A method for producing leather using cell cultures on macroporous polymer scaffolds

Abstract

The present disclosure relates to a method for producing leather comprising culturing cells on a macroporous polymer scaffold. The present disclosure also relates to leather obtained by this method.

Description

[Technical field]

[0001] The present disclosure relates to a method for producing leather comprising culturing cells on a macroporous polymer scaffold. The present disclosure also relates to leather obtained by this method. [Background technology]

[0002] Leather was ranked the number one most resource-intensive material used in the fashion industry, far ahead of cotton or polyester. While animal leather is prized for its durability and beauty, it faces multiple environmental issues, including intensive use of natural resources, animal welfare, and concerns over working conditions. Furthermore, for many applications, leather needs to be free of defaults such as scratches or moth damage. As traditional leather production relies primarily on livestock farming, up to 80% of leather is discarded due to not meeting quality standards.

[0003] With brands under immense pressure to become more ethical and sustainable, there is a need for sustainable alternatives to animal leather that are based on cell biology.

[0004] Synthetic leathers have been developed to address some of these concerns, but they lack the qualities of natural leather. Previous attempts to produce artificial leathers have been described. For example, EP1589098 describes a method for growing fibroblasts seeded on a three-dimensional bioactive scaffold. The scaffold can be made from collagen waste from the tanning process ("split"), particles of pure collagen, particles of collagen waste, or synthetic scaffolds (e.g. made of polymers such as HYAFF). WO2017 / 184967 describes the culture of stem cells or keratinocytes on collagen or PET membranes with 4 μm micropores. However, these culture conditions are not optimal for cell proliferation or collagen production.

[0005] The tanning procedure degrades most of the cellular, molecular and extracellular matrix components. The only components that resist storage and tanning are the collagen and elastin fibers present in the final leather and secreted by the fibroblasts (Sharphouse, JH Leather Technician's Handbook. Leather Producer's Association. p. 104. ISBN 0-9502285-1-6). These collagen and elastin fibers are responsible for the mechanical properties of leather. For this reason, inducing the secretion of extracellular matrix (especially collagen and elastin) by the network of fibroblasts growing on the scaffold is a crucial step in leather development.

[0006] Thus, there remains a need to develop cell culture methods that promote cell proliferation as well as the production of collagen, which is essential for the production of high quality leather. Summary of the Invention

[0007] The inventors have developed a new method for producing high-quality leather by culturing fibroblasts in vitro on macroporous polymer scaffolds, which allows efficient high cell attachment, cell culture and proliferation over long periods of time with minimal stress and damage to the cells, resulting in increased collagen production by the fibroblasts.

[0008] The present invention relates to a method for producing leather comprising: a) culturing fibroblasts in vitro on a macroporous polymer scaffold, said scaffold obtaining a tissue comprising macropores with a diameter comprised between 60 and 500 μm, in particular between 80 and 280 μm, preferably between 100 and 280 μm, more preferably between 120 and 250 μm; b) tanning said tissue obtained in step a), thereby forming said leather.

[0009] The present invention also relates to the tissue obtainable by the culture step a) of the method of the invention, and to the tanned leather obtainable by the method of the invention. [Brief description of the drawings]

[0010] [Figure 1] Schemes of porous scaffolds of PDMS or poly(α-hydroxy acid) based materials for fibroblast culture and leather biofabrication. a) Symmetric scaffolds of PDMS or polyester, b) Asymmetric scaffolds of PDMS or polyester. [Diagram 2] Scheme of symmetric PDMS or asymmetric polyester scaffold fabrication by the SCPL process. [Diagram 3] Surface images of porous scaffolds fabricated by the SCPL process with volumetric mixtures of either PDMS (a,c,d) or polyester (b) acquired by scanning electron microscopy (SEM). a) Symmetric PDMS scaffold fabricated with calibrated inorganic macroporogen (150-250 μm), scale bar: 300 microns; b) Asymmetric polyester scaffold fabricated with calibrated organic macroporogen (average particle size 150 μm), scale bar: 400 microns; c) and d) PDMS scaffolds fabricated with calibrated organic macroporogen (average particle size 150 μm), scale bars: c) 400 microns and d) 100 microns. [Figure 4] Polymer functionalization by plasma treatment. [Diagram 5] Functionalization of plasma-activated PDMS with collagen Col (a), polylysine NH2 (b), or carbohydrate or carbohydrate-derived molecule Glu (c). [Figure 6] Functionalization of polyester-based scaffolds: aminolysis and immobilization of carbohydrate / carbohydrate-derived molecules. [Figure 7] PDMS scaffolds functionalized with collagen I seeded with fibroblasts (fluorescence microscopy). Cell nuclei were stained with DAPI. Scale bar: 300 μm. [Figure 8] 3D reconstruction (0–250 μm) of a PDMS scaffold functionalized with collagen I seeded with fibroblasts, taken by confocal microscopy (LEICA TCS SP8, 20X). Total cell nuclei are stained grey and dead cells are stained green. [Figure 9] PDMS (a,b) and polyester (c) scaffolds were functionalized with a) polylysine, or b,c) polylysine and / or carbohydrate / carbohydrate-derived molecules and seeded with fibroblasts (fluorescence microscopy). Cell nuclei were stained with DAPI. Scale bars: 500 μm (a,b) and 250 μm (c). [Figure 10] Tissue production by fibroblasts seeded on macroporous polymer scaffolds (fluorescence microscopy). Collagen secretion from the cells was observed by fluorescence microscopy using anti-collagen I antibody (Sigma, ref. C2456). Cell nuclei were stained with DAPI (blue) and collagen I with green. Scale bar: 500 μm. [Figure 11] Tanning process of tissue samples to obtain tanned leather. a) The tanning process carried out on lab scale, b) Drying of tanned samples, c) The final result of the obtained tanned leather. [Figure 12] Scanning electron microscopy (SEM) images of the surfaces of tanned leather (a) and German tanned leather (b). The thickness of the extracellular matrix cables was measured using ImageJ with the scale (in microns) provided. Scale bar: 10 microns. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The inventors have developed a method to produce high quality leather utilizing an in vitro culture step of fibroblasts on a macroporous scaffold to promote cell proliferation and collagen secretion, imparting the stiffness, mechanical strength and abrasion resistance found in leather.

[0012] The present disclosure relates to a method for producing leather comprising the steps of: a) culturing fibroblasts in vitro on a macroporous polymer scaffold to obtain a tissue comprising macropores having a diameter comprised between 60 and 500 μm, preferably between 80 and 280 μm, more preferably between 100 and 280 μm, and even more preferably between 120 and 250 μm; b) tanning said tissue, thereby forming said leather.

[0013] According to the method of the present disclosure, the scaffold has macropores with diameters comprised between 60 and 500 μm, preferably between 80 and 280 μm, more preferably between 100 and 280 μm, and even more preferably between 120 and 250 μm, into which cells can enter, thus providing a biological environment favorable for the proliferation and function of fibroblasts.

[0014] According to the present disclosure, the term "scaffold" refers to a three-dimensional support that provides physical and structural support to cells and enables tissue formation.

[0015] For the purposes of this disclosure, the term "pore diameter" refers to the average pore diameter measured by scanning electron microscopy (SEM) using ImageJ software (Rasband, WS, ImageJ, US National Institutes of Health, Bethesda, Maryland, USA, https: / / imagej.nih.gov / ij / , 1997-2018) by averaging the diameters of 5 to 30 pores measured.

[0016] To maintain the mechanical structure of the scaffold while providing a suitable biological environment for cell growth, the macroporous scaffold preferably has a porosity of 65% to 98%, in particular 70% to 98%, 75% to 98%, more preferably 80% to 95%. For the purposes of the present invention, the term "degree of porosity" or "void fraction" refers to the ratio of the volume of voids to the volume of material, measured according to gravimetric methods (Guarino V, et al. 2008 Sep;29(27):3662-70). The volume of the samples was calculated from the measurements of the sample dimensions. Measuring the mass of the samples allowed the estimation of the density of the samples. The density (ρSc) of each sample was the average value obtained from four samples. The porosity of the scaffold was then calculated using the following formula: Porosity=(1-(ρSc / ρPpoly)):100 where ρPpoly is the density of the polymer according to the manufacturer's database.

[0017] Macropore formation in the scaffold can be achieved using several techniques well known in the art, such as solvent casting and particle leaching (SCPL) or phase inversion method.

[0018] The scaffold according to the present disclosure can be designed to exhibit a thickness compatible with leather synthesis, in particular, the scaffold can have a thickness of 0.5 to 2.5 mm.

[0019] The scaffold according to the present disclosure can be designed to exhibit a thickness compatible with the fabrication of different biomaterials. In particular, the scaffold can have a thickness of 0.25 to 4.5 mm, in particular 0.5 to 2 mm.

[0020] According to one particular embodiment, the polymeric support does not have a fibrous structure.

[0021] In certain embodiments, polymers that can be used for the scaffold can be organosilicon-based polymers such as polydimethylsiloxane (PDMS), poly(α-hydroxy acid), or mixtures thereof.

[0022] In certain embodiments, the polymer that can be used for the scaffold can be a polymeric organosilicon compound, such as PDMS, which shows biocompatibility and long-term stability in contact with biological materials.In addition, the high solubility of oxygen in the PDMS matrix allows cells to be supplied with oxygen and prevents hypoxia (Merkel, TC, et al. 2000, J. Polym. Sci. B Polym. Phys., 38: 415-434).In addition, polymeric scaffolds with high oxygen permeability are particularly suitable for promoting gas exchange, nutritional supply and removal of waste products associated with cell metabolism, improving fibroblast proliferation and function in vitro.In certain embodiments, the PDMS is SYLGARD™ 184 PDMS (Dow).

[0023] In certain embodiments, when the scaffold is composed of an organosilicon-based polymer such as PDMS, macropore formation can be achieved by solvent casting and particle leaching (SCPL). For example, the SCPL method can be carried out by mixing the polymer with a catalyst. Then, particles having a specific dimension, referred to herein as porogen, are added to the polymer / catalyst solution, mixed, and cast onto the support for reticulation. When the polymer reticulates, a composite structure is created that contains the porogen along with the polymer. This composite is then placed in a bath that dissolves the porogen, leaving a porous scaffold. The porogen solvent used is selected for its ability to dissolve the porogen and not be a solvent for the polymer.

[0024] Examples of suitable porogens include inorganic salt crystals such as sodium chloride crystals, potassium chloride, sodium sulfate, and amorphous materials such as poly(ethylene glycol) (PEG), polyvinylpyrrolidone, sucrose crystals, gelatin spheres, paraffin spheres, and the like.

[0025] Examples of porogen solvents include chloroform, tetrahydrofuran, dimethylsulfoxide, methanol, and water.

[0026] Solvents for the polymers may in particular be N,N-dimethylformamide, N-methylpyrrolidone, dimethylacetamide, dimethylsulfoxide, chloroform and mixtures thereof.

[0027] The catalyst may be any polymeric organosilicon catalyst known in the art, particularly a PDMS catalyst such as a platinum or palladium catalyst.

[0028] The concentration of the porogen in the solution of the polymer may in particular be from 50% to 90% by weight, in particular from 75% to 85% by weight, relative to the weight of the polymer.

[0029] Said scaffold obtained by the SCPL method is a symmetrical scaffold, with interconnected macropores, preferably with a diameter comprised between 100 and 280 μm, distributed throughout the scaffold.

[0030] By symmetrical scaffold is intended a scaffold in which macropores with a diameter comprised between 60 and 500 μm, preferably between 80 and 280 μm, more preferably between 100 and 280 μm and even more preferably between 120 and 250 μm are homogeneously distributed throughout the thickness of the scaffold. In particular, said scaffold has two macroporous faces, in particular both said faces comprise macropores with a diameter comprised between 60 and 500 μm, preferably between 80 and 280 μm, more preferably between 100 and 280 μm and again more preferably between 120 and 250 μm.

[0031] In another particular embodiment, the polymer that can be used in the scaffold can be a polyester-derived poly(α-hydroxy acid).

[0032] Non-limiting examples of poly(α-hydroxy acid) derived polyesters, particularly biosourced and biodegradable polyesters, that can be used to form the scaffold are polylactic acid (PLA), polyglycolic acid (PGA), copolymers of lactic and glycolic acid (PLGA), homopolymers and copolymers of hydroxy acids such as poly(3-hydroxybutyrate), poly(4-hydroxybutyrate), poly(3-hydroxyvalerate), poly(5-hydroxyvalerate), poly(3-hydroxypropionate), poly(3-hydroxyhexanoate), poly(3-hydroxyoctanoate), poly(3-hydroxyoctodecanoate); polycaprolactone; homopolymers and copolymers of poly(butylene succinate) and poly(butylene adipate); and polyhydroxyalkanoates (PHAs) and their derivatives, polyhydroxyesters of 3-, 4-, 5-, and 6-hydroxyalkanoic acids, and mixtures thereof.

[0033] Polyesters have interesting properties from a mechanical point of view and are compatible with cell cultures. They are biosourced since they are produced from agricultural waste and are completely biodegradable. However, polyesters are hydrophobic and impermeable to oxygen, making molecular diffusion impossible after reticulation.

[0034] Therefore, to facilitate gas exchange, the oxygen impermeable polymer scaffold may further comprise nano- and micropores with diameters of less than 20 μm, particularly between 0.1 μm and 10 μm, more particularly between 2 μm and 8 μm.

[0035] The scaffold, which is composed of an oxygen impermeable polymer, such as a polyester polymer, and contains micropores, can be an asymmetric scaffold or a symmetric scaffold.

[0036] The polyester polymer symmetric scaffold according to the present disclosure can be achieved by any method known to those skilled in the art.As a non-limiting example, the polymer symmetric scaffold can be obtained by a combination of a one-phase inversion process (e.g., NIPS), which allows the formation of micropores that facilitate gas and nutrient exchange, and a solvent casting and particle leaching (SCPL) process, which forms macropores that allow cell growth and tissue formation.

[0037] Phase inversion is a method well known in the art and examples include non-solvent induced phase separation process (NIPS), thermally induced phase separation process (TIPS), vapor induced phase separation process (VIPS), and polymerization induced phase separation (PIPS).

[0038] In particular, the scaffold can be manufactured by the following steps: preparing a solution of the polymer comprising the polymer and at least one solvent for the polymer, as described above; adding a solid porogen to the prepared solution of the polymer under homogeneous stirring; pouring the solution comprising the polymer and porogen onto a solid support; and introducing the solution of the polymer into a non-solvent solution (water); and finally immersing the porogen / polymer complex in a porogen solvent to dissolve the porogen.

[0039] Examples of suitable porogens include inorganic salt crystals, such as sodium chloride crystals and potassium chloride crystals, and amorphous materials, such as poly(ethylene glycol) (PEG), polyvinylpyrrolidone, sucrose crystals, gelatin spheres, and paraffin spheres.

[0040] The concentration of the porogen in the solution of the polymer may in particular be from 50% to 98% by weight, in particular from 80% to 95% by weight, relative to the weight of the polymer.

[0041] When phase inversion occurs at the interface between the polymer and the non-solvent solution, the formation of micropores of different sizes is observed (NIPS) and, following the SCPL process, the formation of macropores is observed. The polyester scaffold obtained by the combination of the NIPS and SCPL processes is a symmetric scaffold with a network of interconnected macropores with diameters comprised between 60 and 500 μm, preferably between 80 and 280 μm, more preferably between 100 and 280 μm, even more preferably between 120 and 250 μm, and micropores with diameters less than 20 μm, in particular between 0.1 and 15 μm, more particularly between 2 and 8 μm, distributed throughout the thickness of the scaffold, as represented in FIG. 1a. In particular, said polyester symmetric scaffold exhibits two macroporous faces, in particular both of said faces containing macropores with diameters between 60 and 500 μm, preferably between 80 and 280 μm, more preferably between 100 and 280 μm, even more preferably between 120 and 250 μm.

[0042] According to the above-mentioned method, the solid substrate onto which said solution of polymer is deposited can be made of glass, metal or plastic resistant to the solvent, such as polytetrafluoroethylene (Teflon®), nylon 6,6 or poly(ethylene terephthalate), among others.

[0043] In another particular embodiment, said polyester scaffold is obtainable by a combination of SCPL and a phase inversion method selected from the group comprising: TIPS, VIPS and PIPS.

[0044] TIPS is a process in which a polymer solution is formed at high temperature using a high boiling point solvent and then cooled to induce phase separation and solidification of the polymer. A microporous scaffold is obtained after extraction of the diluent.

[0045] In the VIPS process, a cast film of polymer and solvent is exposed to an atmosphere of vapor of a non-solvent molecule, typically water. The vapor permeates the film, causing precipitation of the polymer, ultimately forming a symmetric porous scaffold without a dense skin layer. Because the thermodynamic properties of the casting solutions in NIPS and VIPS are similar, VIPS should produce scaffolds with morphology similar to that produced by NIPS.

[0046] The PIPS process is a phase separation that occurs in a multicomponent mixture induced by the polymerization of one or more components. An increase in the molecular weight of the reactive components causes one or more components to become immiscible with each other, resulting in spontaneous phase separation. The morphology of the final phase-separated structure is generally random, since the occurrence and course of phase separation are stochastic.

[0047] In another particular embodiment, the polyester polymer scaffold is an asymmetric scaffold, which can be achieved by any method known to those skilled in the art, said asymmetric scaffold being obtained by a solvent casting and particle leaching (SCPL) process, forming macropores that allow cell growth and tissue formation.

[0048] In particular, the scaffold can be manufactured by the following steps: preparing a solution of the polymer, comprising the polymer and at least one solvent for the polymer, as described above; adding solid porogen to the prepared solution of the polymer under homogeneous stirring; slowly evaporating the solvent to reticulate the polymer, and finally immersing the porogen / polymer complex in a porogen solvent to dissolve the porogen.

[0049] Asymmetric macroporous scaffolds are formed when the solvent evaporates and the porogen dissolves in the porogen solvent. Rapid evaporation of the solvent on top results in a dense nanoporous surface, while slow evaporation of the solvent deep in the polymer solution results in the formation of micropores. Macropores are formed due to the dissolution of the porogen.

[0050] Examples of suitable porogens include inorganic salt crystals, such as sodium chloride crystals or potassium chloride crystals, and amorphous materials, such as poly(ethylene glycol) (PEG), polyvinylpyrrolidone, sucrose crystals, gelatin spheres, paraffin spheres, and the like.

[0051] The concentration of the porogen in the solution of the polymer may in particular be from 500% to 900% by weight, in particular from 700% to 900% by weight, relative to the weight of the polymer.

[0052] The polyester asymmetric scaffold obtained by the SCPL process is an asymmetric scaffold having a network of interconnected macropores with a diameter comprised between 60 and 500 μm, preferably between 80 and 280 μm, more preferably between 100 and 280 μm, and even more preferably between 120 and 250 μm, and micropores with a diameter of less than 10 μm, in particular between 0.1 and 5 μm, and more particularly between 1 and 5 μm, distributed throughout the thickness of the scaffold, as represented in Figure 1b. In particular, said polyester asymmetric scaffold presents a macroporous surface and a nanoporous surface, more particularly a first surface comprising macropores with a diameter of 60 to 500 μm, preferably between 80 and 280 μm, more preferably between 100 and 280 μm, and even more preferably between 120 and 250 μm, and a second, opposite surface comprising only nanopores with a diameter of 10 nm or less, preferably 5 nm or less.

[0053] In order to functionalize the surface of the scaffold designed as above and to enhance the cytocompatibility of the scaffold, bioactive molecules can be grafted onto the surface of the scaffold.The bioactive molecules can be selected from polysaccharides such as cellulose, pectin, pullulan, keratan, hyaluronic acid, chondroitin sulfate, chitosan and heparin; proteins such as fibrinogen and collagen, especially soluble collagen; peptides known for their cell adhesion ability, such as peptides containing arginine-glycine-aspartic acid (RGD) or arginine-glycine-aspartic acid-serine (RGDS) sequence; and mixtures thereof.The bioactive molecules can be grafted by any method known in the art.

[0054] According to one particular embodiment, the bioactive molecule is collagen. Indeed, collagen allows cell adhesion, since collagen specifically interacts with integrin receptors expressed on the surface of skin-derived cells. In order to graft collagen onto the surface of the scaffold, in a particular embodiment, said scaffold can be treated with plasma generated in air or oxygen to add electronegative silanol groups. Collagen is then grafted onto the surface of the scaffold by immersing said scaffold in a solution containing collagen, followed by rinsing, preferably with phosphate buffered saline (PBS).

[0055] According to a preferred embodiment, the bioactive molecules may be carbohydrates and / or carbohydrate-derived molecules, such as glycosaminoglycans (e.g., heparin sulfate, heparin, chondroitin sulfate, dermatan sulfate, keratan sulfate, hyaluronic acid), which play a fundamental role in the organization and homeostasis of the extracellular matrix due to their unique biophysicochemical properties (viscoelasticity, high water-holding capacity, ability to interact specifically or non-specifically with various proteins).

[0056] To graft carbohydrates and / or carbohydrate-derived molecules (e.g., glycosaminoglycans) onto the surface of the scaffold, the scaffold can first be pretreated to provide a positive charge, preferably positive amine groups, on the surface of the scaffold before functionalizing with negatively charged molecules. The positive amine groups can be added by treating the scaffold with plasma generated in oxygen or air and immersing the scaffold in a polylysine solution, treating the scaffold with plasma generated in nitrogen, or by aminolysis reactions. Once the surface of the scaffold has been treated to be positively charged, the scaffold can be functionalized with carbohydrates and / or carbohydrate-derived molecules by immersing the scaffold in a solution containing the carbohydrates and / or carbohydrate-derived molecules followed by rinsing with, for example, ultrapure water.

[0057] According to a preferred embodiment, said positive amine groups are added by aminolysis of the polymer with a solution of at least one aliphatic α,ω-diamine. The aminolysis reaction can in particular be carried out by immersing the polymer scaffold in a solution containing an aliphatic α,ω-diamine, preferably in a solution of 1,6-hexanediamine in propanol or water and mixtures thereof, followed by rinsing, for example, with ultrapure water.

[0058] According to the method of the present disclosure, fibroblasts are seeded onto the polymer scaffold as described above and cultured under appropriate growth conditions, in particular in a medium that allows inducing the proliferation and secretion of extracellular matrix, such as different types of collagen and elastin fibers.

[0059] Fibroblasts according to the present disclosure are cells that synthesize extracellular matrix such as collagen, elastin, glycoproteins, and non-proteoglycan polysaccharides among other extracellular matrix components. Fibroblasts can be mammalian fibroblasts or non-mammalian fibroblasts. The cells can be a single cell type or a combination of multiple cell types. In a preferred embodiment, fibroblasts are the only cells cultured on the scaffold according to the method of the present disclosure, since the only components that resist the storage and tanning process are the collagen and elastin fibers secreted by fibroblasts.

[0060] Numerous media are commercially available and well known to those skilled in the art. The medium may be a minimal medium, which contains, among other things, mineral salts, amino acids, vitamins, and a carbon source essential for the cells, and a buffer system for adjusting the pH. Basal media that can be used in the method according to the invention include, but are not limited to, for example, MEM medium, DMEM / F12 medium, DMEM medium RPMI medium, Ham's F12 medium, IMDM medium, and KnockOut™ DMEM medium (Life Technologies). Depending on the medium used, it may be necessary or desirable to add glutamine, ascorbic acid, growth factors, one or more antibiotics, such as streptomycin, penicillin, and / or antimycotin.

[0061] In another aspect, the present disclosure also relates to a tissue (also called in-vitro skin) obtained by the cell culture step a) as described above, preferably comprising fibroblasts cultured on a macroporous polymer scaffold as described above, comprising macropores with a diameter comprised between 60 and 500 μm, preferably between 80 and 280 μm, more preferably between 100 and 280 μm, even more preferably between 120 and 250 μm.

[0062] In certain embodiments, the scaffold comprises at least one polymer, such as a polymeric organosilicon compound, such as polydimethylsiloxane (PDMS), or a biodegradable polyester.

[0063] In a preferred embodiment, the tissue comprises a scaffold having macropores interconnected by micropores, preferably the micropores having a diameter of less than 20 μm, more preferably less than 10 μm.

[0064] In certain embodiments, the tissue comprises an asymmetric scaffold comprising macropores having a diameter of 80-280 μm, more preferably 100-280 μm, even more preferably 120-250 μm, and micropores having a diameter of less than 10 μm, particularly 0.1 μm to 5 μm, more particularly 1 to 5 μm, distributed throughout the thickness of the scaffold.

[0065] In a more specific embodiment, the tissue comprises a scaffold exhibiting a first surface comprising macropores having a diameter of 80-280 μm, and a second, opposing surface comprising only nanopores having a diameter of 10 nm or less, preferably 5 nm or less.

[0066] In a preferred embodiment, said tissue is obtained after at least 1 week, preferably 2 weeks, 3 weeks or 4 weeks of culture.

[0067] The tissue obtained by the cell culture process described above is then tanned by any method known in the art to create chemical bonds between the elastin and collagen fibers and transform them into leather.

[0068] A variety of tanning processes can be used for tanning leather, including chrome tanning using chromium (III) sulfate tanning agent, tanning using aluminum salts, aldehydes, and organic compounds, vegetable tanning using tannins, and tanning using low molecular weight phenolic and acrylic polymers (hereinafter referred to as "polymer tanning").

[0069] In another aspect, the present disclosure relates to tanned leather obtained by the above-mentioned method. As used herein, the term "leather" or "synthetic leather" refers to a material obtained by tanning or chemical treatment of animal skin or tissue containing collagen, elastin and other components of the extracellular matrix. According to the present disclosure, the tissue (i.e., in vitro skin) is obtained by an in vitro culture process of fibroblasts and contains a dense mesh of cells and extracellular matrix components.

[0070] The present disclosure relates to in-vitro skin tanned leather obtained by the method of the present disclosure.

[0071] The present disclosure also relates to the use of a macroporous scaffold as described above for the manufacture of leather, preferably by tanning tissue obtained from the culture of fibroblasts on said macroporous scaffold, more preferably said scaffold comprising macropores with a diameter comprised between 60 and 500 μm, preferably between 80 μm and 400 μm, between 100 and 350 μm, between 100 and 300 μm, more preferably between 80 and 280 μm, between 100 and 280 μm, even more preferably between 120 and 250 μm. In a particular embodiment, said scaffold is a PDMS or polyester scaffold. In another particular embodiment, said scaffold is an asymmetric or symmetric scaffold as described above.

[0072] Embodiments of the present invention are described in the following specific examples, which are illustrative and should not be construed as limiting. EXAMPLES

[0073] To produce high-quality leather, the inventors recreate skin in the laboratory. To do so, they follow three steps: first, they create a scaffold that allows cells to attach and develop; second, they add cells to this scaffold and allow them to grow and differentiate; and third, they perform an abbreviated tanning procedure of the material, converting the tissue into leather.

[0074] 1. Scaffolding A wide variety of scaffolds have been used in tissue engineering in the medical field. These scaffolds use different structures, manufacturing methods, materials (biological and synthetic) and surface functionalization (Shafiee A, Atala A. Annu Rev Med. 2017 Jan 14;68:29-40). However, only a few studies have been carried out on the use of scaffolds for tissue engineering in the field of leather synthesis.

[0075] The inventors decided to use the macroporous material to fabricate a scaffold for growing cultured fibroblast tissue in the laboratory.

[0076] The synthesis of fibroblastic tissue used in leather synthesis requires polymers that are permeable to oxygen and / or small molecules to facilitate gas exchange, nutrient supply and removal of waste products associated with cellular metabolism. For this reason, we tested two families of polymers for the fabrication of porous scaffolds: 1. Polydimethylsiloxane (PDMS), the most commonly used silicone elastomer in microfluidic applications, as a polymer for creating leather scaffolds due to its biocompatibility and long-term stability in contact with biological materials (Aucoin L, et al. J Biomater Sci Polym Ed. 2002;13(4):447-62). Another important advantage of PDMS is the high solubility of oxygen in the PDMS matrix, which allows oxygenation to cells within the implant and prevents hypoxia (Merkel, TC, et al. (2000). J. Polym. Sci. B Polym. Phys., 38: 415-434). PDMS is hydrophobic, but the surface can be modified to make it hydrophilic by methods such as plasma functionalization. Once PDMS is hydrophilic, it can bind biological and / or chemical molecules, a prerequisite for cell adhesion (Li B, et al. J Biomed Mater Res A. 2006 Dec 15;79(4):989-98).

[0077] 2. Biosourced, biodegradable and bioabsorbable polyesters and polyester derivatives are polymers that have interesting properties from a mechanical point of view, are compatible with cell culture in medical applications, are produced from agricultural waste and are completely biodegradable (Farah, Shady & Anderson, Daniel & Langer, Robert. (2016) Advanced Drug Delivery Reviews. 107. 10.1016 / j.addr.2016.06.012; Kurokawa, N.; Kimura, S.; Hotta, AJ Appl. Polym. Sci. 2018, 135, 45429; Meng, et al. (2016) Journal of Applied Polymer Science. 133. n / an / a. 10.1002 / app.43530). However, they are impermeable to oxygen and diffusion of molecules after reticulation is not possible.

[0078] To solve this, we induced the formation of micropores of about 1-20 microns that allow increased diffusion of oxygen and molecules, created using the "Non-solvent Induced Phase Separation Process" (NIPS) method described in Al Tawil et al. 2018, European Polymer Journal. 105, 370-388, 2018.

[0079] To fabricate macroporous features (pores between 80 and 250 microns) on either PDMS or polyester-based polymers, we used the "solvent casting and particle leaching" (SCPL) method for PDMS and asymmetric polyester scaffolds, and NIPS coupled with SCPL for symmetric polyesters. In SCPL, porosity can be controlled by varying the ratio of particles to solvent, and pore size is determined by the diameter of the porogen in solution (Sola A, et al. Mater Sci Eng C Mater Biol Appl. 2019), while in NIPS, porosity and pore size are controlled by the physicochemical properties of the macroporogen, the polymer / solvent mixture, and the non-solvent.

[0080] For the fabrication of macroporous features we followed different methods depending on the polymer used: 1. For PDMS, we used the SCPL method by either using size-calibrated sodium chloride as described in Pedraza et al. 2012. J Biomater Sci Polym Ed. 2013;24(9):1041-56 or by adding a porogen with appropriate particle size and high water solubility. The resulting scaffold is a symmetric scaffold with interconnected macropores of 150-250 μm in diameter distributed throughout the scaffold.

[0081] 2. For polyester, we used the following combination: a) One "Non-solvent-induced phase separation process" (NIPS) process coupled with one "Solvent casting and particle leaching" (SCPL) process by homogeneously spreading macroporogen particles on the support surface and pouring a polymer / solvent mixture onto the support containing macropores. The resulting porous scaffold is symmetrical with two porous faces containing macropores and micropores. b) Using the SCPL method by adding a porogen with appropriate particle size and high water solubility. The resulting scaffold is an asymmetric scaffold with a porous side containing interconnected macropores with diameters of 150-250 μm and a smooth side without macro / micropores.

[0082] Using the method of fabricating porous geometries of PDMS and polyester, we obtained two different types of support scaffolds: symmetric and asymmetric (FIG. 1a-b).

[0083] SCPL on PDMS and polyester was performed as follows: 1. Symmetric supports in PDMS (Figure 1a) were obtained using the SCPL "solvent casting and particle leaching" method (Figure 2) by mixing porogen with PDMS (Dow Sylgard 184 elastomer) and casting onto the supports; the mixture was incubated at 50 °C to crosslink the polymer. The porogen was removed from the scaffolds by immersion in deionized water for 72 h, changing the water every 24 h.

[0084] 2. Symmetric supports in polyester (Fig. 1a) were fabricated using the “Non-solvent Induced Phase Separation Process” (NIPS) method coupled with the SCPL “Solvent Casting and Particle Infusion” method by a series of steps detailed in Fig. 2. The “Non-solvent Induced Phase Separation Process” NIPS procedure was used to create non-interconnected, surface-parallel micropores (up to 10 microns) when the solvent / polymer mixture was placed into the non-solvent (water).

[0085] 3. An asymmetric support made of polyester (Figure 1b) was fabricated using the SCPL “solvent casting and particle leaching” method by a series of steps detailed in Figure 2. SCPL macropogen creates macropores (up to about 120–160 μm) by dissolving in a non-solvent (water) and induces macropore interconnectivity in the scaffold.

[0086] The physical and structural properties of the scaffolds were observed with a FEI Nova NanoSEM 450 model scanning electron microscope. Representative images of the surface condition of the scaffolds are shown in Figure 3.

[0087] As shown in Figure 3, the PDMS scaffolds are porous at the surface, with a range of different square pore sizes between 100 and 200 microns for the mixtures containing PDMS with calibrated square macroporogens (Figure 3a), and a more homogeneous distribution of round pore sizes with diameters of approximately 160 microns for the mixtures containing round shaped macroporogens (Figure 3c-d). The high magnification images in panel 3d show that the pores are interconnected to the interior of the scaffold for the mixtures containing PDMS with both square and round macroporogens (data not shown). The pixel-to-distance scale was obtained from the provided images (in microns) and used to determine the size of the pores using ImageJ (Rasband, WS, ImageJ, US National Institutes of Health, Bethesda, Maryland, USA, https: / / imagej.nih.gov / ij / , 1997-2018).

[0088] The polyester scaffolds exhibited a highly porous surface with pore sizes of approximately 100-200 microns in diameter (Figure 3b). The pixel-to-distance scale was obtained from the provided images (in microns) and used to determine the size of the pores using ImageJ (Rasband, WS, ImageJ, US National Institutes of Health, Bethesda, Maryland, USA, https: / / imagej.nih.gov / ij / , 1997-2018).

[0089] 2. Functionalization Two different physicochemical and chemical functionalization methods were performed on the PDMS or polyester scaffolds:

[0090] 2.1. Physicochemical methods: Physical functionalization using air / oxygen / nitrogen plasma (Figure 4) is an adaptation of a method currently used for the fabrication of microfluidic chips to PDMS scaffolds (Tang, KC et al. (2006). Journal of Physics: Conference Series. 34. 155).

[0091] The procedure chosen for its efficiency was functionalization with plasma in a clean room equipped with plasma cleaning (Reference: PlasmaACE1 24kHz, PLASMA Instrument). Air / oxygen plasma functionalization of PDMS (Figure 4) adds electronegative silanol groups (Dhananjay Bodas, Chantal Khan-Malek. Chemistry, Volume 123, Issue 1, 2007, Pages 368-373). Nitrogen functionalization of the PDMS surface (Figure 4) adds amine groups to the surface (Chengxin Yang and Yong J. Yuan. Applied Surface Science. Volume 364, 2016, Pages 815-821).

[0092] After plasma functionalization, the scaffolds were immediately incubated with different biological or chemical agents to functionalize their surfaces as follows: 1. Collagen (Figure 5a): Incubated with 250 μg / mL collagen I (50201, ibidi) for 2 h. Excess collagen was removed by three successive washes with 15 ml PBS, then incubated with 15 ml medium and seeded with cells on the same day.

[0093] 2. Polylysine (Figure 5b, left and center): Incubated with 50 mg / mL polylysine (A3890401, Thermo) for 2–3 h. Excess polylysine was removed by three successive washes with deionized water, then incubated with medium and inoculated with cells on the same day.

[0094] 3. Polylysine + carbohydrate / carbohydrate-derived molecule mixture (Figure 5c): Incubated with 50 mg / mL polylysine (A3890401, Thermo) for 2–3 h. Excess polylysine was removed by three successive washes with deionized water, then incubated with a saturating concentration of the mixed carbohydrate / carbohydrate-derived molecule. Excess functionalized molecules were removed by three successive washes with deionized water, then incubated with medium and seeded with cells.

[0095] 4. Carbohydrates / carbohydrate-derived molecules (Figure 5c): Incubated with a mixture of carbohydrates / carbohydrate-derived molecules at saturating concentrations. Excess functionalized molecules were removed by three successive washes with deionized water, and then the culture medium was incubated and seeded with cells.

[0096] Functionalization with collagen adds a biologically active layer (Figure 5a) that allows cell adhesion. Indeed, collagen specifically interacts with integrin receptors expressed on the surface of skin-derived cells (Cedric Zeltz, Donald Gullberg. J Cell Sci 15 February 2016; 129 (4): 653-664).

[0097] Air / oxygen plasma functionalization of PDMS (Figure 4) adds electronegative silanol groups to its surface, which interact with the positively charged (at physiological pH) amine groups of polylysine, resulting in an overall positively charged layer on the surface of the PDMS scaffold (Figure 5b, left).

[0098] A layer of carbohydrate / carbohydrate-derived molecules promoting cell adhesion, proliferation, and collagen secretion can be added to either a) polylysine-coated PDMS scaffolds (Figure 5b) or b) positively charged PDMS scaffolds obtained by nitrogen plasma treatment (Figures 4 and 5c).

[0099] 2.2 Chemical methods The functionalization of the entire surface of the porous polyester scaffold was carried out following a two-step process (Figure 6). The aminolysis reaction of the polyester scaffold was carried out by immersing the polyester scaffold pieces in a solution of hexane-1,6-diamine (HDA) dissolved in a propanol / water mixed solvent for 15 min. The immobilization of carbohydrates / carbohydrate-derived molecules on the polyester scaffold surface was carried out at room temperature by immersing the aminated polyester scaffold in a solution containing said carbohydrates / carbohydrate-derived molecules.

[0100] 3. Cell Culture on the Scaffolds The skin is a flattened and extended anatomical structure that covers the entire exterior of the body and is continuous with the mucous membrane at the level of the external openings of the cavity. It is composed of three layers: the epidermis, the dermis and the subcutaneous tissue. The epidermis is the outermost layer of the skin and is composed of a stratified, keratinized epithelium made up of several layers of cells. The cells that compose this layer are called keratinocytes and have the main function of protecting the skin from the environment.

[0101] The dermis is located under the epidermis and extends to the subcutaneous tissue and is composed of three main types of cells: a) fibroblasts, which are the main cells of the dermis and handle the synthesis of collagen, elastic fibers, reticular fibers, and extracellular matrix materials, and b) immune cells: histiocytes are tissue macrophages that reside in the connective tissue and assist the immune system. Mast cells are inflammatory cells that reside around blood vessels in the dermis (Brown TM, Krishnamurthy K. 2021 May 10. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan). In addition to these cells, the dermis is composed of a complex and abundant substance called the extracellular matrix (ECM), whose main components are collagen and elastin secreted by fibroblasts (Schwarz RI. Biochem Biophys Rep. 2015 Sep;3:38-44).

[0102] For the production of classical cowhide leather, the tanning procedure completely removes the outer layer (epidermis) and the inner layer (hypodermis) of the skin, leaving only the dermis, which is the only component of leather in the final state (Sharphouse, JH Leather Technician's Handbook. Leather Producer's Association. P. 104. ISBN 0-9502285-1-6). The slaughter of the animal and the subsequent storage dehydrates and degrades most of the cells, molecules and extracellular matrix components. Among the components of the extracellular matrix found in leather in the final state, the only components that survive storage and tanning are the collagen and elastin fibers. Collagen and elastin fibers are secreted by fibroblasts (Sharphouse, JH Leather Technician's Handbook. Leather Producer's Association. P. 104. ISBN 0-9502285-1-6). These collagen and elastin fibres provide the stiffness, mechanical strength and abrasion resistance found in leather.

[0103] Since the final cowhide leather contains only the dermis, whose main components are collagen and elastin fibers secreted by fibroblasts, the inventors decided to culture a fibroblast cell line to obtain dermal tissue that is particularly suitable for the synthesis of leather.

[0104] Fibroblasts are seeded onto the functionalized scaffold, allowing them to proliferate under optimal conditions and to be induced to secrete extracellular matrix (collagen, elastin, etc.).

[0105] 3.1 Cell proliferation a. Biological functionalization (collagen) To investigate the proliferation and viability of cells in the fabricated porous polymer scaffolds functionalized with biologically active molecules (biological functionalization), PDMS scaffolds functionalized with collagen I (see Figure 4a,c,d) (Figure 5a) were seeded with fibroblasts for 7 days, fixed and stained with the nuclear marker DAPI staining protocol. Images of the seeded scaffolds were made by fluorescence microscopy (Figure 7). As shown in Figure 7, biological functionalization allowed for very dense attachment of cells to the fabricated macroporous polymer scaffolds.

[0106] The proliferation and viability of cells on the fabricated macroporous polymer scaffolds are investigated over time. PDMS scaffolds functionalized with biologically active molecules (collagen I) were seeded with fibroblasts under standard culture conditions for 4 weeks, fixed, and then stained using the HCS LIVE / DEAD® Green Kit (Invitrogen, ref. H10290). The HCS LIVE / DEAD® Green Kit measures cytotoxicity using a non-fluorescent, cell-impermeable compound that exhibits strong fluorescence enhancement when bound to DNA. Dead cells allow the infiltration of Image-iT® DEAD Green™ viability stain, while the assay also employs a cell-permeable nuclear segmentation tool that stains both live and dead cells, reflecting the total cell number in the sample. The 4-week stained samples were observed under a confocal microscope (OCCIGEN Imaging-Cytometry Platform, Genopole). Figure 8 shows that while the total cell density was high, few dead cells (less than 2%) were observed up to 250 μm of the basal layer. These results indicate that collagen I functionalized (biologically functionalized) PDMS scaffolds allow efficient high cell attachment, cell culture and long-term cell proliferation while minimizing stress and damage to the cells.

[0107] b. Chemical functionalization (polylysine and carbohydrate / carbohydrate-derived molecules, Figure 5b-c).

[0108] Cells were seeded on PDMS and polyester scaffolds coated with a chemical surface functional layer (polylysine or carbohydrate / carbohydrate-derived molecules) and the proliferation potential of fibroblasts was evaluated using chemical molecules instead of collagen I or other biologically derived proteins. Indeed, chemical functionalization with the above-mentioned molecules is several orders of magnitude cheaper than using biologically derived proteins such as collagen or fibronectin.

[0109] PDMS scaffolds functionalized with polylysine or carbohydrate / carbohydrate-derived molecules were seeded with fibroblasts for 7 days, fixed and stained with the nuclear marker DAPI staining protocol. Images of the seeded scaffolds were made by fluorescence microscopy (Figure 9a-b).

[0110] PDMS scaffolds functionalized with both polylysine (Figure 9 a) and carbohydrate / carbohydrate-derived molecules (Figure 9 b) were able to attach cells at very high densities, in the same order of magnitude as observed with biologically functionalized scaffolds (collagen I, Figure 7 ).

[0111] Polyester scaffolds functionalized with polylysine or carbohydrate / carbohydrate-derived molecules were seeded with fibroblasts for 7 days, fixed, and stained with the nuclear marker DAPI staining protocol. Images of the seeded scaffolds were observed by fluorescence microscopy (Figure 9c). Polyester scaffolds functionalized with both polylysine (data not shown) or carbohydrate / carbohydrate-derived molecules (Figure 9c) were both able to attach cells at very high densities.

[0112] These results demonstrate efficient attachment, culture and proliferation of cells when chemical agents are used for functionalization, opening up the use of animal-free, economically viable reagents for the functionalization of PDMS or polyester scaffolds.

[0113] 3.2 Induction of extracellular matrix (ECM).

[0114] As explained above, the tanning procedure degrades most of the cellular, molecular and extracellular matrix components. The only components that resist storage and tanning are mostly collagen and elastin fibers present in the final leather and secreted by fibroblasts (Sharphouse, JH Leather Technician's Handbook. Leather Producer's Association. P. 104. ISBN 0-9502285-1-6). These collagen and elastin fibers are responsible for the mechanical properties of leather. For this reason, inducing the secretion of extracellular matrix (especially collagen, elastin) by a network of fibroblasts growing on the scaffold is a crucial step in leather development.

[0115] Collagen secretion experiments were performed to evaluate the ability of fibroblasts seeded on functionalized scaffolds to secrete extracellular matrix. To evaluate this, fibroblasts were seeded on functionalized scaffolds, cultured for 4 weeks, fixed, and co-stained with the nuclear marker DAPI and anti-collagen I antibody (Sigma, ref. C2456). Images of seeded scaffolds were made by fluorescence microscopy (Figure 10).

[0116] 4.Tanning process The tissue obtained after culturing on the scaffold was subjected to a tanning process to obtain leather. The whole process can be conveniently divided into three different steps for each type of leather (Figure 11): a) a simplified beamhouse operation process to soak the tissue, remove the cell culture medium and chemically prepare the tissue for tanning, b) tanning the tissue using any method known in the art, such as chrome tanning, aluminum tanning, aldehyde tanning, organic tanning, synthetic tanning and / or vegetable tanning, inducing the formation of chemical bonds between extracellular matrix chains in the tissue (Figure 11a), and c) drying the tanned leather (Figure 11b) and obtaining our tanned leather (Figure 11c).

[0117] The physical and structural properties of the extracellular matrix (ECM) collagen and elastin fibers of the obtained tanned leather were compared with those found in animal tanned leather. We observed its surface condition by scanning electron microscopy (SEM) with an FEI Nova NanoSEM 450 model scanning electron microscope, obtained the scale (in microns) from the images provided, and determined the thickness of the ECM cables using ImageJ software. Images of the surface ECM fibers of our tanned leather (Figure 12a) and of the German bull tanned leather (Figure 12b) are shown in Figure 12. The thickness of the extracellular matrix (ECM) collagen and elastin fibers of the rabbit leather obtained according to the method of the present invention is similar to that obtained in high-quality German bull tanned leather, in the expected range between 150 and 750 microns.

Claims

1. Leather manufacturing method, including the following steps: a) A step of obtaining tissue by culturing fibroblasts in vitro on a macroporous polymer scaffold, wherein the scaffold includes macropores with a diameter of 60 to 500 μm. b) A step of tanning the tissue to form the leather.

2. The method according to claim 1, wherein the scaffold includes macropores with a diameter of 80 to 280 μm.

3. The method according to claim 1, wherein the scaffolding has at least one surface having a porosity of 65% to 98%.

4. The method according to claim 1, wherein the scaffold comprises at least one polymer.

5. The method according to claim 4, wherein the scaffold comprises a polymer that is at least a polymer organosilicon compound or a biodegradable polyester.

6. The method according to claim 4, wherein the scaffold comprises a polymer in which at least polydimethylsiloxane (PDMS).

7. The method according to claim 4, wherein the scaffold comprises a polymer that is at least polyester.

8. The method according to claim 4, wherein the scaffold comprises a polymer that is at least biodegradable and bioabsorbable polyester.

9. The method according to claim 1, wherein the macropores are interconnected with micropores having a diameter of less than 20 μm.

10. The method according to claim 1, wherein the scaffolding is a symmetrical scaffolding containing macropores distributed over the thickness of the scaffolding.

11. The method according to claim 10, wherein the scaffold has two macroporous surfaces, and both surfaces contain macropores with a diameter in the range of 60 to 500 μm.

12. The method according to claim 10, wherein the scaffold comprises polydimethylsiloxane (PDMS) and the scaffold is obtained by solvent casting and particle leaching (SCLP) method.

13. The method according to claim 10, wherein the scaffold comprises polyester and is obtained by a solvent casting and particle leaching (SCLP) method and a non-solvent-induced phase separation process (NIPS).

14. The method according to claim 1, wherein the scaffold is an asymmetrical scaffold comprising macropores with a diameter of 60 to 500 μm and micropores with a diameter of less than 10 μm distributed throughout the entire thickness of the scaffold.

15. The method according to claim 14, wherein the scaffold has a first surface containing macropores with a diameter of 60 μm to 500 μm, and a second opposite surface containing only nanopores with a diameter of 10 nm or less.

16. The method according to claim 15, wherein the scaffold comprises biodegradable polyester and the scaffold is obtained by solvent casting and particle leaching (SCLP).

17. The method according to claim 1, wherein a bioactive molecule is grafted onto the surface of the scaffold.

18. The method according to claim 17, wherein the bioactive molecule is collagen, carbohydrates, or carbohydrate-derived molecules such as glycosaminoglycans.

19. A tissue obtained after step a) of the method according to any one of claims 1 to 18, comprising fibroblasts cultured on a macroporous scaffold as defined in any one of claims 1 to 18.

20. Tanned leather that can be obtained by the method described in any one of claims 1 to 18.

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