of a cell layer to be placed and / or grown on the scaffold. In some embodiments, the scaffold may have the length and / or width of a cell layer described herein. In some embodiments, the scaffold may include pore sizes less than 1 nanometer. In some embodiments, the scaffold may include pore sizes greater than 1 nanometer. In some embodiments, the scaffold may include pore sizes between 10 μm and 900 μm.

[0083] In some instances, pore size can affect tissue ingrowth, as described in Example 3. In some instances, larger pore sizes lead to better tissue growth (FIGS. 15A-F). In some instances, smaller pores result in more weight contribution from the fibers, creating the illusion of heavier, stronger skin (FIGS. 16A-F).

[0084] 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 include a front surface in contact with a filler and a back surface in contact with a filler, the filler separating the front surface from the back surface. In some embodiments, the separation of the front surface from the back surface by the filler may form gaps to allow for nutrient entry, waste removal, cell adhesion, cell proliferation, 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 comprise terry, frieze, velvet, corduroy, bettin, or any combination thereof. In some embodiments, the scaffold may comprise multiple layers of scaffold material. In some embodiments, the multiple layers of scaffold material may comprise a combination of different form factors. In some embodiments, the different form factors may comprise a nonwoven material, a woven material, a needlepunched material, a three-dimensional structure, or any combination thereof. In some embodiments, the scaffold may comprise a three-layer composite, where the three-layer composite may comprise two outer layers and one inner layer. In some embodiments, the two outer layers may comprise surface layer properties, and the inner layer may comprise bulk properties. In some embodiments, the multiple layers may comprise multiple layers of thin material. In some embodiments, the multiple layers may be fused together, held together by intertwining, laminated together, sewn together, glued together, woven together, baked together, or any combination thereof.

[0085] In some embodiments, a nonwoven, needle-punched material may include a first plurality of pores. In some embodiments, the scaffold may include a nonwoven construction that may create the first plurality of pores. In some embodiments, the first plurality of pores may include an average pore size of about 30-70 μm. In some embodiments, the first plurality of pores may include an average pore size of about 80-120 μm. In some embodiments, the first plurality of pores may include an average pore size of about 50 μm or about 100 μm. In some embodiments, the scaffold may include a second plurality of pores created by needle-punching.

[0086] In some embodiments, the scaffold may comprise terry cloth, which may comprise an all-in-one woven fabric that includes high porosity sections (fiber loops) and a stable backbone (woven mesh base) that allows for the two main structural needs of a scaffold: a high porosity environment for tissue growth and mechanical support for the end product's application mechanism.

[0087] In some embodiments, the scaffold can include a spacer fabric. In some embodiments, the spacer fabric can include both a stable backbone and a high porosity environment. In some embodiments, the core can be high porosity and high volume, allowing for extensive tissue growth. In some embodiments, the two distinct surfaces can be different from each other, allowing for further specification of function. In some embodiments, the upper surface can be highly porous and dissolvable, allowing for a smooth, fiber-free upper surface, while the lower surface can be made of a stable material to provide mechanical support.

[0088] In some embodiments, the cell layer may not be formed on a scaffold. In some embodiments, the dermal layer may not be formed on a scaffold (e.g., a collagen matrix). In some embodiments, the cruelty-free leather does not include a scaffold. In some embodiments, the cells may be formed on a scaffold and then substantially removed from the scaffold prior to tanning. In some embodiments, the scaffold may comprise a dissolvable scaffold, where the scaffold may be contacted with a solvent capable of dissolving the scaffold.

[0089] In some embodiments, the solvent provided herein can be any suitable solvent that dissolves the scaffold or contains the degrading agent necessary to degrade or dissolve the scaffold. In some embodiments, the solvent comprises water. In some embodiments, the solvent comprises an organic solvent. In some embodiments, the solvent comprises acetone, benzylamine, ethyl acetate, or a combination thereof. In some embodiments, the solvent comprises acetone. In some embodiments, the solvent comprises ethyl acetate. In other embodiments, the solvent comprises benzylamine. In some embodiments, the solvent comprises benzene, acetonitrile, ethanol, diethyl ether, methylene chloride, 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 to about 50°C. In some embodiments, the solvent can be at a temperature of 25°C to about 125°C. In some embodiments, the solvent can be at a temperature of about 25°C to about 100°C.

[0090] 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 polyhydroxyalkanoate (PHA), polyhydroxybutyrate (PHB), polycaprolactone (PCL), or oxidized alginate. In some embodiments, the thermoplastic polymer comprises polyhydroxyalkanoate (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.

[0091] In some embodiments, the thermoplastic polymer comprises polyvinyl alcohol (PVA). In some embodiments, the solvent comprises water. In some embodiments, the thermoplastic polymer can comprise polyvinyl alcohol (PVA), where the solvent can comprise water.

[0092] In some embodiments, the scaffold comprises polylactic acid (PLA). In some embodiments, the solvent comprises a benzyl group, an ethyl group, a haloalkane, or a combination thereof. In some embodiments, the scaffold may comprise polylactic acid (PLA), wherein the solvent may comprise a benzyl group, an ethyl group, a haloalkane, or a combination thereof. In some embodiments, the solvent may comprise benzylamine.

[0093] In some embodiments, the scaffold may comprise a bio-based material, such as those provided elsewhere herein. In some embodiments, the solvent provided herein comprises a cellulase (e.g., for degrading the bio-based material). In some embodiments, the scaffold may comprise a bio-based material and the solvent may comprise an enzyme that degrades the bio-based material. In some embodiments, the scaffold may comprise cellulose and the solvent may comprise a cellulase.

[0094] In some embodiments, removing scaffold material from the cell and scaffold composition comprises using a dissolution method. In some embodiments, dissolution may comprise contacting the scaffold with a solvent. In some embodiments, the solvent may comprise benzylamine, hot water, enzymes, or any combination thereof. In other embodiments, the solvent comprises an organic solvent. In some embodiments, the organic solvent comprises acetone, benzylamine, or ethyl acetate. In some embodiments, a solvent may be applied to the scaffold at some stage during growth or post-processing to remove the scaffold. In some embodiments, the temperature of the solvent is varied during the dissolution method. In some embodiments, the temperature of the solvent is below the boiling point of the solvent. In some embodiments, the temperature of the solvent is 150°C or less (e.g., 140°C or less, 130°C or less, 120°C or less, 110°C or less, 100°C or less). 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 from about 25°C to about 100°C. In some embodiments, the temperature of the solvent is from about 40°C to about 50°C.

[0095] In some embodiments, removing the scaffold material from the cell and scaffold composition can include using a degradation method. In some embodiments, the scaffold degrades over time via hydrolysis. In some embodiments, the scaffold degrades over time via 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 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.

[0096] In some embodiments, lysing may comprise contacting the scaffold with a lysing agent. In some embodiments, the solvent may comprise the lysing agent. In some embodiments, the lysing agent may comprise an enzyme. In some embodiments, the lysing agent comprises cellulase. In some embodiments, the enzyme may comprise cellulase. In some embodiments, the enzyme may comprise cellulase to digest cellulose. In some embodiments, the lysing agent comprises lipase. In some embodiments, the lysing agent comprises alginate lyase. In some embodiments, the scaffold comprises calcium alginate and the lysing agent comprises alginate lyase. In other embodiments, the scaffold comprises an ester-containing polymer and the lysing agent comprises an esterase. In some embodiments, the scaffold comprises an ester-containing polymer and the lysing agent comprises a lipase.

[0097] In some embodiments, the dissolution agent comprises a chelator. In some embodiments, the dissolution agent comprises ethylenediaminetetraacetic acid (EDTA). In some embodiments, the scaffold comprises alginate and the dissolution agent comprises ethylenediaminetetraacetic acid (EDTA).

[0098] In some embodiments, the dissolution agent comprises a caustic. In some embodiments, the dissolution agent comprises a strong acid. In some embodiments, the strong acid comprises hydrochloric acid, nitric acid, hydroiodic acid, perchloric acid, chloric acid, or a combination thereof. In some embodiments, the strong acid comprises hydrochloric acid. In some embodiments, the strong acid comprises nitric acid. In some embodiments, the strong acid comprises perchloric acid. In some embodiments, the strong acid comprises hydroiodic acid. In some embodiments, the strong acid comprises chloric acid. In other embodiments, the dissolution agent comprises a strong base. In some embodiments, the strong base comprises lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, strontium hydroxide, barium hydroxide, or a combination thereof. In some embodiments, the strong base comprises sodium hydroxide. In some embodiments, the strong base comprises lithium hydroxide. In some embodiments, the strong base comprises potassium hydroxide. In some embodiments, the strong base comprises calcium hydroxide. In some embodiments, the strong base comprises strontium hydroxide. In some embodiments, the strong base comprises barium hydroxide. In some embodiments, the dissolution agent may comprise a chemical such as NaOH to decompose PLA. In some embodiments, the dissolution agent comprises an oxidizing agent that partially or completely decomposes the material. In some embodiments, the oxidizing agent is sodium periodate. In some embodiments, the agent can directly decompose the scaffold material into smaller chunks that can then be more easily solvated and removed from the cell and scaffold composition. In some embodiments, the scaffold removal process can occur at any stage of the manufacturing process. In some embodiments, the scaffold removal process can be performed after the initiation of tissue growth, during the tissue growth period, after tissue growth is completed, but before, during, or after the beam house and tanning process to convert the tissue into leather. In some embodiments, disclosed herein are compositions 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.

[0099] In some embodiments, the scaffold may further comprise polyglycolic acid (PGA), polybutylene succinate (PBS), bioabsorbable synthetic polymers, cellulose, acetate, acrylic, fiber, linen, rayon, velvet, modacrylic, olefin polyester, saran, vinyon, 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 comprise bast fibers, where the bast fibers may comprise flax, hemp, linen, jute, ramie, kenaf, sisal, or any combination thereof.

[0100] In some embodiments, any one of the scaffolds provided herein may comprise a thickness of about 0.1 mm to about 4 mm. In some embodiments, the scaffold may comprise a thickness of about 1 mm to about 3 mm. In some embodiments, the scaffold may comprise a thickness of about 1 mm. In some embodiments, the scaffold may comprise a thickness of about 2 mm. In some embodiments, the scaffold may comprise a thickness of about 3 mm.

[0101] In some embodiments, any one of the scaffolds provided herein may comprise fibers comprising a dtex of about 0.1 dtex to about 50 dtex. In some embodiments, the scaffold comprises fibers comprising a dtex of at least 0.1 dtex (e.g., at least 1 dtex, at least 2 dtex, at least 5 dtex, at least 10 dtex, at least 20 dtex, at least 30 dtex, at least 40 dtex). In some embodiments, the scaffold comprises fibers comprising a dtex of at most 50 dtex (e.g., at most 40 dtex, at most 30 dtex, at most 20 dtex, at most 15 dtex, at most 10 dtex). In some embodiments, the scaffold comprises a dtex of about 0.1 dtex to about 30 dtex. In some embodiments, the scaffold comprises a dtex of about 1 dtex to about 10 dtex. In some embodiments, the scaffold may comprise fibers comprising a dtex of about 6.7 dtex. In some embodiments, the scaffold comprises fibers comprising a dtex of about 1.7 dtex. In some embodiments, the scaffold comprises fibers comprising a dtex of about 3.3 dtex.

[0102] In some embodiments, any one of the scaffolds provided herein can comprise fibers comprising a diameter of about 1 μm to about 100 μm. In some embodiments, the scaffold comprises fibers comprising 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 comprising 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 comprising a diameter of about 1 μm to about 70 μm. In some embodiments, the scaffold comprises fibers comprising a diameter of about 10 μm to about 50 μm. In some embodiments, the scaffold comprises fibers comprising a diameter of about 10 μm to about 100 μm.

[0103] In some embodiments, disclosed herein are compositions comprising an at least partially coated scaffold in contact with an extracellular matrix comprising collagen, wherein the scaffold is at least partially coated with a coating comprising Matrigel, vitronectin, fibronectin, soy protein extract, pea protein extract, corn protein extract, synthetically generated peptide, RNA-binding glycine-rich (RBG) protein, polylysine, synthetic protein, RGD peptide, polylysine, polyarginine, polyornithine, recombinant protein, oligomer, polymer, glycidyltrimethylammonium chloride (GTMAC), carbohydrate-binding module, cellulose-binding domain, starch-binding domain, or any combination thereof. In some embodiments, at least partially coating the scaffold with wheat protein or pea protein, such as those described in Example 5, increases the percentage of cells on the scaffold compared to untreated controls (Figure 5). In some embodiments, conjugation of pea (e.g., sweet pea) protein is more effective than wheat flour protein (Figure 5, Figures 6A-F).

[0104] In some embodiments, the scaffold is at least partially coated with a carbohydrate-binding module. In some instances, the carbohydrate-binding module is a protein domain found in carbohydrate-active enzymes, such as glycoside hydrolases. In some instances, the carbohydrate-binding module has carbohydrate-binding activity. In some instances, the carbohydrate-binding module is selected from CBM3, CBM4, CBM6, CBM9, CBM17, CBM20, CBM21, CBM25, CBM28, CBM32, and CBM49. In some instances, 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 instances, the enzyme does not hydrolyze the scaffold.

[0105] In some embodiments, the scaffold can be modified to include functional groups. In some instances, the functional groups are naturally present on the scaffold. In other instances, the functional groups are the result of modification. In some embodiments, the scaffold includes an amine hydroxyl group, a sulfhydryl group, a tyrosyl group, an amine, a sulfate, an aldehyde, a hydrazide, a diazirine, an aryl azide, an acrylate, an epoxide, or a carboxylic acid group. In some embodiments, the scaffold includes an amine hydroxyl group. In some embodiments, the scaffold includes a sulfhydryl group. In some embodiments, the scaffold includes a tyrosyl group. In some embodiments, the scaffold includes a carboxylic acid group. In some embodiments, the scaffold includes an amine. In some embodiments, the scaffold includes a sulfate. In some embodiments, the scaffold includes an aldehyde. In some embodiments, the scaffold includes a hydrazide. In some embodiments, the scaffold includes a diazirine. In some embodiments, the scaffold includes an aryl azide. In some embodiments, the scaffold includes an acrylate. In some embodiments, the scaffold includes an epoxide. In some embodiments, the scaffold is at least partially coated with GTMAC, hi some embodiments, the GTMAC provides primary amines on the surface of the scaffold.

[0106] In some embodiments, the coating can include modifications. In some embodiments, the modifications can include reductive modification, additive modification, or a combination thereof. In some embodiments, the modifications can include reductive modification, additive modification, or a combination thereof. In some embodiments, reductive modification can include using a base to slightly cleave molecules to generate more reactive species to increase adhesion or to generate some surface roughness. In some embodiments, additive modification can include adding functional ammonium groups with glycidyl trimethylammonium chloride (GTMAC) to increase the surface positive charge and increase adhesion, or grafting polymer brushes to adjust the charge, density, stiffness, 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, as provided by at least partial coating of GTMAC. In some embodiments, at least partial coating with a coating comprising GTMAC increases the surface charge of the scaffold.

[0107] In some embodiments, the modification comprises hydrolysis. In some embodiments, the modification comprises hydrolysis to expose chemically active groups such as hydroxyl, carboxylic acid, ketone, etc. In some embodiments, the modification exposes hydroxyl groups. In some embodiments, the modification exposes carboxylic acid groups. In some embodiments, the modification exposes ketone groups. In some embodiments, the hydrolysis is completed at elevated pressure. In some embodiments, the hydrolysis is completed under autoclave conditions. In some embodiments, the hydrolysis is completed via an enzymatic reaction. In some embodiments, the hydrolysis is completed under acidic conditions. In some embodiments, the hydrolysis is completed under basic conditions.

[0108] In some embodiments, the modification comprises oxidation, hi some embodiments, the modification comprises oxidation with sodium periodate or other suitable oxidizing agent.

[0109] In some embodiments, the modification comprises a Maillard reaction, as described in Example 5. In some examples, the scaffold is immersed in a protein-rich solution under autoclave conditions to treat the surface. In some examples, the Maillard reaction aids in the conjugation of pea protein to lyocell (FIG. 20C), which improves cell spreading compared to unmodified scaffolds (FIG. 20B). In some examples, the scaffolds herein are oxidized with sodium periodate, as described in Example 5. In some examples, a combination of modification methods is used, such as a combination of Maillard reaction and oxidation, which can result in cell spreading (FIG. 20F).

[0110] In some embodiments, in any of the compositions provided herein, the collagen is associated with a scaffold. In some embodiments, associated comprises bonding. In some embodiments, bonding comprises covalent or non-covalent bonding. In some embodiments, in any of the compositions provided herein, the collagen is bound to a scaffold. In some embodiments, the association of the collagen with the scaffold comprises cross-linking between the collagen and the scaffold.

[0111] In some embodiments, collagen associates with, e.g., binds to, the scaffold via non-specific adsorption. In some instances, non-specific adsorption includes van der Waals interactions, hydrogen bonding, depletion interactions, electrostatic interactions, or a combination thereof. In some embodiments, collagen associates with the scaffold via van der Waals interactions. In some embodiments, collagen associates with the scaffold via hydrogen bonding. In some embodiments, collagen associates with the scaffold via depletion interactions. In some embodiments, collagen associates with the scaffold via electrostatic interactions. In some instances, at least partially covering the surface with a coating as described elsewhere herein can increase the strength of non-specific adsorption. In other embodiments, modifying the scaffold, such as modifying as described elsewhere herein, increases the strength of non-specific adsorption of collagen to the scaffold.

[0112] In some embodiments, the collagen is associated with the scaffold via covalent interactions. In some embodiments, the collagen is associated with the scaffold via covalent bonds. In some instances, modifications, such as those described elsewhere herein, allow for covalent interactions, such as covalent bonding of the collagen to the scaffold.

[0113] 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 is associated with, e.g., forms a covalent bond with, the scaffold via EDC / NHS coupling, such as between an NHS ester and a carbodiimide. In some embodiments, the fibroblasts and / or fibroblast-like cells comprise a carbodiimide. In some embodiments, the fibroblasts and / or fibroblast-like cells comprise an N-hydroxysuccinimide ester (NHS ester). In some embodiments, the fibroblasts and / or fibroblast-like cells are associated with, e.g., forms a covalent bond with, the scaffold via EDC / NHS coupling.

[0114] In some embodiments, the scaffold comprises a click chemistry moiety. 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 moiety. In some embodiments, the collagen comprises an azide. In some embodiments, the scaffold comprises an alkyne. In some embodiments, the collagen is associated with the scaffold, e.g., forms a covalent bond with the scaffold, via a click chemistry reaction, such as between an azide and an alkyne. In some embodiments, the fibroblasts and / or fibroblast-like cells comprise a click chemistry moiety. In some embodiments, the fibroblasts and / or fibroblast-like cells comprise an azide. In some embodiments, the fibroblasts and / or fibroblast-like cells comprise an alkyne. In some embodiments, the fibroblasts and / or fibroblast-like cells are associated with the scaffold, e.g., forms a covalent bond with the scaffold, via a click chemistry reaction, such as between an azide and an alkyne.

[0115] 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 via coupling of a Michael donor and a Michael acceptor. In some embodiments, the fibroblasts and / or fibroblast-like cells comprise a Michael donor. In some embodiments, the fibroblasts and / or fibroblast-like cells comprise a Michael acceptor. In some embodiments, the fibroblasts and / or fibroblast-like cells associate with the scaffold via 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.

[0116] 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 is associated with the scaffold, e.g., by forming a covalent bond with the scaffold via coupling of the thiol and the maleimide. In some examples, the collagen is associated with the scaffold in the presence of (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (sulfo-SMCC), where both the scaffold and the collagen comprise a thiol. In some embodiments, the fibroblasts and / or fibroblast-like cells comprise a thiol. In some embodiments, the fibroblasts and / or fibroblast-like cells comprise a maleimide. In some embodiments, the fibroblasts and / or fibroblast-like cells are associated with the scaffold, e.g., by forming a covalent bond with the scaffold via coupling of the thiol and the maleimide. In some examples, fibroblasts and / or fibroblast-like cells associate with the scaffold in the presence of (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (sulfo-SMCC), where both the scaffold and the fibroblasts and / or fibroblast-like cells contain thiols.

[0117] In some embodiments, collagen is associated with the scaffold, such as by forming a covalent bond with the scaffold, via a Maillard reaction, such as a reaction between a reducing sugar and a protein. In some embodiments, fibroblasts and / or fibroblast-like cells are associated with the scaffold, such as by forming a covalent bond with the scaffold, via a Maillard reaction, such as a reaction between a reducing sugar and a protein.

[0118] In some embodiments, the scaffold may comprise a thermoplastic polymer that can be subsequently substantially removed from the extracellular matrix prior to tanning. In some embodiments, the thermoplastic polymer may comprise polyvinyl alcohol (PVA), wherein the method may comprise substantially removing the PVA prior to tanning by contacting the PVA with water having a temperature of about 18°C ​​to about 90°C. In some embodiments, the thermoplastic polymer may comprise polylactic acid (PLA), wherein the method may comprise substantially removing the PLA prior to tanning by contacting the PLA with a solvent to remove the PLA. In some embodiments, the solvent may comprise a benzyl group, an ethyl group, a haloalkane, a benzylamine, or any combination thereof. In some embodiments, the scaffold may comprise a dissolvable scaffold, wherein the dissolvable scaffold can be substantially removed by contacting the scaffold with a solvent. In some embodiments, the method of manufacturing a scaffold may comprise needle-punching the scaffold using a needle loom to entangle fibers into the nonwoven scaffold material. In some embodiments, the needle loom may comprise barbed needles. In some embodiments, needle punching can create pores in the scaffold material. In some embodiments, a method of manufacturing a scaffold can include at least partially coating the scaffold with a coating comprising Matrigel, vitronectin, fibronectin, a protein extract from soybean, a protein extract from pea, a protein extract 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 a combination thereof.

[0119] In some embodiments, a composition comprising a scaffold in contact with an extracellular matrix is ​​disclosed herein. In some embodiments, the extracellular matrix may include collagen. In some embodiments, the scaffold and cell composition may include at least one component of natural skin, such as melanocytes, hair follicles, sweat glands, and nerve endings. In certain cases, cruelty-free leather can be distinguished from normal natural skin by the absence of at least one of these components. In some embodiments, a scaffold and cell composition leather having at least one cell exhibiting an abnormal phenotype or altered genotype may include all of these components.

[0120] Disclosed herein in some embodiments is a composition comprising 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 isolated immortalized animal cells. In some embodiments, the isolated immortalized animal cells can be propagated beyond the Hayflick limit. In some embodiments, the isolated immortalized animal cells can be propagated for more than 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 partially decellularized can include a substantial absence of intact cells in the composition.

[0121] In some embodiments, additional components can be added to the scaffold and cell composition. Such additional components can include myoepithelial cells, ductal cells, secretory cells, alveolar cells, Langerhans cells, Merkel cells, adhesions, mammary glands, or any mixture thereof. In some embodiments, the cruelty-free leather can include one or more of the following: nerve cells, connective tissue (including bone, cartilage, cells that differentiate into bone-forming cells and chondrocytes, and lymphatic tissue), epithelial cells (including endothelial cells that form the lining of cavities and blood vessels or channels, exocrine epithelial cells, epithelial absorptive cells, keratinizing epithelial cells, and extracellular matrix-secreting cells), and undifferentiated cells (such as embryonic cells, stem cells, and other progenitor cells).

[0122] In some embodiments, the scaffold and cell composition may comprise a hair follicle. The hair follicle may comprise one or more structures, including a dermal papilla, matrix, root sheath, bulge, internal cisterna, flank muscle, sebaceous gland, apocrine sweat gland, or any combination thereof. The hair follicle may comprise one or more hair follicle cells, including dermal papilla cells, outer root sheath cells, or any combination thereof. In some embodiments, the hair follicle may reside in the epidermal layer. In some embodiments, the hair follicle may reside in the dermal layer. In some embodiments, the hair follicle cells may be differentiated 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%, or 100% of the hair follicle cells may be differentiated from induced pluripotent stem cells.

[0123] In some embodiments, the scaffold and cell composition may be free of hair, blood vessels, sebaceous glands, hair follicles, oil glands, nerves, or any combination thereof, immediately before or immediately after tanning. Cell type

[0124] In some embodiments, at least a portion of one or more cells in the cruelty-free leather can be differentiated from precursor cells, such as stem cells. In some embodiments, the cruelty-free leather can be produced from engineered cells or tissues comprising engineered cells as disclosed herein. In some embodiments, the fibroblasts in the cruelty-free leather can be differentiated from stem cells. In some embodiments, the keratinocytes in the cruelty-free leather can be differentiated from stem cells. In some embodiments, the melanocytes in the cruelty-free leather can be differentiated from stem cells.

[0125] In some embodiments, the stem cells may 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 may be totipotent, pluripotent, or multipotent. In some embodiments, the stem cells may include adult stem cells, umbilical cord blood stem cells, or a combination thereof. Embryonic stem cells may be obtained from fertilized embryos that may be less than one week old. Induced pluripotent stem cells may be obtained by inducing expression of one or more of Oct3, Oct4, Sox2, Klf4, TERT, Bmil, CcnD1, Cdk4, SV40 large T antigen, c-Myc, or fragments of these genes in any somatic cell. In some embodiments, the somatic cell may include an adult somatic cell. In some embodiments, the somatic cell may include a fibroblast. In some embodiments, the exogenous vector may include or encode a gene that induces pluripotency. In some embodiments, the exogenous vector may include a plasmid. In some embodiments, induced pluripotent stem cells can be obtained by an active protein product or a biologically active fragment thereof. In some embodiments, one or more other genes can be induced to reprogram somatic cells into induced pluripotent stem cells. In some embodiments, the genes for inducing pluripotency can include NANOG, UTF1, LIN28, SALL4, NR5A2, TBX3, ESSRB, DPPA4, SV40LT, REM2, MDM2, and cyclin D1. In some embodiments, the genes can be derived from humans. In some embodiments, the genes can be derived from mammals, birds, reptiles, amphibians, fish, invertebrates, or any combination thereof.

[0126] 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, electrical delivery, 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, the somatic cells can comprise adult somatic cells. In some embodiments, the somatic cells can be transfected with a vector to deliver a gene that induces pluripotency. In some embodiments, the vector can comprise a viral vector. In some embodiments, the vector can comprise a retroviral vector. In some embodiments, the gene that induces pluripotency can include Oct3, Oct4, Sox2, Klf4, TERT, Bmil, 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, the somatic cells can comprise adult somatic cells. In some embodiments, somatic cells can be transfected with an extrachromosomal vector. In some embodiments, the extrachromosomal vector can comprise a plasmid. In some embodiments, the extrachromosomal vector can deliver Oct3, Oct4, Sox2, Klf4, TERT, Bmil, CcnD1, Cdk4, SV40 large T antigen, c-Myc, any fragment thereof, or any combination thereof.

[0127] Disclosed herein in some embodiments are compositions comprising isolated cells that can be directed to the production of cruelty-free leather, cruelty-free leather, isolated epidermal layers, isolated dermal layers, layered structures, products made therefrom, methods of manufacturing the same, or any combination thereof. Disclosed herein in some embodiments are methods and compositions comprising isolated cells. In some embodiments, the isolated cells can comprise cells derived from an animal. In some embodiments, the cells can be derived from a mammal, a bird, a reptile, an amphibian, a fish, an invertebrate, or any combination thereof. In some embodiments, the cells may be derived from antelope, bear, beaver, bison, wild boar, camel, caribou, cat, cattle, deer, dog, elephant, moose, fox, giraffe, goat, rabbit, human, horse, ibex, kangaroo, lion, llama, lynx, mink, moose, oxen, peccary, pig, rabbit, rhinoceros, seal, sheep, lamb, squirrel, tiger, whale, wolf, yak, or zebra. In some embodiments, the animal may be a primate, bovine, ovine, porcine, equine, canine, feline, rodent, lagomorph, fish, bird, or reptile. In some embodiments, the cells may be derived from an avian species. In some embodiments, the avian species may include a chicken, duck, emu, goose, grouse, ostrich, pheasant, pigeon, quail, or turkey. In some embodiments, the cells may be derived from a reptile, such as a turtle, snake, lizard, amphibian, crocodile, or alligator. In some embodiments, the cells may be derived from an amphibian. In some embodiments, the amphibian may include a frog, toad, salamander, or newt. In some embodiments, the cells may be derived from a fish.In some embodiments, the fish may include anchovy, sea bass, catfish, carp, cod, eel, flounder, pufferfish, grouper, haddock, halibut, herring, mackerel, mahi-mahi, manta ray, marlin, orange roughy, perch, barracuda, pollock, salmon, sardine, shark, sea bream, flounder, stingray, swordfish, tilapia, trout, tuna, or Atlantic rock shrimp. In some embodiments, the isolated cells may be taken from a biopsy. In some embodiments, the isolated cells may include isolated animal cells. In some embodiments, the isolated animal cells may include primate cells, bovine cells, ovine cells, porcine cells, equine cells, canine cells, feline cells, rodent cells, avian cells, marsupial cells, reptilian cells, or lagomorph cells. In some embodiments, the isolated animal cells may be genetically engineered cells. The cells may include a cell line having a plurality of cells. In some embodiments, the isolated animal cells may be immortalized cells. In some embodiments, the cells may include human cells, fibroblasts, stem cells, or any combination thereof. In some embodiments, the cells may be adipose tissue-derived cells (e.g., adipocytes), chondrocytes, osteocytes, osteoblasts, myofibroblasts, satellite cells, myoblasts, myocytes, keratinocytes, corneocytes, 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 may produce extracellular matrix (ECM). In some embodiments, ECM proteins may include collagen, type I collagen, type III collagen, elastin, fibronectin, laminin, or any combination thereof. In some embodiments, engineered tissue may include the cells described herein. In some embodiments, the isolated animal cells may comprise isolated engineered animal cells. In some embodiments, the isolated engineered animal cells may comprise genetically engineered cells. In some embodiments, the genetically engineered cells may comprise an exogenous polynucleotide. In some embodiments, the genetically engineered cells may comprise a gene for collagen production.In some embodiments, the collagen gene may be P4HA, P4HB, COL1A1, COL1A2, COL2A1, COL3A1, or any combination thereof. In some embodiments, the collagen gene may have an altered promoter that may alter, e.g., increase or decrease, expression of the collagen gene. In some embodiments, the collagen gene may be derived from a human. In some embodiments, the collagen gene may be derived from an animal, mammal, bird, reptile, amphibian, fish, invertebrate, or any combination thereof. In some embodiments, the exogenous gene or genes may cause immortalization. In some embodiments, the exogenous gene is hTERT, TERT, Bmil, CcnD1, a mutant of Cdk4, Cdk4, TAg (SV40 large T), SV40, c-myc, H-ras, Ela, c-mycER. 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 cells may include immortalized cells, tissues developed therefrom, or any combination thereof. In some embodiments, the exogenous polynucleotide may encode: (i) a polypeptide that can interact with a tumor suppressor protein or a fragment thereof and 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 may be measured by an in vitro assay. In some embodiments, the immortalized cells may have random mutations or multiple mutations. In some embodiments, the mutations may be generated by UV mutagenesis, chemical mutagenesis, or any combination thereof. In some cases, the immortalized cells may have targeted mutations, for example, targeted mutations may be generated by a CRISPR system. In some embodiments, the mutations may be in cell cycle genes, oncogenes, metabolic genes, or any combination thereof. In some embodiments, the immortalized cells 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 cells may have increased or decreased expression of oncogenes or genes involved in the control of cell proliferation.In some embodiments, the immortalized cells have undergone 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. The immortalized cells may be propagated beyond 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 1000 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 may comprise the finite number of cell doublings that a primary cell can propagate. In some embodiments, the immortalized cells can be propagated beyond the Hayflick limit. In some embodiments, the immortalized cell line can be propagated sufficiently to produce at least 1 million square feet of cruelty-free leather per year.

[0128] In some embodiments, in any of the compositions provided herein, the scaffold may be seeded with any suitable collagen-producing cells. In some embodiments, the scaffold may be seeded with primary cells. In some embodiments, the scaffold provided herein may be seeded with collagen-producing cells including fibroblasts, keratinocytes, melanocytes, epithelial cells, comeocytes, Langerhans cells, basal cells, or combinations thereof. In some embodiments, the scaffold provided herein may be seeded with fibroblasts. In some embodiments, the scaffold provided herein may be seeded with keratinocytes. In some embodiments, the scaffold provided herein may be seeded with melanocytes. In some embodiments, the scaffold provided herein may be seeded with epithelial cells. In some embodiments, the scaffold provided herein may be seeded with comeocytes. In some embodiments, the scaffold provided herein may be seeded with Langerhans cells. In some embodiments, the scaffold provided herein may be seeded with basal cells. How to Make Cruelty-Free Leather

[0129] Some embodiments herein disclose methods for producing the compositions disclosed herein, including seeding isolated animal fibroblasts or fibroblast-like cells onto a scaffold to form a composition. In some embodiments, the 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 adjacently arranging a plurality of multicellular bodies, where the plurality of multicellular bodies can fuse to form a planar layer. In some embodiments, the cells can be grown three-dimensionally. In some embodiments, the cells can be grown in suspension. In some embodiments, forming the cell layer involves using a scaffold. In some embodiments, the cell layer can be formed by disposing a plurality of isolated cells, multicellular bodies, or a combination thereof, on a scaffold. In some embodiments, the forming step can include disposing or placing the multicellular bodies, or seeding 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 positioned adjacent to one another horizontally and / or vertically. In some embodiments, the formation of the cell layer can be performed without a scaffold. In some embodiments, the cell layer can be formed on a scaffold, which can then 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, allowing cell culture medium to contact all surfaces of the multicellular bodies and / or layers during placement and subsequent fusion. In some embodiments, the support substrate can be made from natural biomaterials such as collagen, fibronectin, laminin, and other extracellular matrices. In some embodiments, the support substrate can be made from synthetic biomaterials such as hydroxyapatite, alginate, agarose, polyglycolic acid, polylactic acid, and copolymers thereof.In some embodiments, the support substrate can be 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 creation of a planar layer. In some embodiments, the support substrate can be elevated above a non-permeable surface, such as a portion of a cell culture environment (e.g., a Petri dish, cell culture flask, etc.) or a bioreactor. In some embodiments, a permeable, elevated support substrate can help prevent premature cell death, promote cell proliferation, and facilitate the fusion of multicellular bodies to form a layer. In some embodiments, the cell layer can include a dermal layer. In some embodiments, the cruelty-free leather can include a dermal layer, or at least a partially decellularized portion thereof. In some embodiments, the dermal layer can be an engineered dermal equivalent, e.g., 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 dermal layer 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 beta fibroblast, or a combination thereof. In some embodiments, the dermal layer may contain other types of cells, such as immune cells, macrophages, adipocytes, or a combination thereof. In some embodiments, the dermal layer 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 dermal layer may contain matrix components in addition to cells. In some embodiments, the matrix component may include any one or more of collagen, elastin, extrafibrous matrix, extracellular gel-like substances composed primarily of glycosaminoglycans, proteoglycans, glycoproteins, or any combination thereof.In some embodiments, the extracellular gel-like substance, primarily composed of glycosaminoglycans, may include hyaluronan. In some embodiments, the dermal layer may include a matrix support. In some embodiments, the matrix support may be a scaffold. In some embodiments, the matrix support may include a contracted collagen gel. In some embodiments, the pure collagen matrix may be a polyglycolic acid mesh covered with a silastic membrane (C-GAG), or a collagen and glycosaminoglycan matrix covered with a silastic membrane (C-GAG), a biopolymer, or any combination thereof. In some embodiments, the biopolymer may include chitosan. In some embodiments, the matrix may be seeded with fibroblasts. In some embodiments, seeding with fibroblasts can generate organotypic models. In some embodiments, the cell layer may include naturally occurring dermis, keratinocytes, or a combination thereof. In some embodiments, the naturally occurring dermis may be harvested from allogeneic cadaver skin. In some embodiments, the keratinocytes may form a sheet of keratinocytes. In some embodiments, the cell layer may comprise freeze-dried defatted dermis from cadaver skin to support the keratinocyte sheet.

[0130] In some embodiments, the methods provided herein may further include growing cells under agitated conditions. In some embodiments, agitated conditions result in greater glucose consumption, as described in Example 1. In some embodiments, the methods provided herein may further include growing cells under static conditions.

[0131] In some embodiments, the methods provided herein further comprise seeding the cells at any suitable seeding density determined by one of skill in the art. In some embodiments, the cells are 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 / cm2 , 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 In some embodiments, the cells are seeded at a density of at most 2M cells / cm. 2 (e.g., at most 1.5M cells / cm 2 , at most 1.25M cells / cm 2 , 1M cells / cm 2 , at most 800k cells / cm 2 , at most 600k cells / cm 2 , at most 400k cells / cm 2 , at most 200k cells / cm 2 , at most 100k cells / cm 2 In some embodiments, the cells are seeded at a density of 200k cells / cm. 2 In some embodiments, the cells are seeded at a density of about 30k cells / cm. 2 to approximately 2M cells / cm 2 In some embodiments, the cells are seeded at a density of about 62.5k cells / cm. 2 to approximately 1M cells / cm 2 In some embodiments, the cells are seeded at a density of about 62.5k cells / cm. 2 to approximately 200k cells / cm 2 In some instances, lower seeding densities (e.g., 30k cells / cm) are used. 2 ) results in higher collagen production than higher seeding densities (Figure 24).

[0132] In some embodiments, the methods provided herein further comprise incubating for any suitable period of time. In some embodiments, the methods comprise 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 methods comprise 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 methods comprise incubating for about 1 week to about 12 weeks. In some embodiments, the methods comprise incubating for about 4 weeks to about 8 weeks. In some embodiments, the methods comprise incubating for about 4 weeks. In some embodiments, the methods comprise incubating for about 6.5 weeks. In some embodiments, the methods comprise incubating for about 8 weeks.

[0133] In some embodiments, the methods provided herein further comprise soaking the scaffold in a protein-containing solution (e.g., serum) prior to culturing. In some embodiments, soaking comprises soaking the scaffold in fetal bovine serum. In some embodiments, soaking can be important for cell adhesion and proliferation. In some instances, soaking allows for the adsorption of adhesive proteins to the surface, which can increase cell adhesion.

[0134] In some embodiments, the thickness of the dermis layer can be designed to suit the function or use of cruelty-free leather. In some embodiments, the dermis layer can have a thickness of about 0.01 mm to about 50 mm. In some embodiments, the dermis layer can have a thickness of about 0.01 mm to about 10 mm, about 0.01 mm to about 8 mm, about 0.01 mm to about 5 mm, about 0.02 mm to about 5 mm, about 0.05 mm to about 5 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 2 mm, about 0.1 mm to about 1 mm, about 0.1 mm to about 0.8 mm, or about 0.1 mm to about 0.5 mm. In some embodiments, the dermis layer can have a thickness of about 0.02 mm to about 5 mm. For example, the dermis layer can have a thickness of about 0.1 mm to about 0.5 mm. In some embodiments, the dermis layer can have a thickness of about 0.2 mm to about 0.5 mm. In some embodiments, the thickness 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 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 dermal layer can have a thickness of at least about 50 mm.

[0135] In some embodiments, the length of the dermal layer can be designed to accommodate the function or use of cruelty-free leather. In some embodiments, the dermal layer can have a length of about 0.01 mm to about 50 mm. In some embodiments, the dermal layer can have a length of about 0.01 mm to about 10 mm, about 0.01 mm to about 8 mm, about 0.01 to about 5 mm, about 0.02 to about 5 mm, about 0.05 to about 5 mm, about 0.1 to about 5 mm, about 0.1 to about 2 mm, about 0.1 to about 1 mm, about 0.1 to about 0.8 mm, or about 0.1 to about 0.5 mm. In some embodiments, the dermal layer can have a length of about 0.02 mm to about 5 mm. For example, the dermal layer can have a length of about 0.1 mm to about 0.5 mm. In some embodiments, the dermal layer can have a length of about 0.2 mm to 0.5 mm. In some embodiments, the length 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 length 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 length of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 700, or 1000 mm. In some embodiments, the dermal layer can have a length 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 length of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400 m.

[0136] In some embodiments, the width of the dermis layer can be designed to suit the function or use of cruelty-free leather. In some embodiments, the dermis layer can have a width of about 0.01 mm to about 50 mm. In some embodiments, the dermis layer can have a width of about 0.01 mm to about 10 mm, about 0.01 mm to about 8 mm, about 0.01 to about 5 mm, about 0.02 to about 5 mm, about 0.05 to about 5 mm, about 0.1 to about 5 mm, about 0.1 to about 2 mm, about 0.1 to about 1 mm, about 0.1 to about 0.8 mm, or about 0.1 to about 0.5 mm. In some embodiments, the dermis layer can have a width of about 0.02 mm to about 5 mm. In some embodiments, the dermis layer can have a width of about 0.1 mm to about 0.5 mm. In some embodiments, the dermis layer can have a width of 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, or 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.

[0137] In some embodiments, cruelty-free leather may include one or more dermal layers. In some embodiments, cruelty-free leather may 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, where cruelty-free leather may include two or more dermal layers, a dermal layer may be placed on top of another dermal layer. In some embodiments, cruelty-free leather may include two dermal layers, for example, a first dermal layer and a second dermal layer. In some embodiments, a first dermal layer may be placed on top of a second dermal layer.

[0138] In some embodiments, the dermal layer, or at least a partially decellularized portion thereof, may be layered, e.g., having multiple sublayers. In some embodiments, the sublayers may have different compositions, e.g., different concentrations of fibers. In some embodiments, the sublayers of the dermal layer, or at least a partially decellularized portion thereof, may have different thicknesses, densities, or combinations thereof. In some embodiments, the dermal layer, or at least a partially decellularized portion thereof, may have a papillary dermis layer, a reticular dermis layer, at least a partially decellularized portion of either of these, or any combination thereof. In some embodiments, the papillary dermis layer, or at least a partially decellularized portion thereof, may include loose areolar connective tissue, loosely aligned fibers, at least a partially decellularized portion thereof, or any combination thereof. In some embodiments, the loosely aligned fibers may include collagen fibers. In some embodiments, the reticular dermis layer may include dense irregular connective tissue including collagen fibers and dermal elastic fibers.

[0139] In some embodiments, the dermal layer, or at least a partially decellularized portion thereof, can comprise a free collagen matrix or lattice, which can be contractile in all directions and homogeneous. In some embodiments, fibroblasts (e.g., immortalized bovine fibroblasts), and, where appropriate, other types of dermal cells, can be distributed within a continuous collagen gel. In some embodiments, the dermal equivalent can comprise at least one matrix of type I collagen within which fibroblasts can be distributed. In some embodiments, the dermal equivalent can also comprise other extracellular matrix components. In some embodiments, the extracellular matrix components can include collagen, e.g., type IV collagen, laminin, entactin, fibronectin, proteoglycans, glycosaminoglycans, or hyaluronic acid. In some embodiments, the dermal layer can comprise type IV collagen and laminin, entactin, or a combination thereof. In some embodiments, the concentrations of these various components can be adjusted. For example, in some embodiments, the concentration of laminin can be about 1% to about 15% of the final volume. In some embodiments, the concentration of type IV collagen can be about 0.3% to about 4.5% of the final volume. In some embodiments, the concentration of entactin can be about 0.05% to about 1% of the final volume. In some embodiments, the collagen can be bovine collagen, rat collagen, fish collagen, any other source of natural collagen, or genetically engineered collagen that allows contraction in the presence of fibroblasts, or any combination thereof. In some embodiments, the collagen can be from a non-natural source. In some embodiments, the matrix can be a tension-free collagen gel obtained by both horizontal and vertical contraction without imposing preferential organization of fibroblasts. In some embodiments, the matrix, also referred to as "free," can be unattached to a support, its volume can be modified without restriction, and it can be given various thicknesses and diameters.In some embodiments, the thickness of the dermis equivalent may be at least 0.05 cm, and in some embodiments, may be approximately 0.05 to 2 cm. In some embodiments, the thickness may also be increased without impairing the beneficial properties of the skin equivalent or cruelty-free leather. In some embodiments, the thickness may be from about 3 mm to about 20 cm or more. In some embodiments, the cruelty-free leather may include only the dermis layer.

[0140] In some embodiments, the cell and scaffold composition may comprise an epidermal layer (e.g., an artificial epidermal layer). In some embodiments, the epidermal layer may be an engineered epidermal equivalent, e.g., an artificial epidermal layer formed in vitro.

[0141] 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 of the epidermal layer may comprise epidermal keratinocytes, basal keratinocytes, proliferating basal keratinocytes, differentiated suprabasal keratinocytes, or any combination thereof.

[0142] 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, e.g., keratinocytes that may not be differentiated. In some embodiments, the epidermal layer may further comprise not only fully differentiated keratinocytes, but also partially differentiated keratinocytes. In some embodiments, one or more epidermal layers in a cruelty-free leather may transition from undifferentiated basal keratinocytes to fully differentiated keratinocytes as they progress from the dermal-epidermal junction where basal keratinocytes may be localized.

[0143] In some embodiments, basal keratinocytes express hemidesmosomes, which may help secure the epidermal and dermal layers together. In some embodiments, basal keratinocytes may also play a role in regenerating skin. In some embodiments, the epidermal layer in the cruelty-free leather herein may have basal keratinocytes that perform these functions. In some embodiments, cruelty-free leather containing such basal keratinocytes may be capable of regeneration. In some embodiments, the distinction between basal keratinocytes and differentiated keratinocytes in one or more epidermal layers of cruelty-free leather may be that epidermal keratinocytes along the basement membrane zone (BMZ) may have both E-cadherin and P-cadherin, while differentiated keratinocytes located away from the BMZ may express only E-cadherin.

[0144] In some embodiments, the basal keratinocytes of the epidermal layer can be aligned in a layer that directly contacts the dermal layer, serving as a boundary between differentiated keratinocytes and fibroblasts. In other cases, there may be gaps between the basal keratinocytes and the dermal layer. Furthermore, there may be gaps between the basal keratinocytes and other basal keratinocytes, and gaps may remain between the differentiated keratinocytes and the dermal layer. In these latter cases, where gaps may exist between the basal keratinocytes or differentiated keratinocytes and the dermal layer, the dermal and epidermal layers may not be in uniform contact with each other, but may be adjacent to each other. In some embodiments, the dermal and epidermal layers may be adjacent in that there may be generally fluid between the dermal and epidermal layers, but there is substantially no other intervening layer, such as a cell layer, collagen, matrix, or other support.

[0145] In some embodiments, keratinocytes in the epidermal layer may express one or more markers, including, but 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 (e.g., TAp63), laminin beta 3 (LAMB3), and keratin 18 (KRT18).

[0146] In some embodiments, the thickness of the skin layer can be designed to suit the function or use of cruelty-free leather. In some embodiments, the skin layer can have a thickness of about 0.001 mm to about 10 mm. In some embodiments, the skin 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 mm, 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 may have a thickness of about 0.01 mm to about 2 mm. In some embodiments, the epidermal layer may have a thickness of about 0.1 mm to about 0.22 mm. In some embodiments, the thickness of the epidermal layer may 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 may 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 may be the thickness of the epidermal layer and the basement membrane substitute.

[0147] In some embodiments, the length of the skin layer can be designed to suit the function or use of cruelty-free leather. In some embodiments, the skin layer can have a length of about 0.01 mm to about 50 mm. In some embodiments, the skin layer can be about 0.01 mm to about 10 mm, about 0.01 mm to about 8 mm, about 0.01 to about 5 mm, about 0.02 to about 5 mm, about 0.05 to about 5 mm, about 0.1 to about 5 mm, about 0.1 to about 2 mm, about 0.1 to about 1 mm, about 0.1 to about 0.8 mm, or about 0.1 to about 0.5 mm. In some embodiments, the skin layer can have a length of about 0.02 mm to about 5 mm. In some embodiments, the skin layer can have a length of about 0.1 mm to about 0.5 mm. In some embodiments, the skin layer can have a length of about 0.2 mm to 0.5 mm. In some embodiments, the length of the skin 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 skin 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 skin layer can have a length of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 700, or 1000 mm. In some embodiments, the skin layer can have a length of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700 cm. In some embodiments, the skin layer can have a length of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400 m.

[0148] In some embodiments, the width of the skin layer can be designed to suit the function or use of cruelty-free leather. In some embodiments, the skin layer can have a width of about 0.01 mm to about 50 mm. In some embodiments, the skin layer can have a width of about 0.01 mm to about 10 mm, about 0.01 mm to about 8 mm, about 0.01 to about 5 mm, about 0.02 to about 5 mm, about 0.05 to about 5 mm, about 0.1 to about 5 mm, about 0.1 to about 2 mm, about 0.1 to about 1 mm, about 0.1 to about 0.8 mm, or about 0.1 to about 0.5 mm. In some embodiments, the skin layer can have a width of about 0.02 mm to 5 mm. In some embodiments, the skin layer can have a width of about 0.1 mm to 0.5 mm. In some embodiments, the skin layer can have a width of about 0.2 mm to 0.5 mm. In some embodiments, the width of the skin 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 skin 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 skin layer can have a width of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 700, or 1000 mm. In some embodiments, the skin 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 skin layer can have a width of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400 m.

[0149] In some embodiments, the epidermal layer can be stratified, e.g., have multiple sublayers. In some embodiments, the sublayers can have different cellular compositions, e.g., different types of keratinocytes. In some embodiments, the sublayers can include engineered cells. In some embodiments, the sublayers of the epidermal layer can have different thicknesses and / or densities. In some embodiments, the epidermal layer can have one or more of a keratinized layer (stratum corneum), a clear / translucent layer (stratum lucidum), a granular layer (stratum granulosum), a spinous layer (stratum spinosum), a basal / germinativum layer (stratum basale / germinativum), or any combination thereof. In some embodiments, the epidermal layer can include a functional epidermal permeability barrier (e.g., an organized lipid bilayer in the stratum corneum). In some embodiments, the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, or stratum basale / stratum germinativum may have a thickness of about 0.0001 mm to about 5 mm. In some embodiments, the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, or stratum basale / stratum germinativum may 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 / stratum germinativum may 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.

[0150] In some embodiments, the epidermal layer may further comprise cells that produce pigment, such as melanin. In some embodiments, such pigment-producing cells may be melanocytes. In some embodiments, melanocytes in the epidermal layer may express one or more markers. In some embodiments, such markers may include, but are not limited to, SRY-box containing gene 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 may not comprise an epidermal layer.

[0151] Provided herein are methods for producing any one of the compositions provided herein. In some embodiments, the method comprises at least partially covering a scaffold with a coating comprising Matrigel, vitronectin, fibronectin, a protein extract from soybean, a protein extract from pea, a protein extract 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, GTAMC, a carbohydrate-binding module, a cellulose-binding domain, a starch-binding domain, or a combination thereof. In some embodiments, the method further comprises seeding isolated animal fibroblasts or fibroblast-like cells onto the scaffold to form a composition. In some embodiments, the collagen, fibroblasts, and / or fibroblast-like cells are associated with the scaffold by any of the means described elsewhere herein, such as nonspecific adsorption, covalent interaction, or specific adsorption.

[0152] In some embodiments, methods for producing cruelty-free leather can include forming and tanning a cell and scaffold composition. In some embodiments, the method can include further processing the cell and scaffold composition, for example, to achieve the thickness and texture of natural leather. In some embodiments, tanning can occur after at least partial decellularization of the cell layer. In some embodiments, tanning can make cruelty-free leather resemble natural leather, which can be a durable and flexible material made by tanning animal hides and skins, often bovine hides. Tanning, as used herein, can refer to the process of treating animal skin to produce leather or the process of treating a composition disclosed herein to produce cruelty-free leather. Tanning can be performed in a variety of ways, including vegetable tanning (e.g., using tannins), chrome tanning (chromium salts, including chromium sulfate), aldehyde tanning (using glutaraldehyde or oxazolidine compounds), syntanning (synthetic tannins using aromatic polymers), bacterial staining, and the like. In some embodiments, tanning can be metal-free. In some embodiments, tanning can be an environmentally friendly process. In some embodiments, tanning can be performed to convert proteins in the hides, skin, or compositions disclosed herein into a stable material that will not decay, while allowing the material to maintain flexibility. In some embodiments, chromium can be used as a tanning material. In some embodiments, tanning can include chromium, aluminum, zirconium, titanium, iron, sodium aluminum silicate, formaldehyde, glutaraldehyde, oxazolidine, isocyanate, carbodiimide, polycarbamoyl sulfate, tetrakishydroxyphosphonium sulfate, sodium p-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]benzenesulfonate, pyrogallol, catechol, syntan, or any combination thereof. In some embodiments, tanning can be performed on engineered cells in a scaffold.In some embodiments, the tissue to be tanned may include a fiber or multiple fibers (e.g., polyester fiber, synthetic fiber, natural fiber). In some embodiments, the pH of the cell layer or layered structure may be adjusted (e.g., lowered; e.g., to about pH 2.8-3.2) to facilitate tanning. In some embodiments, the pH may be raised ("basified" to a slightly higher level, e.g., about pH 3.8-4.2) after tanning. 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, tanning may be performed on tissue (e.g., tissue from engineered cells). In some embodiments, tanning may also be performed on a layered structure, e.g., a layered structure including at least a dermal layer. In some cases, tanning may also be performed on synthetic leather. In some embodiments, tanning can be performed after forming a cell layer, such as a dermal layer or an epidermal layer, hi some embodiments, tanning can be performed after forming a layered structure.

[0153] In some embodiments, the methods provided herein may further comprise processing. Processing may be performed on one or more cell layers, such as one or more cell layers on a scaffold. (Further) processing may be performed on (e.g., tanned) hides. In some embodiments, the processing may be selected from the group consisting of curing, soaking, bating, pickling, deacidifying, thinning, retanning, lubricating, crusting, wetting, wringing, shaving, rechroming, neutralizing, dyeing, fat-recoring, filling, peeling, stuffing, whitening, fixing, setting, drying, conditioning, beating, staking, buffing, finishing, oiling, brushing, padding, impregnating, spraying, roller coating, curtain coating, polishing, plating, embossing, ironing, burnishing, tumbling, and combinations thereof.

[0154] In some embodiments, cruelty-free leather may comprise collagen and extracellular matrix components produced by cells of the dermal and / or epidermal layers disclosed herein. In some embodiments, cruelty-free leather may comprise at least a partially decellularized dermal and / or epidermal layer as disclosed herein. In some embodiments, cruelty-free leather does not comprise an epidermal layer. In some embodiments, cruelty-free leather may also 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, cruelty-free leather may comprise at least a portion of engineered cells (e.g., cells containing a molecular switch). Cruelty-free leather may comprise immortalized cells. In some embodiments, cruelty-free leather may comprise isolated cells. In some embodiments, cruelty-free leather may comprise a cell line.

[0155] In some embodiments, cruelty-free leather may include hair. In some embodiments, cruelty-free leather may include hair in one or more layered structures. In some embodiments, cruelty-free leather may include fur. In some embodiments, the hair (e.g., fur) may be natural, synthetic, or a combination thereof. In some embodiments, the hair (e.g., fur) may grow from cells in the cruelty-free leather or may be added to the cruelty-free leather from an exogenous source. In some embodiments, cruelty-free leather may be hairless.

[0156] Cruelty-free leather may include at least a portion of a prokaryotic cell, a eukaryotic cell, or a combination thereof. In some embodiments, cruelty-free leather may include at least a portion of a bacterial cell, such as E. coli. In some embodiments, cruelty-free leather may include at least a portion of a eukaryotic cell (e.g., bovine cell, porcine cell, human cell, Saccharomyces cerevisiae).

[0157] In some embodiments, at least a portion of one or more cells of the cruelty-free leather may be genetically engineered cells. The term "genetically engineered" may refer to an artificial modification to the nucleic acid content of a cell. Thus, genetically engineered cells may include cells that contain an insertion, deletion, and / or substitution of one or more nucleotides in the genome of the cell, as well as modifications that include the introduction of a self-replicating extrachromosomal nucleic acid inserted into the cell. Genetically engineered cells also include those in which transcription of one or more genes is altered, e.g., increased or decreased.

[0158] In some embodiments, the thickness of the leather may be reported in millimeters, ounces, or irons. In some embodiments, 1 ounce is equivalent to 1 / 64 inch or 0.0156 inch or 0.396 mm. In some embodiments, 1 iron is equivalent to 1 / 48 inch or 0.0208 inch or 0.53 mm. How to use

[0159] Disclosed herein in some embodiments is a method comprising grafting a composition disclosed herein onto a patient in need of a skin graft for the treatment of lost or damaged skin. In some embodiments, the patient may have a laceration, bruise, lesion, sore, burn, wound, surgical wound, surgically removed skin, necrotic skin, or any combination thereof. Disclosed herein in some embodiments is a method comprising tanning a composition disclosed herein to produce cruelty-free leather. Disclosed herein in some embodiments is a method of using the cruelty-free leather disclosed herein as a substitute for conventional leather in leather products. Disclosed herein is a leather product comprising the cruelty-free leather disclosed herein. In some embodiments, the leather goods may include watch straps, belts, suspenders, packaging, shoes, boots, footwear, gloves, clothing, bags, clutches, wallets, coin purses, billfolds, key pouches, credit card cases, pencil cases, backpacks, cases, purses, saddles, saddlery, whips, luggage, travel goods, rucksacks, portfolios, briefcases, briefcases, attaché cases, pet supplies, leashes, collars, hunting and fishing supplies, gun cases, cutlery cases, firearm holsters, stationery, writing implements, book covers, camera cases, eyeglass cases, cigarette cases, cigar cases, jewelry cases, cell phone holsters, sporting goods, balls, basketballs, soccer balls, footballs, or combinations 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 a trunk, a suitcase, a travel bag, a beauty case, a toiletry item, or any combination thereof. Disclosed herein in some embodiments is a method of using a composition as disclosed herein for the treatment of a disease or illness.Disclosed herein in some embodiments are kits comprising a composition as disclosed herein or a leather product as disclosed herein. [Example] Example 1. Cell adhesion to nylon and bamboo / cotton scaffolds

[0160] To evaluate cell adhesion to various scaffold materials, bovine dermal fibroblasts were attached to 1-ply or 2-ply nylon scaffolds or 40:60 bamboo / cotton scaffolds under static and rocking growth conditions for 17 days. Cell proliferation was assessed by measuring glucose levels before each administration. Generally, rocking conditions resulted in greater glucose consumption. The bamboo / cotton blend consumed the most glucose under both static and rocking conditions, while glucose consumption on rocking 2-ply nylon scaffolds steadily increased over time. Microscopic observations, including cells stained with calcein-AM and Hoechst staining (see Figures 1A, 1B, 1C, 1E, 1F, 1G, 1I, and 1J), showed that cell adhesion on the 1-ply nylon surface was lower than that on the 2-ply nylon surface, despite clear extracellular matrix formation on the scaffold. Microscopic observations, seen in Figures 1D, 1H, and 1K, showed that cells attached to bamboo / cotton scaffolds grew best under both rocking and static conditions, exhibiting spindle-like cell growth. These data, as compared with nylon, suggest that woven materials may not provide sufficient surface area or flexibility for good cell adhesion. Alternatively, nonwoven materials, such as bamboo / cotton blends, have shown significantly improved cell adhesion, and nonwoven nylon, such as needle-punched nylon scaffolds, have been shown to increase cell adhesion compared to simple meshes, further enhancing the benefits of nonwoven materials. Example 2. Conditions for dissolving the scaffold

[0161] Poly(vinyl alcohol) (PVOH) and poly(lactic acid) (PLA) were tested as dissolvable scaffolds for fibroblast adhesion, preventing fiber protrusion and improving tissue quality. The conditions required for dissolving PVOH scaffolds were deionized water or DMEM at 95°C for 1 minute. The conditions required for dissolving PLA were overnight in organic solvents such as benzylamine or ethyl acetate. Successful dissolution of the scaffolds was determined by observing visual changes and assessing scaffold removal and potential damage to the hide, as well as the weight of the scaffolds before and after dissolution, as seen in Table 1. Dissolving PLA in benzylamine overnight resulted in a 72% weight loss of the scaffold, while dissolving it in ethyl acetate under similar conditions resulted in a 93% weight loss of the scaffold. The effect of dissolution conditions on hide was evaluated on salted rawhide (pre-tanning) and tanned but un-liquidated hide (post-tanning).

[0162] Exposing untanned hides to high temperatures (95°C) on a PVOH scaffold resulted in tissue destruction. However, exposing tanned hides to high temperatures (95°C) on a PVOH scaffold resulted in approximately 100% scaffold removal, albeit not complete destruction, while still reducing hide quality. Longer dissolution times (45 minutes or 1.5 hours) at lower dissolution temperatures in water (90°C) resulted in 27.3% and 46.7% scaffold removal, respectively, but the denaturation temperature of tanned hides is approximately 70°C, resulting in damage to the hide. Due to the lower denaturation temperature, a lower dissolution temperature of 40-50°C may be desirable to avoid damage to the hide. However, exposure to benzylamine and ethyl acetate did not significantly alter hides on a PLA scaffold before and after tanning. This data highlights the ability to dissolve PLA and PVOH scaffolds under various conditions to help prevent fiber protrusion from the tissue and improve tissue quality. [Table 1] Example 3. Effect of scaffold pore size on tissue quality

[0163] A series of PLA scaffold materials were tested with different pore sizes, including 55 μm, light punch 55 μm (55LP), 80 μm, and 100 μm, and tissue quality was evaluated as a function of pore size in a cardholder format. Scaffolds were assembled into frames and placed in a bioreactor. Seeding solution (2.75x10 6Scaffolds were seeded with 1000 cells / mL and allowed to stand, while rocking conditions were initiated after 1 week. After seeding, cells were not fed for 2 days, and then fed 7 days a week with complete medium changes. The medium was sampled daily. Tissues were grown for 27 days, and tissue biopsies were taken using a sterile 4 mm biopsy punch. No significant differences in sulfated glycosaminoglycans (sGAGs) were observed after 24 or 96 hours (n = 2 biopsies), and no significant differences in collagen or total protein concentrations were observed between scaffolds with different pore sizes (n = 3 biopsies) (Figure 31). The 55LP scaffold exhibited the highest bending stiffness of the raw and raw hides, indicating that increasing thickness and moment of inertia increase the stiffness of the material. Scaffolds with smaller pore sizes were heavier both before and after incubation, resulting in a tanned hide that felt more robust, as seen in (Figure 13). However, as seen in Figure 13, the calculated net weight of tissue content for all scaffolds was similar regardless of pore size. Histological examination was performed on trichrome-stained tissue cross sections. Generally, larger pore sizes resulted in better tissue ingrowth, as seen in Figures 15A-F, where the average pore size was 100 μm, compared to Figures 16A-F, where the average pore size was 50 μm. As seen in Figures 14A-F, the 55LP scaffold, with the smallest pore size and greatest starting thickness, exhibited poor tissue ingrowth. Generally, as seen in Figures 2A, 2B, 2C, and 2D, the larger pore-sized materials exhibited more inconsistent and light spots when scanned, but PLA scaffolds generally exhibited increased skin consistency compared to pressed PET scaffolds. The calculated tissue weight was similar across all conditions, regardless of pore size. Histological examination was performed on trichrome-stained tissue cross sections. Generally, larger pore sizes result in better tissue ingrowth, as seen in Figure 3A where the pores are 100 μm compared to Figure 3B where the pores are 50 μm. Scaffolds with larger pores result in better tissue ingrowth, but smaller pore sizes result in a greater weight contribution from the fibers, creating the illusion of a heavier, stronger skin, demonstrating the importance of the porosity of the scaffold material. Example 4. Effect of PLA immersion

[0164] PLA scaffolds were tested to determine whether soaking in fetal bovine serum (FBS) was necessary for proper cell adhesion and proliferation. Soaking in FBS allows sticky proteins to adsorb onto the surface, thus promoting cell adhesion. Two boxes containing five square scaffolds made of PLA with a 1.7 dtex and 60 μm pore size were prepared, along with a control box containing PET. One set of five scaffolds was filled with pure FBS to cover the channels and soaked overnight at 37°C, while the other set and the control (PET scaffolds) were not (see Figure 4A). After aspirating the FBS, fibroblasts were seeded into all channels. These scaffolds were harvested at various times. They were biopsied using a sterile biopsy punch once on day 1, twice on day 2, and twice on day 4, and the cultured cells were saved for analysis. Biopsies from days 2 and 4 were used for further analysis. Three of these biopsies were used for DNA assays, and one was used for calcein imaging. The DNA assay showed that FBS-soaked PLA scaffolds resulted in the highest initial cell adhesion compared to non-soaked PLA scaffolds (Figure 4B), thus demonstrating the beneficial effects of channel soaking and protein adsorption on the surface. Example 5. Protein-Scaffold Conjugates for Cell Adhesion and Proliferation

[0165] Poor cell adhesion to biomaterials after seeding can lead to cell death, reduced viability, and poor collagen production, which can be addressed by improving the cytocompatibility of the biomaterial surface. As observed in Example 2, conventional cell culture proteins can be applied to enhance cell adhesion. Low-cost, cruelty-free options were also evaluated. Proteins extracted from sweet pea and wheat flour were conjugated to PLA and lyocell and compared with the respective unmodified scaffolds to determine whether functionalization of the scaffold surface could enhance cell adhesion and proliferation. Proteins were conjugated to PLA using a carbodiimide crosslinker (EDC / NHS) via a bond between the activated carboxylic acid of PLA and the amine of the protein. Attempts to enhance protein-to-PLA conjugation included adding carboxyl groups to PLA prior to protein conjugation. Proteins were conjugated to lyocell via the Maillard reaction in an autoclave. Autoclaving was performed once, twice, or four times. Cell culture conditions were as follows: 500k / cm in tissue culture-treated 48-well plates using DMEM HG containing 10% FBS. 2Cell adhesion and proliferation were measured by MTT and DNA assays, demonstrating that protein conjugation to lyocell, but not PLA, significantly enhanced cell adhesion and proliferation compared to unmodified controls. Figure 5 highlights that the percentage of cells on pea protein, wheat protein, and untreated (NT) scaffolds increased after 1 and 4 days of lyocell surface modification. As seen in Figure 6D, fluorescence images after 1 day show cell aggregation on unmodified lyocell, whereas spindle-like growth occurred after conjugation with pea protein (as seen in Figure 6E), highlighting the improved cytocompatibility after modification. Conjugation of pea protein or wheat protein to PLA resulted in little change in the spindle-like cell growth seen in the micrographs of Figures 6A, 6B, and 6C. Furthermore, conjugates of sweet pea protein were shown to be more effective than wheat flour protein (as seen in Figures 5, 6B, 6C, 6E, and 6F). These examples demonstrate the potential for enhancing the cytocompatibility of scaffold surfaces by treatment with biocompatibility-enhancing groups such as these or other polypeptide-containing groups. Furthermore, chemical treatments to tailor surface properties may increase cell affinity or tissue outcomes, which can be achieved through various synthetic approaches, for example, treatment with sodium hydroxide or glycidyltrimethylammonium chloride (GTMAC).

[0166] In another example, pea and wheat proteins were first extracted and sterilized. They were incubated with PLA- and lyocell-based scaffolds. Cells were then seeded onto biopsy punches of these scaffolds and cultured in well plates. The scaffolds were imaged with calcein AM, and cell number and activity were measured using DNA and MTT assays, respectively. Figures 25A-B show an example of the difference between spread cells and rounded, aggregated cells, the former being a preferred morphology indicating cellular affinity for the substrate. No changes in cell morphology, as seen in Figures 17A-C, or cell activity, as seen in Figure 18, were observed with PLA. However, as seen in Figure 18, pea protein treatment on lyocell increased cell activity by 80%, while wheat protein treatment increased it by 40%. Furthermore, as seen in Figures 17D-F, cell morphology changed from round to spindle-like, indicating increased affinity for the surface upon treatment.

[0167] To determine whether the utilization of these low-cost proteins could be improved, additional conjugation methods were tested, including the Maillard reaction, surface activation, and EDC / NHS conjugation. The Maillard reaction was approximated by autoclaving the scaffold while immersed in a protein-rich solution. In some cases, the scaffold surface was activated by oxidation with sodium periodate, and the scaffold was then either seeded directly or conjugated with proteins via the Maillard reaction. Proteins were conjugated to PLA using a carbodiimide crosslinker (EDC / NHS) via a bond between the activated carboxylic acid of PLA and the amine of the protein. The Maillard reaction assisted the conjugation of pea protein to lyocell, as seen in Figure 20C, and was found to improve cell spreading compared to unmodified scaffolds, as seen in Figure 20B. The use of EDC / NHS components to covalently bond pea protein to PLA was also found to increase cell viability by over 50% compared to unmodified PLA, as seen in Figure 21. In this case, previous cold coatings were observed without any change in cell viability, as seen in Figure 18. Oxidation with lyocell alone did not result in long-term cell attachment. While a large spike in cell viability can be seen on day 1 after seeding, this activity significantly decreased by day 4, as seen in Figure 21. This decrease in cell viability can also be supported by the lack of cell spreading in the calcein image taken on day 4, as seen in Figure 20E. However, the combination of both oxidation and Maillard reaction using pea protein on lyocell resulted in cells that appeared very well spread, as seen in Figure 20F, although this increased cell spreading was not reflected in the number of viable cells seen in Figure 21.

[0168] The Maillard reaction was further investigated by adjusting the number of autoclave cycles. One, two, or four autoclave cycles were performed and compared with pure cold immersion without autoclave cycles. As seen in Figure 19, one and two autoclave cycles resulted in the highest cell activity on day 4 after seeding, demonstrating over three times the activity compared to the untreated condition. Cold immersion significantly increased cell activity on day 1, but this activity significantly decreased on day 4. This decrease in activity is likely due to the detachment of protein groups from the scaffold, followed by a subsequent decrease in activity. The increased initial activity compared to one, two, or four autoclave cycles is likely due to higher initial protein quality due to fewer denaturing autoclave cycles.

[0169] These examples of activating scaffold surfaces and conjugating them with protein sources demonstrate the potential for enhancing cytocompatibility by treating the scaffold surface. Furthermore, chemical treatments to tailor surface properties may enhance cell affinity or tissue outcomes. Although not shown here, other methods that can be used to activate the surface of fibers include treatment with sodium hydroxide or glycidyltrimethylammonium chloride (GTMAC). Example 6. Effect of seeding density on tissue outcome

[0170] To optimize the seeding density of bovine dermal fibroblasts, the effect of cell seeding density on tissue outcome was evaluated. Optimization was performed using small-scale PET scaffolds. 125kJ / cm 2 , 250k / cm 2 , 500k / cm 2 , and 1M / cm 2 Seeding density (minimum 30k / cm 2 , max 1M / cm 2) were examined after a culture time of 6.5 weeks (minimum 4 weeks, maximum 8 weeks), which is the time for preserving samples for collagen analysis and scanning electron microscope (SEM) imaging. Collagen concentration was assessed after 24 or 96 hours of digestion following biopsy with an 8 mm biopsy punch. No statistically significant differences in collagen concentration were observed between any of the seeding densities after any time period. 2 , 125k / cm 2 , and 62.5 kJ / cm 2 The seeding density was 220cm. 2 These seeded scaffolds were cultured for 8 weeks and treated similarly to assess collagen production. After 24 or 96 hours of digestion, 65kJ / cm2 2 and 125k / cm 2 There was no statistically significant difference in the amount of collagen produced between areas seeded at a seeding density of 125k / cm (Figures 22 and 23). 2 and 500k / cm 2 There was a difference between the areas seeded at a seeding density of 30kJ / cm and the areas seeded at a seeding density of 100kJ / cm. As the seeding density increased, the amount of collagen produced increased. 2 10 shows that substantially higher soluble collagen was measured when a seeding density of 100 μg / ml was used. Example 7. Tissue and leather growth process

[0171] The cell source (immortalized / isolated cells) is thawed and expanded until the desired cell number is reached. The scaffold material is prepared on a frame and sterilized along with the bioreactor vessel. Cells are seeded onto the scaffold material in the bioreactor (500k cells / cm). 2The engineered animal hide is then cultured in a medium (cloth) and tissue growth medium is added, which is periodically replaced. To convert the grown tissue into leather, the engineered animal hide is removed from the growth medium and frame and optionally washed. Optionally, the hide can be subjected to salting, demineralization, calcification, or baiting. The hide is then subjected to tanning to produce leather, which can then be optionally processed (i.e., adding fat solution, adding dyes, adding retanning agents). Example 8. Effect of fiber diameter and sterilization parameters on fiber strength

[0172] Fiber diameter can have a significant impact on the texture of the resulting leather, particularly its roughness. Furthermore, material roughness and fiber diameter can affect cell behavior. A series of PLA fiber diameters ranging from 1 to 40 dtex were investigated. As seen in Table 2, increasing fiber thickness from the control (100 gsm) to 6.7 dtex decreased areal density, collagen, and DNA content. [Table 2] Example 9. Cross-sectional histological images of cell and scaffold compositions

[0173] Figures 7A and 7B show histological images of 5 μm sections of tissue stained with trichrome stain on a scaffold, with labels highlighting dense tissue, fibers, nuclei, and non-dense tissue. Figure 8A shows a histological image of a 5 μm section of natural bovine hide stained with trichrome stain, with labels indicating the location of the grain and dermis. Figure 8B shows a histological image of a 5 μm section of the superficial layer (grain) of bovine hide stained with trichrome stain. Figure 9A shows a fluorescent microscopy image of fibroblasts at 4x zoom after addition of calcein AM. Live cells fluoresce green, and the arrows highlight the spreading of cells, indicating affinity to the scaffold. Figure 9B shows a fluorescent microscopy image of fibroblasts at 10x zoom after addition of calcein AM. Live cells fluoresce green, and the arrows highlight the spreading of cells, indicating affinity to the scaffold. The second arrow highlights the fibers illuminated with white light. Figure 10A shows a fluorescent microscopy image of fibroblasts at 10x zoom after addition of calcein AM, with live cells fluorescing green. Cell spreading and adhesion highlight good affinity with the scaffold. Figure 10B shows a fluorescent microscopy image of fibroblasts at 10x zoom after addition of calcein AM, with live cells fluorescing green. Cells appear rounded and clumped, indicating poor affinity with the scaffold. Figure 11 shows a histological image of a 5 μm section of tissue stained with trichrome stain, with labels indicating the slide overview, scale bar, cursor position, and tissue thickness. Example 10. Effect of basic fiber materials on tissue outcomes

[0174] Cell adhesion and tissue growth have been attempted with a variety of different materials. In this example, various fiber types were tested first for cell adhesion and then for tissue growth. Various sustainable biobased materials, as well as less sustainable but commercially available materials, were tested. All materials tested here were needle-punched nonwoven materials. PET and nylon materials served as controls. Two different PLA materials were tested: one simply known as PLA and the other known as 50-50. The 50-50 material contains two types of PLA fibers, each comprising 50% by mass. Half of the fibers are the standard high-melting-point PLA used in the first sample type. The other half are special core-sheath fibers, with a core made of a high-melting-point polymer and an outer layer, or sheath, made of a low-melting-point polymer. This special fiber structure allows the sheath material to be easily melted or softened by heat treatment while maintaining the integrity of the core, making the needle-punched nonwoven more easily processable into desired shapes. Two types of viscose materials with different cross sections, circular and trilobal, were tested, designated regular viscose and trilobal viscose, respectively. Viscose is a cellulose fiber made by dissolving wood or other cellulosic fibers in a caustic sulfide solution and then reconstituting the dissolved pulp into fibers. Finally, lyocell materials were tested. Unlike viscose, lyocell is dissolved using a solvent process, which significantly reduces hazardous waste compared to viscose.

[0175] As seen in Figure 26, initial adhesion studies showed high glucose consumption after 7 days of culture on PET, nylon, PET, and 50-50 scaffolds. Significant glucose consumption and cell activity are achieved only if cells have sufficient affinity for the substrate; thus, glucose consumption, like MTT, can be used as an indirect measure of cell affinity for the substrate. Cellulose-based materials consumed less glucose than the previously mentioned materials. Among the cellulosic materials, cotton showed the highest consumption, but as seen in Figure 26, cotton was inferior to the other materials mentioned above. Under these culture conditions, cells did not find high affinity for the cellulosic surface. However, when the culture medium was switched, cells were observed to adhere to the lyocell-based material.

[0176] Subsequent tissue culture experiments tested additional materials. Scaffolds containing polyvinyl alcohol, also known as PVOH or PVA, were tested, as well as three types of Lyocell scaffolds. These three new Lyocell scaffolds used various weights and fiber diameters, including 1.7 dtex, 3.3 dtex, and 6.7 dtex. Additionally, an alginate-containing scaffold was tested. Another scaffold included a Vicryl mesh, which contains PLGA fibers woven into a mesh. Total collagen content and collagen content normalized by total protein content were assessed for each tissue grown. The PET hPL condition achieved the highest collagen per biopsy, as seen in Figure 27, demonstrating that both media conditions, in addition to the scaffold condition, influenced the deposited tissue. Tissues grown on PLA and PVOH had the next highest collagen content, followed by PET in FBS and then Vicryl mesh. Lyocell materials with different fiber weights exhibited significantly different scores from each other. The 3.3 dtex scaffold appeared to produce the most collagen of the three, while the 1.7 dtex and 6.7 dtex scaffolds appeared to have the least amount of tissue deposited. Notably, the alginate scaffold produced the least amount of collagen. Next, small tissue squares were tanned and marked to distinguish them from each other, as seen in Figure 28. hPL PET appeared to produce the most collagen and stained the deepest. Example 11. Effect of scaffold shape factors on tissue outcome

[0177] Herein, a number of biomaterial scaffold form factors are tested to evaluate their effect on tissue formation. The control used in this experiment is a simple needle-punched nonwoven material that is heated and plated to a predetermined thickness on one side, leaving the other side smooth; this condition is referred to as the "Pressed Control." 3M Thinsulate Material Type G was tested here and contains three main layers: two spunbond and one airlaid nonwoven, sandwiched together in a spunbond, then airlaid, then spunbond layer. The material is polyester and is likely coated with various materials to maximize its thermal insulation. The material is also sewn together at intervals with lines of stitching; for the purposes of this experiment, sutures were used to create a more uniform thickness throughout the material. This condition will be referred to as "Thinsulate." Next is a towel-like material made from PET material called "Secant Towel." This material can be seen in detail in Figures 41A-B. Here, the looped fiber pattern resulting from the flat knit is visible, suggesting it is a form of terrycloth. Next is a thicker material containing two outer fabrics separated by fibers running at regular intervals between them. These fiber types are commonly known as spacer fabrics and are referred to here as "Secant Spacer." Next is "Fibertex," another needle-punched nonwoven. However, this "non-press control" fabric is not pressed, and unlike the control, which contains a pair of PET fibers, this fabric consists of only a single Lyocell fiber. Next is a "Double Stack of Pressed," in which two "Press Control" fabrics are laminated together, one on top of the other, with the rough sides facing inward and the smooth sides facing outward. The two pieces of fabric are laminated together by stitching.Next is the "Smooth Facing Laminate," in which a Vicryl mesh sheet is laminated to the top of the "Press Control" fabric by stitching, adjacent to the smooth side of the "Press Control" fabric. Next is the "Rough Facing Laminate," which is the same as the "Smooth Facing Laminate" except for the placement of the mesh and "Press Control" fabric, with the rough side of the "Press Control" fabric adjacent to the mesh. Next is the "Autoclaved Laminate," which is the same as the "Rough Facing Laminate," except the scaffolds are laminated together and then autoclaved; in this case, the other side is sterilized before assembly. Finally, there is the "Absorbable only," in which two sheets of Vicryl mesh are laminated together so that the entire scaffold contains a knitted absorbent material.

[0178] After the full incubation period on cardholder-scale materials, the skins were analyzed for biochemical content and physical properties. When normalized by the volume of biopsies taken, the "absorbent only" condition had the highest collagen concentration at the 96-hour digestion time point, as seen in Figure 32. However, this result can be discounted by the fact that its thickness, and therefore the volume of the biopsy, was smaller than any of the other conditions, as seen in Figure 33. Thinsulate, Fibertex, and rough laminate therefore have the next highest collagen density among the shape factors tested, as seen in Figure 32. Looking at other physical and biochemical characteristics, Secant Towel had the highest DNA content, and Secant Spacer had the highest thickness. In contrast, the pressed double stack had higher DNA, thickness, and weight content compared to the other conditions. In Figure 34, Fibertex is directly compared to the pressed control condition, and it can be seen that the thickness, weight, hydroxyproline, and DNA content are all very similar. This is understandable given that the scaffold samples have the most similar shape factors. In Figure 35, collagen values ​​after 4 and 8 weeks of tissue growth are compared between the pressed control and Fibertex. Unlike the hydroxyproline measurements at 8 weeks, soluble collagen is seen to be slightly lower in Fibertex at 8 weeks but higher at 4 weeks. Because Fibertex has a slightly different internal structure compared to the pressed control, differences in growth rate may be expected.

[0179] In addition to biochemical changes, these different shape factors have also been shown to affect how tissue forms in three dimensions. Histology can be used to observe how tissue forms differently in different scaffolds. The pressed control exhibited relatively even tissue distribution throughout the thickness of the scaffold, with slightly denser tissue deposition on one surface, as shown in Figure 36. In contrast, the pressed double stack of scaffolds had a very clear gradient of tissue deposition, with higher density at the edges and almost none in the center, as seen in Figure 37, and even completely empty areas. The pressed double stack of scaffolds was much thicker than the pressed control, suggesting an effect of material thickness. The secant towel material, like the pressed control, yielded tissue with relatively continuous tissue deposition, as seen in Figure 38. Unlike the pressed control, the secant towel appears to have a distinct surface pattern on one side, with a wavy surface. There is a high density of fibers near the peaks and few, if any, fibers in the valleys. The local collagen distribution of the pressed control and secant towel can be compared. In the pressed control, the collagen deposition is very planar, as seen in Figures 39A-B, whereas in the secant towel, the collagen deposition is much more random, and the arrangement appears more circular and isotropic in comparison, as seen in Figures 40A-B. Smooth- and rough-surface laminates can be distinguished by the nature of the two outer surfaces that result from the tissue deposition. In the smooth-surface laminate, the rough side of the pressed control faces outward, and a cross-section of the tissue shows one smooth and one rough side, as seen in Figure 42. In contrast, in the rough-surface laminate, the rough side of the pressed control faces inward, and a cross-section of the tissue shows two smooth outer surfaces, as seen in Figure 43. The autoclaved laminate, like the rough-surface laminate in Figure 44, shows two smooth sides, but with a dense layer of collagen on one side. This is due to differences in the properties of Vicryl after autoclaving, which may have hydrolyzed prior to seeding. Fibertex exhibits a slightly different texture deposition compared to the pressed control.Where gaps exist in the tissue cross-section, some discontinuous tissue formation is observed; however, outside these gaps, relatively continuous tissue deposition is observed, as seen in Figure 45. The Thinsulate sandwich structure exhibits slightly thicker tissue cross-sections, but relatively continuous tissue accumulation throughout the entire thickness. Despite denser fiber networks on the top and bottom surfaces from the spunbond layer, good tissue penetration is observed through the surface, and no impermeable layer appears to be present in Figure 46. Finally, the absorbent tissue in Figure 47 exhibits extremely dense tissue formation not seen in any of the other tissues. Unlike the other materials, this material is initially very thin, highly cytocompatible, and dissolved away by the end of the culture. All of these factors likely contributed to the high tissue density observed through both the histological and biochemical data. To aid interpretation and better understand the anatomical structure of the histological images, please refer to Figure 11.

[0180] Throughout this experiment, various shape factors were tested, including needle-punched nonwovens, three-layer sandwiches, mesh and nonwoven laminates, terry cloth, spacer fabrics, and pure knit meshes. Other shape factors, such as woven meshes as seen in Example 1, were also tested. The diverse effects these shape factors can have on tissue formation can be seen. The hollow tissue center in the double-stack case, the incredibly dense tissue in the absorbent-only case, and the continuous tissue despite the absence of fibers in towel-like fabrics can all be exploited to achieve desired tissue properties. This data demonstrates that shape factors can influence tissue outcomes. Example 12. Effect of scaffold adhesion factors on leather results

[0181] Surface modification of polylactic acid (PLA) and PLA scaffolds was tested to evaluate the effectiveness of covalent and noncovalent adhesion factors on the surface. Polylactic acid (PLA) scaffolds were coated with glycidyltrimethylammonium chloride (GTAMC), which allows the introduction of amines (PLA-NH2) onto the polymer surface, which can undergo nonspecific adsorption to collagen. Polylactic acid scaffolds were coated with pea protein and hydrolyzed under acidic conditions, exposing hydroxyl groups, carboxylic acids, and ketones (PLA-hyd), which can then undergo nonspecific adsorption to collagen. Polylactic acid scaffolds were coated with pea protein and oxidized using sodium periodate (PLA-ox), which allows for increased nonspecific collagen adsorption. Fibroblasts were seeded on each of the three scaffolds and the PLA control and allowed to proliferate to generate extracellular matrices containing collagen. Optionally, the scaffolds were removed by dissolution in benzylamine, ethyl acetate, or acetone. The resulting material is examined, for example, by histology and microscopy, to determine the resulting collagen density and distribution, taking into account the aforementioned scaffold modifications. The resulting material is tanned to produce cruelty-free leather, and the thickness and strength of the hides are compared.

[0182] In another example, a polylactic acid (PLA) scaffold is coated with a chemically modifiable polymeric material. In one example, the polymer-coated PLA is further modified by the addition of the carbodiimide linker EDC (PLA-EDC). The PLA-EDC is seeded with fibroblasts, and the fibroblasts are allowed to proliferate, resulting in the formation of an extracellular matrix containing collagen. The collagen is modified with N-hydroxysuccinimide. The collagen and the carbodiimide linker PLA-EDC are chemically crosslinked, resulting in a covalently bonded scaffold-polymer material. In another example, the polymer-coated PLA is further modified by the addition of azide (-N3) (PLA-N3). The PLA-N3 is seeded with fibroblasts, and the fibroblasts are allowed to proliferate, resulting in an extracellular matrix containing collagen. The collagen is then modified with an alkyne and crosslinked to the azide via a click chemistry reaction, resulting in a covalently crosslinked scaffold-collagen material. The PLA-N3-collagen and PLA-EDC-collagen materials are then optionally decellularized to examine collagen density and distribution before being compared to a PLA control via histology and microscopy. The resulting material is optionally tanned to produce a cruelty-free leather, and the thickness and strength of the hide are compared to a PLA control. Example 13. Skin strength and thickness

[0183] The average double tear strength and thickness of hides prepared with various scaffolds were measured and compared to a typical bovine hide crust. The average double tear strength was measured using the ISO 3377-2 method. The measurement results can be found in Table 3. [Table 3]

[0184] The resulting dual tear strength and thickness may indicate that the compositions provided herein, including scaffolds in contact with extracellular matrix, and the methods provided herein, may result in scaffolds with tunable strength simply by adjusting the scaffold material. Furthermore, this data suggests that when compared to bovine hide crusts, the compositions provided herein may have similar strength to natural hide, but in thinner materials.

[0185] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous 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 can be employed in practicing the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby. [Brief explanation of the drawings]

[0186] [Figure 1]Figure 1A shows a microscopic image of unstained fibroblasts adhered to a 1-ply nylon scaffold under rocking conditions. Figure 1B shows a microscopic image of unstained fibroblasts adhered to a 2-ply nylon scaffold under rocking conditions. Figure 1C shows a microscopic image of unstained fibroblasts adhered to a 40:60 bamboo / cotton scaffold under rocking conditions. Figure 1D shows a microscopic image of fibroblasts adhered to a 1-ply cotton scaffold under rocking conditions. Figure 1E shows a microscopic image of calcein-AM stained fibroblasts adhered to a 1-ply nylon scaffold under rocking conditions. Figure 1F shows a microscopic image of calcein-AM stained fibroblasts adhered to a 2-ply nylon scaffold under rocking conditions. Figure 1G shows a microscopic image of calcein-AM stained fibroblasts adhered to a 40:60 bamboo / cotton scaffold under rocking conditions. Figure 1H shows a microscopic image of fibroblasts stained with calcein-AM adhered to a 1-ply cotton scaffold under rocking conditions. Figure 1I shows a microscopic image of fibroblasts stained with Hoechst stain adhered to a 1-ply nylon scaffold under rocking conditions. Figure 1J shows a microscopic image of Hoechst stained fibroblasts adhered to a 2-ply nylon scaffold under rocking conditions. Figure 1K shows a microscopic image of Hoechst stained fibroblasts adhered to a 40:60 bamboo / cotton blend scaffold under rocking conditions. Figure 1L shows a microscopic image of Hoechst stained fibroblasts adhered to a 1-ply cotton scaffold under rocking conditions. [Figure 2] Figure 2A shows scan data for a skin grown on a lightly punched PLA scaffold with a 55 μm pore size. Figure 2B shows scan data for a skin grown on a PLA scaffold with a 55 μm pore size. Figure 2C shows scan data for a skin grown on a PLA scaffold with a 80 μm pore size. Figure 2D shows scan data for a skin grown on a PLA scaffold with a 100 μm pore size. [Figure 3] Figure 3A shows histological images of 5 μm cross sections of tissue stained with trichrome stain when the scaffold is PLA with an average pore size of approximately 100 μm, and Figure 3B shows histological images of 5 μm cross sections of tissue stained with trichrome stain when the scaffold is PLA with an average pore size of approximately 50 μm. [Figure 4A]Figure 4A shows a schematic diagram of the experiment performed to investigate the effect of FBS-soaked PLA scaffolds on cell adhesion and proliferation. [Figure 4B] FIG. 4B shows sample cells / biopsies grown on non-serum-soaked PET controls compared to serum-soaked and non-soaked PLA. [Figure 5] Figure 5 shows the percentage of cells on unmodified, pea protein-conjugated, and wheat protein-conjugated PLA and Lyocell scaffolds measured by MTT assay after 1 and 4 days, where NT is untreated, Pea is pea protein, and Wheat is wheat protein. [Figure 6] Figure 6A shows a fluorescence microscopy image of fibroblasts after 1 day growing on an unmodified PLA scaffold. Figure 6B shows a fluorescence microscopy image of fibroblasts after 1 day growing on a PLA scaffold conjugated with pea protein. Figure 6C shows a fluorescence microscopy image of fibroblasts after 1 day growing on a PLA scaffold conjugated with wheat protein. Figure 6D shows a fluorescence microscopy image of fibroblasts after 1 day growing on an unmodified lyocell scaffold. Figure 6E shows a fluorescence microscopy image of fibroblasts after 1 day growing on a lyocell scaffold conjugated with pea protein. Figure 6F shows a fluorescence microscopy image of fibroblasts after 1 day growing on a lyocell scaffold conjugated with wheat protein. [Figure 7] Figures 7A and 7B show histological images of 5 μm cross-sectional tissue stained with trichrome stain on the scaffold, with labels highlighting dense tissue, fibers, nuclei, and less dense tissue. [Figure 8] Figure 8A shows a histological image of a 5 μm cross section of bovine hide stained with trichrome stain, with the grain and corium locations labeled. Figure 8B shows a histological image of a 5 μm cross section of the superficial layer (grain) of bovine hide stained with trichrome stain. [Figure 9]Figure 9A shows a 4x zoom fluorescence microscopy image of fibroblasts after addition of calcein AM. Live cells fluoresce green, and the arrows highlight cell extensions, indicating affinity to the scaffold. Figure 9B shows a 10x zoom fluorescence microscopy image of fibroblasts after addition of calcein AM. Live cells fluoresce green, and the arrows highlight cell extensions, indicating affinity to the scaffold. The second arrow highlights fibers illuminated with white light. [Figure 10] Figure 10A shows a 10x zoom fluorescence microscope image of fibroblasts after addition of calcein AM, with live cells fluorescing green. Cell spreading and adhesion highlight high affinity to the scaffold. Figure 10B shows a 10x zoom fluorescence microscope image of fibroblasts after addition of calcein AM, with live cells fluorescing green. Cell rounding and clumping indicate low affinity to the scaffold. [Figure 11] Figure 11 shows a histological image of a 5 μm section of tissue stained with trichrome stain, with labels indicating the slide overview, scale bar, cursor position, and tissue thickness. [Figure 12] Figure 12 shows the residual glucose levels measured from the last four feeds from days 10 to 17 of tissue culture in fresh medium, 40:60 bamboo / cotton, 1-ply nylon, 2-ply nylon, and 1-ply cotton scaffolds under static and rocking conditions. [Figure 13] Figure 13 shows basic physical data for tissue on 100 gsm (blue), 50 gsm (red), 55 μm pore size light punch (55LP) (green), pressed PET (PPET) (purple), and VL1 on PPET scaffolds (blue). Physical data includes harvest thickness, crust thickness, harvest weight, raw material weight, crust weight, and calculated tissue-only weight. [Figure 14] Figures 14A and 14B show histological images of tissue grown on a light punch scaffold with a 55 μm pore size, with a 5 μm cross section stained with trichrome stain. [Figure 15]Figures 15A, 15B, 15C, 15D, 15E, and 15F show histological images of 5 μm cross sections of tissue grown on 50 gsm scaffolds stained with trichrome stain. [Figure 16] Figures 16A, 16B, 16C, 16D, 16E, and 16F show histological images of 5 μm cross sections of tissue grown on 100 gsm scaffolds stained with trichrome stain. [Figure 17] Figure 17A shows a fluorescence microscope image of fibroblasts grown on an unmodified PLA scaffold after 4 days. Figure 17B shows a fluorescence microscope image of fibroblasts grown on a PLA scaffold conjugated with pea protein after 4 days. Figure 17C shows a fluorescence microscope image of fibroblasts grown on a PLA scaffold conjugated with wheat protein after 4 days. Figure 17D shows fibroblasts grown on an unmodified lyocell scaffold after 4 days. Figure 17E shows fibroblasts grown on a lyocell scaffold conjugated with pea protein after 4 days. Figure 17F shows fibroblasts grown on a lyocell scaffold conjugated with wheat protein after 4 days. [Figure 18] FIG. 18 shows the cell viability at day 1 (blue) and day 4 (red) 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), normalized to the viability of fibroblasts grown on unmodified PLA (PLA NT). [Figure 19]FIG. 19 shows the relative cell viability at day 1 (blue) and day 4 (red) of fibroblasts grown on unmodified lyocell (Lyo), lyocell conjugated with pea protein by cryoadsorption (Lyo pea cryo), lyocell conjugated with pea protein after one autoclave cycle (Lyo pea auto 1), lyocell conjugated with pea protein after two autoclave cycles (Lyo pea auto 2), and lyocell conjugated with pea protein after four autoclave cycles (Lyo pea auto 4) relative to fibroblasts grown on unmodified PLA (PLA NT). [Figure 20] Figure 20A shows a fluorescence microscopy image of fibroblasts grown on an unmodified PLA scaffold after 1 day. Figure 20B shows a fluorescence microscopy image of fibroblasts grown on an unmodified lyocell scaffold after 4 days. Figure 20C shows a fluorescence microscopy image of fibroblasts grown on a lyocell scaffold conjugated with pea protein, where the pea protein was conjugated via autoclaving after 4 days. Figure 20D shows a fluorescence microscopy image of fibroblasts grown on a PLA scaffold conjugated with pea protein after 4 days. Figure 20E shows a fluorescence microscopy image of fibroblasts grown on an oxidized lyocell scaffold after 4 days. Figure 20F shows a fluorescence microscopy image of fibroblasts grown on an oxidized lyocell scaffold functionalized with pea protein after 4 days. [Figure 21] FIG. 21 shows the relative cell viability of fibroblasts grown on PLA conjugated with pea protein (PLA Pea), Lyocell conjugated with pea protein (Lyocell Pea autoclaved), oxidized Lyocell conjugated with pea protein (Lyocell Pea oxidized), oxidized Lyocell, and unmodified Lyocell scaffolds, compared to cells grown on unmodified PLA, after 1 day (blue) and 4 days (red). [Figure 22]FIG. 22 shows the mean collagen concentrations after 24 or 96 hours of digestion obtained from tissue biopsies with seeding densities of 125k cells / cm2, 250k cells / cm2, 500k cells / cm2, and 1M cells / cm2. [Figure 23] Figure 23 shows the collagen concentration (wet weight) of tissue obtained after 24 hours (blue) or 96 hours (orange) of digestion at seeding densities of 62.5k cells / cm2, 125k cells / cm2, and 500k cells / cm2. [Figure 24] Figure 24 shows the collagen concentration per unit wet weight in tissue obtained after 24 hours (orange) and 96 hours (blue) of digestion for seeding densities of 500k cells / cm, 125k cells / cm, 60k cells / cm, and 30k cells / cm. [Figure 25] Figure 25A shows a fluorescence microscope image of fibroblasts that have successfully attached and spread onto the scaffold, and Figure 25B shows a fluorescence microscope image of fibroblasts that have failed to attach and clumped onto the scaffold. [Figure 26] Figure 26 shows the glucose concentration (g / L) after 1 day (blue), 3 days (red), and 7 days (green) for cell cultures growing on control scaffolds and nylon, PLA, 50:50 PLA:Bioc PLA, cotton, trilobal viscose, regular viscose, and lyocell scaffolds. [Figure 27] FIG. 27 shows the protein content (blue) and collagen content normalized to biopsy (red) of tissues grown on PET scaffolds in hPL medium, and on 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 FBS medium. [Figure 28] Figure 28 shows tanned skins obtained from tissue growth on various scaffolds. The tissues in the top row, from left to right, are hPL PET, FBS PET, PVOH (not shown), PLA in the top row, and 1.7 dtex lyocell, 3.3 dtex lyocell, 6.7 dtex lyocell, alginate, and nylon mesh in the bottom row. [Figure 29] Figure 29 shows a control skin exposed to high temperatures (left), a skin exposed to 95°C to remove the scaffold after tanning (center), and a skin exposed to 95°C to remove the scaffold before tanning (right). [Figure 30] Figure 30a shows pre-tanned skin (left) and post-tanned skin (right) after the skin was treated with benzylamine. Figure 30b shows pre-tanned skin (left) and post-tanned skin (right) after the skin was treated with ethyl acetate. Figure 30c shows skin that was not treated with benzylamine or ethyl acetate. [Figure 31] Figure 31 shows the collagen concentration (blue) and total protein concentration (red) of biopsy tissue cultured on a 55 μm pore size scaffold (55-1), a 55 μm pore size Light Punch (55-LP-1) scaffold, an 80 μm pore size scaffold (80-1), and a 100 μm pore size scaffold (100-1). [Figure 32] Figure 32 shows collagen content normalized by volume after 24 hours (blue) or 96 hours (red) of digestion from tissue grown on pressed control (pressed PET), Thinsulate, Secant Towel, Secant Spacer, Fibertex, pressed PET double stack, Smooth Facing Laminate, Rough Facing Laminate, autoclaved laminate, and absorbable-only scaffold materials. [Figure 33] Figure 33 shows potential normalization factors including DNA (μg), thickness (mm*5), and weight (mg*10) for tissue grown on pressed control (pressed PET), Thinsulate, Secant Towel, Secant Spacer, Fibertex, pressed PET double stack, smooth laminate, rough laminate, autoclaved laminate, and absorbable-only scaffold materials. [Figure 34]FIG. 34 shows the thickness, weight, hydroxyproline content, and DNA content from tissue grown on pressed control (PET) (blue) and Fibertex (red). [Figure 35] Figure 35 shows the collagen content of biopsies after 4 weeks of growth or 8 weeks of growth for tissue grown on pressed control (PET) (blue) and Fibeltex (red) after 24 or 96 hours of digestion. [Figure 36] FIG. 36 shows histological images of 5 μm cross sections of tissue grown on pressed PET scaffolds stained with trichrome stain. [Figure 37] FIG. 37 shows histological images of 5 μm cross sections of tissue grown on double layer pressed PET scaffolds stained with trichrome stain. [Figure 38] FIG. 38 shows a histological image of a 5 μm cross section of tissue grown on a knitted towel-like material stained with trichrome stain. [Figure 39] Figures 39A and 39B show histological images of 5 μm sections of tissue stained with trichrome stain showing the planar, aligned collagen arrangement. [Figure 40] Figures 40A and 40B show histological images of 5 μm cross sections of tissue grown on knitted towel-like material stained with trichrome stain. [Figure 41] 41A and 41B show images of a knitted towel-like material into which tissue can grow. [Figure 42] FIG. 42 shows histological images of 5 μm cross sections of tissue grown on Vicryl and pressed laminate scaffolds stained with trichrome stain. [Figure 43] FIG. 43 shows histological images of 5 μm cross sections of tissue grown on Vicryl and rough-pressed laminate scaffolds stained with trichrome stain. [Figure 44] Figure 44 shows histological images of 5 μm cross sections of tissue grown on Vicryl and autoclaved pressed laminate scaffolds stained with trichrome stain. [Figure 45]Figure 45 shows a histological image of a 5 μm cross section of tissue grown on a Fibertex scaffold stained with trichrome stain. [Figure 46] Figure 46 shows a histological image of a 5 μm cross section of tissue grown on a Thinsulate scaffold stained with trichrome stain. [Figure 47] FIG. 47 shows histological images of 5 μm cross sections of tissue grown on Vicryl-only scaffolds stained with trichrome stain.

Claims

1. A composition comprising a scaffold in contact with an extracellular matrix comprising collagen, said scaffold comprising a needle-punched nonwoven material.

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

3. 1. A composition comprising a scaffold in contact with an extracellular matrix comprising collagen, wherein the scaffold comprises polycaprolactone (PCL), polylactic acid (PLA), polylactic-polyglycolic acid (PLGA), polyethylene terephthalate (PET), nylon, polyethylene (PE), polyethylene furanoate (PEF), polypropylene (PP), polyvinyl alcohol (PVA), cotton, bast fibers, viscose, modal, lyocell, vegetable protein fibers, bio-based materials, viscose, cellulose, alginate fibers, thermoplastic starch, or combinations thereof.

4. 1. A composition comprising an at least partially coated scaffold 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 extract from soybean, a protein extract from pea, a protein extract 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, said scaffold in contact with a solvent capable of dissolving said scaffold.

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

7. The composition of any one of claims 2 to 6, wherein the scaffold comprises a needle-punched nonwoven material.

8. 10. The composition of claim 1 or claim 7, wherein the needlepunched nonwoven material comprises a first plurality of pores.

9. The composition of claim 8 , wherein the scaffold comprises a nonwoven construction creating the first plurality of pores.

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

11. 10. The composition of claim 9, wherein the first plurality of pores comprises an average pore size of about 80 to 120 μm.

12. 10. The composition of claim 9, wherein the first plurality of pores comprises an average pore size of about 50 μm or about 100 μm.

13. The composition of any one of claims 8 to 12, wherein the scaffold comprises a second plurality of pores created by needle punching.

14. 6. The composition of any one of claims 1-3 or 5, wherein the scaffold is at least partially coated with a coating comprising matrigel, vitronectin, fibronectin, a protein extract from soybean, a protein extract from pea, a protein extract 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.

15. 15. The composition of claim 4 or 14, wherein the scaffold comprises an amine hydroxyl group, a sulfhydryl group, a tyrosyl group, or a carboxylic acid group.

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

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

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

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

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

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

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

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

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

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

26. 24. The composition of claim 23, wherein the modification comprises hydrolysis.

27. 27. The composition of claim 26, wherein the modification exposes chemically active groups including hydroxyl, carboxylic acid, ketone, or combinations thereof.

28. 24. The composition of claim 23, wherein the modification comprises oxidation.

29. 29. The composition of claim 28, wherein the oxidation occurs with sodium periodate.

30. 30. The composition of any one of claims 23 to 29, wherein the scaffold comprises an amine, a carboxylic acid, a sulfate, an aldehyde, a hydrazide, a sulfhydryl, a diazirine, an aryl azide, an acrylate, or an epoxide.

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

32. 32. The composition of claim 31 , wherein the scaffold comprises a primary amine.

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

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

35. The composition of any one of claims 1 to 34, wherein the collagen is associated with the scaffold via non-specific adsorption.

36. 36. The composition of any one of claims 1 to 35, wherein the collagen is associated with the scaffold via van der Waals interactions.

37. The composition of any one of claims 1 to 36, wherein the collagen is associated with the scaffold via hydrogen bonds.

38. The composition of any one of claims 1 to 37, wherein the collagen is associated with the scaffold via depletion interactions.

39. The composition of any one of claims 1 to 38, wherein the collagen is associated with the scaffold via electrostatic interactions.

40. The composition of any one of claims 1 to 34, wherein the collagen is associated with the scaffold via covalent interactions.

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

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

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

44. 44. The composition of any one of claims 41 to 43, wherein the collagen is associated with the scaffold via EDC / NHS coupling.

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

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

47. 47. The composition of any one of claims 45 or 46, wherein the collagen is associated with the scaffold via a click chemistry reaction.

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

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

50. 50. The composition of any one of claims 48 or 49, wherein the collagen is associated with the scaffold via Michael donor and Michael acceptor coupling.

51. The composition of any one of claims 48 to 50, wherein the Michael donor comprises an enolate.

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

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

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

55. 55. The composition of any one of claims 53 or 54, wherein the collagen is associated with the scaffold via thiol and maleimide coupling.

56. 56. The composition of any one of claims 53-55, wherein the collagen is associated with the scaffold in the presence of (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (sulfo-SMCC), and the scaffold and collagen comprise a thiol.

57. 35. The composition of any one of claims 1 to 34, wherein the collagen is associated with the scaffold via a Maillard reaction.

58. 58. The composition of any one of claims 1-4, or 6-57, wherein the scaffold comprises a dissolvable scaffold, the scaffold being in contact with a solvent capable of dissolving the scaffold.

59. 60. The composition of claim 5 or 58, wherein the temperature of the solvent is below the boiling point of the solvent.

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

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

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

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

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

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

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

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

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

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

70. 69. The composition of any one of claims 58 to 68, wherein the scaffold dissolves over time via hydrolysis.

71. 71. The composition of any one of claims 58 to 70, wherein the scaffold degrades over time via hydrolysis in neutral aqueous solution.

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

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

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

75. 73. The composition of claim 72, wherein the dissolution agent comprises a strong acid.

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

77. 73. The composition of claim 72, wherein the lysis agent comprises a strong base.

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

79. 73. The composition of claim 72, wherein the dissolution agent comprises an oxidizing agent.

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

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

82. 72. The composition of 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. 83. The composition of claim 82, wherein the solvent comprises a cellulase.

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

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

86. 86. The composition of claim 82 or 85, wherein the solvent comprises an esterase.

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

88. 83. The composition of claim 82, wherein the scaffold comprises calcium alginate.

89. 89. The composition of claim 82 or 88, wherein the solvent comprises an alginate lyase.

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

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

92. 92. The composition of claim 91, wherein the spacer dough comprises a front surface in contact with a filler and a back surface in contact with the filler, the filler separating the front surface from the back surface.

93. 93. The composition of claim 92, wherein separation of the front surface from the back surface by the filler creates a gap that allows for nutrient entry, waste removal, cell adhesion, cell growth, or any combination thereof.

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

95. 95. The composition of any one of claims 2 or 90-94, wherein the three-dimensional woven material comprises terry, fleece, velvet, corduroy, bettin, or any combination thereof.

96. 96. The composition of any one of claims 1 to 95, wherein the scaffold comprises multiple layers of scaffold material.

97. 97. The composition of claim 96, wherein the multiple layers of the scaffolding material comprise a combination of different form factors.

98. 98. The composition of claim 97, wherein the different form factors comprise a nonwoven material, a woven material, a needlepunched material, a three-dimensional structure, or any combination thereof.

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

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

101. The composition of any one of claims 96 to 100, wherein the multiple layers comprise multiple layers of thin material.

102. 102. The composition of any one of claims 96-101, wherein the multiple layers are fused together, held together by entanglement, laminated together, stitched together, glued together, woven together, baked together, or any combination thereof.

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

104. 104. The composition of claim 103, wherein the isolated animal cell is an isolated animal fibroblast or fibroblast-like cell.

105. 105. The composition of any one of claims 103 to 104, wherein the isolated animal cell is an isolated immortalized animal cell.

106. 106. The composition of claim 105, wherein the isolated immortalized animal cells are capable of growing beyond the Hayflick limit.

107. 107. The composition of claim 105 or claim 106, wherein the isolated immortalized animal cell is capable of being propagated for more than about 40 cell divisions, about 50 cell divisions, or about 60 cell divisions.

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

109. 108. The composition of any one of claims 103 to 107, wherein the isolated animal cell is a human cell.

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

111. 111. The composition of any one of claims 1 to 110, which is at least partially decellularized.

112. 112. The composition of claim 111, wherein being at least partially decellularized comprises being substantially free of intact cells in the composition.

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

114. 114. The composition of 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. 115. The composition of any one of the preceding claims, 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. 116. The composition of any one of claims 1-115, wherein the scaffold comprises bast fibers, including flax, hemp, linen, jute, ramie, kenaf, sisal, or any combination thereof.

117. 117. The composition of any one of the preceding claims, wherein the scaffold has a thickness of from about 0.1 mm to about 4 mm.

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

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

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

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

122. 122. The composition of any one of the preceding claims, wherein the scaffold comprises fibers having a dtex of about 6.7 dtex.

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

124. 124. A method of making the composition of any one of claims 1 to 123, comprising seeding isolated animal fibroblasts or fibroblast-like cells onto a scaffold to form the composition.

125. 124. A method of making a composition according to any one of claims 1 to 123, wherein the scaffold comprises a thermoplastic polymer that is subsequently substantially removed from the extracellular matrix prior to tanning.

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

127. 126. The method of claim 125, wherein the thermoplastic polymer comprises polylactic acid (PLA) and the method comprises substantially removing the PLA prior to tanning by contacting the PLA with a solvent to remove the PLA.

128. 124. A method of making the composition of any one of claims 1 to 123, wherein the scaffold comprises a dissolvable scaffold, and wherein the dissolvable scaffold is substantially removed by contacting the scaffold with a solvent.

129. 124. A method of making the composition of any one of claims 1 to 123, comprising needlepunching the scaffold using a needle loom to entangle fibers into a nonwoven scaffold material.

130. 130. The method of claim 129, wherein the needle loom comprises barbed needles.

131. 130. The method of claim 129, wherein the needlepunching creates pores in the scaffold material.

132. 124. A method of making the composition of any one of claims 1-123, the method comprising at least partially coating the scaffold with a coating comprising Matrigel, vitronectin, fibronectin, a protein extract from soybean, a protein extract from pea, a protein extract 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, GTAMC, a carbohydrate-binding module, a cellulose-binding domain, a starch-binding domain, or a combination thereof.

133. 133. The method of claim 132, further comprising seeding isolated animal fibroblasts or fibroblast-like cells onto the scaffold to form the composition.

134. 124. A method comprising transplanting a composition according to any one of claims 1 to 123 into a patient in need of a skin graft for the treatment of lost or damaged skin.

135. 135. The method of claim 134, wherein the patient has a laceration, a bruise, a lesion, a sore, a burn, a wound, a surgical wound, surgically excised skin, necrotic skin, or any combination thereof.

136. 124. A method comprising tanning a composition according to any one of claims 1 to 123 to produce cruelty-free leather.

137. 124. Cruelty-free leather made by tanning a composition according to any one of claims 1 to 123.

138. Methods include: a) seeding 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), polylactic-polyglycolic acid (PLGA), polyethylene terephthalate (PET), nylon, polyethylene (PE), polyethylene furanoate (PEF), polypropylene (PP), polyvinyl alcohol (PVA), cotton, bast fiber, viscose, modal, lyocell, vegetable protein fiber, alginate fiber, thermoplastic starch, or any combination thereof; b) expanding the cells to produce a composition comprising an extracellular matrix; and c) tanning the composition comprising said extracellular matrix to form cruelty-free leather.

139. 138. Use of the cruelty-free leather of any one of claims 136-137 as a substitute for conventional leather in leather products.

140. 140. The method of claim 139, wherein the leather product comprises a watch strap, a belt, a suspender, packaging, a shoe, a boot, footwear, a glove, a garment, a bag, a clutch, a wallet, a purse, a billfold, a key pouch, a credit card case, a pencil case, a backpack, a case, a purse, a saddle, a tack, a whip, luggage, travel goods, a rucksack, a portfolio, a briefcase, a briefcase, an attaché case, a pet product, a lead, a collar, hunting and fishing supplies, a gun case, a cutlery case, a firearm holster, stationery, a writing implement, a book cover, a camera case, an eyeglass case, a cigarette case, a cigar case, a jewelry case, a cell phone holster, a sporting goods, a ball, a basketball, a soccer ball, a football, or a combination thereof.

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

142. 142. The leather product of claim 141, comprised in a watch strap, belt, suspenders, packaging, shoe, boot, footwear, glove, garment, bag, clutch, wallet, purse, billfold, key pouch, credit card case, pencil case, backpack, case, purse, saddle, saddlery, whip, luggage, travel goods, rucksack, portfolio, briefcase, attache case, pet supplies, leash, collar, hunting and fishing supplies, gun case, cutlery case, firearm holster, stationery, writing implement, book cover, camera case, eyeglass case, cigarette case, cigar case, jewelry case, cell phone holster, sporting goods, ball, basketball, soccer ball, football, or combination thereof.

143. 143. The method of claim 140 or the leather product of claim 142, wherein the garment comprises a top, bottom, outerwear, or any combination thereof.

144. 143. The leather product of claim 140 or claim 142, wherein the bag comprises a handbag with or without a shoulder strap.

145. 143. The method of claim 140 or the leather product of claim 142, wherein the luggage comprises a trunk, a suitcase, a travel bag, a beauty case, a toiletry item, or a combination thereof.

146. 124. A method of using a composition according to any one of claims 1 to 123 for the treatment of a disease or disorder.

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

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