WO2022003652A1 - Poly(ℇ-caprolactone)-collagen/tgf-β3 scaffold - Google Patents

Abstract

The present invention relates to a poly(ℇ-caprolactone)-collagen (PCL/COL) scaffold, which comprises capsules of transforming growth factor β3 (TGF-β3), and to a method for producing same by means of electrospinning. The structure of the invention forms a polymer matrix for the adhesion, maintenance and proliferation of Wharton's jelly mesenchymal stromal cells (WG-MSC), as a therapeutic strategy in tissue regeneration.

Description

POLY (ℇ-CAPROLACTONE) -COLLAGEN / TGF-β3 SCAFFOLD FIELD OF THE INVENTION The present invention relates to the general field of tissue engineering and regenerative medicine, particularly to a poly ℇ-caprolactone (PCL) and collagen ( COL) comprising transforming growth factor (TGF-β3), with activity in the regeneration or repair of tissue. BACKGROUND OF THE INVENTION Tissue engineering is a science that has been described as a strategy to design and manufacture living tissues using biomaterials, involving the combination of cells, bioactive molecules and biomaterials that provide structural support and supply of biological agents, for the design and development of functional biological structures suitable for the replacement, repair or regeneration of damaged tissues or organs as well as the renewal of parts lost due to disease, cancer and trauma (C. Rovera et al. Mechanical behavior of biopolymer composite coatings on plastic films by depth-sensing indentation – A nanoscale study, Journal of colloid and interface science, 512, 638- 646 (2018). https://doi.org/10.1016/j.jcis.2017.10.108). Conventional therapies for tissue regeneration that are based on autografts and / or allografts to cover the affected area and reduce the healing processes, present limitations that, to mention a few, focus on the lack of available tissue to meet the current demand for patients who need it, treatment based on immunosuppressants that put the patients' lives at risk, risk of infection, immune rejection and appearance of cosmetic and functional defects (FJ O'brien. Biomaterials & scaffolds for tissue engineering. Materials today, 14, 88-95 (2011). Https://doi.org/10.1016/S1369-7021(11)70058-X, JW Su et al. Closure of a large tracheoesophageal fistula using AlloDerm, The Journal of thoracic and cardiovascular surgery, 135, 706-707 (2008). https://doi.org/10.1016/j.jtcvs.2007.11.014). Cell Therapies As an alternative to the use of grafts, cell therapy is developed that focuses on the repair of injured tissues and consists of the transplantation of individual cells to a recipient tissue in sufficient quantities for them to survive and restore tissue function (VF Segers et al. Stem-cell therapy for cardiac disease. Nature, 451, 937 (2008). https://doi.org/10.1038/nature06800). Within the specific lineages and multipotent cells that are used are epidermal stem cells characterized in that they can regenerate epidermis, sebaceous glands and eight different types of epithelial cells of the hair follicle. Since epidermal stem cells have the potential to regenerate the skin, several clinical trials have focused on them to offer an alternative treatment option for major skin lesions, resulting from extensive burns, infection, or trauma. Additionally, cultured human keratinocytes can differentiate and form a functional skin barrier that can be transplanted into patients suffering from severe burn injuries. Currently, they represent the most widely used cellular products in the world, for example, in the pharmaceutical market, several types of cellular products have been developed for skin repair in the form of confluent or pre-confluent autologous or allogeneic keratinocytes, with applications not only for the treatment of severe burns, but also for cases of venous or diabetic ulcers. On the other hand, chondrogenic cells or chondrocytes are the only cells present in the articular cartilage, they are responsible for synthesizing all the components of the articular matrix and have the capacity for adaptive deformation in response to mechanical forces. They are used for the effective treatment of circumscribed cartilage injuries (dissecting osteochondritis, patellar chondropathy, ankle chondromalacia, osteochondral injuries). Otherwise mesenchymal stromal cells (CEM) are primitive multipotent cells, with fibroblast morphology, originating from the mesodermal germ layer, which have the ability to differentiate into osteocytes, chondrocytes, adipocytes both in vivo and in vitro. These cells have immunosuppressive properties and through paracrine signaling they favor angiogenesis and cell repair processes. In addition, the fact that they can be differentiated into cartilage, bone or fat, makes their use possible in the treatment of diseases or injuries that affect these tissues. Wharton's gelatin mesenchymal stromal cells (CEM-GW) in particular stimulate other cells to begin creating a wide variety of processes such as new tissue formation, healing, tissue repair, bone, skin, and ligament healing. Although CEM-GW present innumerable characteristics that classify them as a possible therapeutic strategy, their application has also presented limitations. In particular, cells that are transplanted by injection or infusion are poorly retained at the transplanted site, generating a very low graft rate and therefore reducing the effectiveness of the treatment (Y. Tabata. Biomaterial technology for tissue engineering applications, Journal of the Royal Society interface, 6, S311-S324 (2009). Https://doi.org/10.1098/rsif.2008.0448.focus, WM Saltzman et al. Building drug delivery into tissue engineering design, Nature Reviews Drug Discovery, 1, 177 ( 2002). Https://doi.org/10.1038/nrd744, H. Naderi et al. Critical issues in tissue engineering: biomaterials, cell sources, angiogenesis, and drug delivery systems, Journal of biomaterials applications, 26, 383-417 ( 2011). Https://doi.org/10.1177/0885328211408946). Strategies that favor the retention of EMF at the site have therefore been studied, such as, for example, the use of fibrin clots or collagen supports. Additionally, once the stability of the cells is achieved, it is essential to guarantee proliferation. and CEM-GW secretion profiles, which can be achieved by maintaining cell viability and inducing the production of various factors. Scaffolds as dermal substitutes Scaffolds designed for the purpose of being used in tissue regeneration or repair, in particular as ideal dermal substitutes, should have the following characteristics (A.-TN Truong et al. Comparison of dermal substitutes in wound healing utilizing a nude mouse model, Journal of burns and wounds, 4 (2005), DW Hutmacher, Scaffolds in tissue engineering bone and cartilage, Biomaterials, 21, 2529-2543, (2000), S. MacNeil, Biomaterials for tissue engineering of skin, Materials Today , 11, 26-35, (2008), AC Colorado et al. Analysis of biomaterials for use in skin tissue engineering: review, Biomedical Engineering Journal, 7, 11-23 (2013)): - Three-dimensional and highly porous structures with a network of interconnected pores for cell growth and the transport of nutrients and metabolic flow waste; - biocompatible and bioresorbable with a controllable degradation and resorption rate to adapt to cell / tissue growth in vitro and / or in vivo; - a suitable surface chemistry for cell attachment, proliferation and differentiation; - mechanical properties to match those of the tissues at the implantation site. Transforming Growth Factor (TGF-β3) The TGF-β1, 2 and 3 family of isoforms play a number of crucial roles in dermal wound repair and regeneration, including regulation of cell proliferation, differentiation, migration, the invasion and chemotaxis of the epithelial and fibroblastic tissue compartments, as well as the proliferation, migration and invasion of cells endothelial cells and mediation in the formation of new blood vessels (M. Valluru et al. Transforming growth factor-β and endoglin signaling orchestrate wound healing, Frontiers in physiology, 2, 89, (2011). https://doi.org/ 10.3389 / fphys.2011.00089). TGF-β isoforms are present in all three classic stages of dermal wound repair. In the inflammatory stage, TGF-βs participate as potent chemoattractants and mediators of inflammation for various types of immune cells, including neutrophils and other polymorphonuclear cells. In the migration stage, the three isoforms of TGF-β promote re-epithelialization, stimulating the synthesis of extracellular matrix and promoting the recruitment of fibroblasts adjacent to the wound; in this stage, TGF-β1 promotes keratinocyte migration. Finally, in the remodeling stage, TGF-β1 induces wound contracture, collagen I and III synthesis, while TGF-β3 regulates this production to minimize scar formation (R. Gilbert et al. Signaling by transforming growth factor beta isoforms in wound healing and tissue regeneration, Journal of developmental biology, 4, 21 (2016). https://doi.org/10.3390/jdb4020021). The transforming growth factor (TGF-β3) binds to receptor proteins on the surface of epithelial and endothelial cells, mainly, where said union triggers the transmission of signals within the cell, controlling various cellular activities, such as growth , proliferation, differentiation, motility and controlled cell death. Protein is also involved in blood vessel formation, regulation of bone growth, wound healing, and immune system function. TGF-β3 in turn acts as a transduction factor, signal transduction processes allow cells to respond to the influences of the environment that surrounds them. Thus, cells modulate all their functions, from the most general to other functions such as factor secretion, which is why the marked increase in TGF-β2 in the scaffolds where this encapsulated factor remains is attributed to the presence of TGF-β3. Various developments have been described for wound healing and tissue regeneration, related to constructs based on natural or synthetic polymers, with or without incorporation of cells. For example, patent US9655995B2 discloses a three-dimensional nanofiber scaffold and methods for using said scaffolds in repairing damaged tissue, particularly cardiac tissue. Said nanofiber scaffold is composed of a biodegradable polymer such as polycaprolactone or poly (ℇ-caprolactone), which is electrospun to create a nanofiber mat that is subsequently divided into strips arranged in one or more layers that are woven in a basketwork configuration. using the noobing technique, that is, where at least two of the nanofiber layers are stacked orthogonally to each other and mimic the structure of native tissue. On said scaffold, one or more relevant cells are seeded that are selected from adult, embryonic, pluripotent or primary stem cells, which adhere, proliferate and organize, imitating the structural and mechanical properties of a tissue, particularly cardiac. Patent US10034737B2 refers to a biomaterial to be used as an implant, which comprises a nanofibrous scaffold made of polymers such as poly (ε-caprolactone) preferably electrospun or collagen, coated with a pair of multilayer drops of polyanions and polycations that incorporate a therapeutic molecule , for example, transforming growth factor; allowing the sustained release of said molecule at the implantation site of the biomaterial and optionally, living cells such as osteoblasts, chondrocytes, mesenchymal stem cells, among others, which are found within an alginate or collagen hydrogel and are deposited on the nanofibrous scaffold covered. Other authors, for example, Ghosal et al (Ghosal et al. 2016. Structural and surface compatibility study of modified electrospun poly (ε-caprolactone) (PCL) composites for skin tissue engineering. AAPS PharmSciTech 18, 72-81 (2017). https://doi.org/10.1208/s12249- 016-0500-8) have addressed the structural and surface compatibility of modified poly (ε-caprolactone) compounds for skin tissue engineering. In this study, biodegradable PCL nanofibers coated with collagen or titanium dioxide are developed, prepared using the electrospinning technique. Additionally, Lizarazo et al (Lizarazo et al. 2019. Poly (ɛ-caprolactone) / collagen electrospun scaffolds with potential use in skin tissue regeneration. Science in Development; 10 (2): 197-208), analyzes electrospun scaffolds of poly (ℇ- caprolactone) / collagen with potential use in regeneration of skin tissue, where constructs were developed from the mixture of polycaprolactone (PCL) and type I collagen, whose objective was the evaluation of the influence of collagen on the fibers of PCL. For this purpose, PCL / COL formulations were used to obtain a polymeric scaffold matrix for the evaluation of its ability to allow adhesion of Wharton's gelatin mesenchymal stromal cells (CEM-GW). The article concludes that the addition of collagen favors the adhesion processes of the (CEM-GW), and that it is possible to obtain a scaffold with adequate cellular support properties as a candidate for the treatment of dermal wounds. However, despite research and development in the field, tissue loss, either due to certain pathologies or due to different traumas, caused by accidents, continues to motivate the development of therapies in the area of tissue engineering to through which the damaged tissue can be restored and / or regenerated, greatly reducing the use of autografts, the problems with the immune response of allografts, the favoring of the effectiveness of cell therapy and the contribution to the acceleration of the healing processes. Therefore, there is still a need for products developed from suitable biomaterials that provide cells with support with structural integrity and supply of biological agents, that favor a tissue-specific microenvironment to replace damaged tissue, induce regeneration and restoration. of functionality appropriate cell tissue, and regulate cell functions and accelerate healing processes. Therefore, to guarantee an ideal therapy for wound healing by means of a product that promotes rapid healing, that presents a high porosity to achieve the correct vascularization, that is bio-active and that contributes to a minimum formation of scars regardless of the cause of tissue loss, it still represents a challenge. BRIEF DESCRIPTION OF THE INVENTION The present invention relates to a poly (ℇ-caprolactone) -collagen (PCL / COL) scaffold comprising transforming growth factor-β3 (TGF-β3) capsules in order to mimic the extracellular matrix of the skin, of high importance in skin re-epithelialization and its production method by means of the electrospinning technique. The scaffold of the present invention makes it possible to create a microenvironment conducive to cell adhesion and proliferation, particularly Wharton's gelatin mesenchymal stromal cells as a therapeutic strategy in tissue regeneration. BRIEF DESCRIPTION OF THE FIGURES FIG. 1A Scaffold structure and layered configuration comprising the encapsulation of the transforming growth factor (TGF-β3). FIG. 1B Scaffold structure and layered configuration comprising the encapsulation of the transforming growth factor (TGF-β3) and additionally an outer layer of cells. FIG. 2 Scanning electron microscopy for evaluation of the morphology of the electrospun PCL membrane. Fibers obtained with the precursor polymer (PCL) are evidenced, which presented an average fiber diameter of 1.12 µm ± 0.15 µm. FIG. 3A-H Scanning electron microscopy for evaluation of the morphology of PCL / COL1-8 membranes, respectively. Fibrous structures of sub-micron size randomly oriented are evident. The increase in elongation forces and the greater bending instability resulted in fibers of smaller diameter with respect to the PCL membrane, due to the presence of the collagen protein characterized by being positively charged. FIG. 4 Release of transforming growth factor β3 vs. time. Capsules on the scaffold (PCAT) and capsules in suspension (Capsules + TGF-β3). TGF-β3 (PCAT) had a release duration of 8 days, compared to suspension capsules that showed a factor release in 5 days. FIG. 5 Cell proliferation analysis of CEM-GW cells in PC, PCA, PCAT scaffolds, after an incubation period of 1, 3, 5 and 7 days of culture. The asterisks denote the statistically significant difference at p <0.05 between the different scaffold formulations with a significance level of 0.009 for a two-way analysis of variance. FIG. 6A-C Graphs of the secretion profiles of the transforming growth factors β by CEM-GW grown on the PC, PCA, PCAT scaffolds. The production of TGF- β1, TGF- β2 and TGF- β3, respectively by the CEM-GW cultured on the scaffolds is shown, evidencing that the secretion profiles of the TGF-β2 isoform vary substantially in the presence of TGF-β3. . FIG. 7A-F– Graphs of the secretion profiles of the factors angiopoietin I, EGF, FGF-b, HGF, PDGFaa, VEGFaa, respectively by CEM-GW grown on PCL, PCA, PCAT scaffolds. It is evidenced that the secretion of FGF-b increases considerably due to the surface conformation of the polymer in the scaffold. electrospinning, while the production of the other factors is not affected by the structure of the scaffold. DETAILED DESCRIPTION OF THE INVENTION The present invention corresponds to a scaffold obtained from different biomaterials, particularly the mixture of polyester poly (ℇ-caprolactone) (PCL) and collagen (COL) that provides a polymeric scaffold matrix for factor immobilization of transforming growth β3 (TGF-β3), for the adhesion, maintenance and proliferation of cells, as a potential therapeutic strategy in the regeneration of cutaneous tissue. For purposes of interpreting this description, the following definitions will apply and where appropriate, terms used in the singular form will also include the plural form. Terms used in the description have the following meanings unless the context clearly indicates otherwise: Scaffold: A construction or structure that provides a physical framework in three-dimensional space in which cells are seeded and is essential to support cells in vitro with a pleasant environment, which should be similar to its natural conditions in vivo, providing an extracellular matrix type microenvironment (ECM) and characterized by comprising materials that actually represent the tissue-specific ECM. As such, scaffold materials allow protection of biologically active substances or cells from the biological environment. In the present invention without being limited thereto, scaffolds can be seeded with the desired cells and incorporated with tissue growth factors to enhance cell functionality. Biomaterials: set of materials used to reproduce the function of living tissues in the biological systems that make up a scaffold, which are mechanically functional and physiologically acceptable as they are temporarily or permanently implanted. in the body and trying to restore the existing defect and, in some cases, achieve tissue regeneration. The examples of biomaterials used here for the generation of scaffolds are characterized by their biocompatibility, they can be of natural origin, for example, without being limited to collagen, gelatin, elastin, chitosan, hyaluronic acid or synthetics such as poly (lactide-glycolide) (PLGA ), poly (ℇ-caprolactone) (PCL). Collagen (COL): natural protein-type biomaterial composed of three chains that form a triple helix, whose mechanical, chemical and biological properties include its great stability, high tensile strength, extensibility, formation of molecular aggregates, water retention, ability to form cross-linking bonds, degradation by collagenases, tissue reabsorption, semipermeability, interaction with different molecules, reduced antigenicity, promotes cell adhesion, interacts with platelets and produces the activation of the components of the blood coagulation system. Alginate: natural biomaterial of the anionic and hydrophilic polysaccharide type, whose chemical structure is characterized by the fact that it contains monomer blocks of β-D-mannuronic acid (M) and α-L-guluronic acid (G) linked (1-4) and exhibits properties biocompatibility, biodegradability, non-antigenicity and chelating capacity, which is why it is used as a support matrix or administration system for tissue repair and regeneration. Polycaprolactone (PCL): biodegradable polyester-type synthetic biomaterial that is composed of a sequence of methylene units, between which ester groups are formed and is degraded by hydrolysis of its ester bonds under physiological conditions (such as in the human body) by what is used as an implantable biomaterial. Mesenchymal stromal cells (CEM): correspond to a group of adult stem cells isolated from tissues such as bone marrow, umbilical cords and adipose tissue, which differentiate into cells of their own lineages or atypical lineages in some cases, such as osteocytes , chondrocytes, adipocytes, myocytes and even hepatocytes and neurons. Multipotent epidermal stem cells: cells found in niches of the interfollicular epidermis, sebaceous glands and in the outer envelope of the root of the hair follicle, called the “bulge”. This reserve of epithelial stem cells is capable of regenerating epidermis, sebaceous glands and eight different types of epithelial cells of the hair follicle. Human keratinocytes: cells that make up most of the epidermis, in humans they represent about 95% of epidermal cells. They have an ectodermal origin and are organized to form a keratinized flat stratified epithelium. The proximity of the numerous desmosome-like junction complexes that exist between keratinocytes allows the cohesion and integrity of the epidermis to be maintained. These cells produce keratin and also produce cytokines that are soluble molecules with regulatory functions of epithelial cells and dermal cells. Keratinocytes form the 4 layers of the epidermis namely the basal layer, the stratum spinosum, the stratum granulosa and the horny layer. Chondrocytes: they are the only cells present in the articular cartilage, they synthesize and secrete the organic components of the extracellular matrix, which are basically collagen, hyaluronic acid, proteoglycans and glycoproteins, and according to the characteristics of the matrix, hyaline and fibrous cartilage can be distinguished. Transforming Growth Factor-β (TGF-β): A cytokine involved in cellular processes such as hematopoiesis, proliferation, angiogenesis, differentiation, migration, and cell apoptosis. In particular, the TGF-β1 and TGF-β2 isoforms present in adult wounds are involved in all phases of healing resulting in the formation of a scar, while embryonic wounds express high levels of TGF-β3, that repairs a wound and does not generate a scar, that is, it induces a regenerative response. Electrospinning: a technique for making ultrafine fibers from natural and synthetic polymers that have diameters ranging from submicron sizes to nanometric scales. In the electrospinning process, the polymer solution is subjected to an electric field up to a critical value, where the repulsive electrical forces exceed the surface tension forces, generating the evaporation of the solvent and collecting only one polymer fiber. TFE: 2,2,2-trifluoroethanol, it is the organic compound with the chemical formula CF3CH2OH, the three fluorine molecules give alcohol a high ionization constant and a very strong hydrogen bonding capacity, which makes it a solvent organic widely used in electrospinning process. HFP: 1,1,1,3,3,3-hexafluoro-2-propanol, is a polar solvent with high ionizing power, ideal for dissolving polyamides and esters. It is used as a polar solvent and exhibits strong hydrogen-bonding properties, dissolving substances that are hydrogen-bonding acceptors, such as amides, ethers, and a wide range of polymers, including those that are not soluble in the more common organic solvents. PCL / COL membranes The scaffolds of the present invention are formed by membranes of different mixtures of poly (ℇ-caprolactone) -collagen. In one embodiment, the collagen used is type I collagen. It is known in the state of the art that the manufacture of scaffolds generally uses collagen concentrations that vary between 50 and 75% with respect to the PCL, which are characterized by producing scaffolds. with a significant decrease in physical characteristics such as resistance and rigidity given the high proportion of natural polymer with respect to synthetic polymer. Likewise, the large amounts of collagen contribute to the formation of large domains within the fibers and the PCL concentrates on the periphery of the itself, masking the collagen inside and thereby eliminating its biological properties. (VY Chakrapani et al. Electrospinning of type I collagen and PCL nanofibers using acetic acid, Journal of Applied Polymer Science, 125, 3221-3227 (2012). Https://doi.org/10.1002/app.36504, Zhang, Y ., et al. Characterization of the surface biocompatibility of the electrospun PCL-collagen nanofibers using fibroblasts. Biomacromolecules, 6 (5), 2583-2589 (2005). https://pubs.acs.org/doi/10.1021/bm050314k, Powell et al. Engineered human skin fabricated using electrospun collagen – PCL blends: morphogenesis and mechanical properties. Tissue Engineering Part A, 15 (8), 2177-2187 (2009). Https://www.liebertpub.com/doi/10.1089 /ten.tea.2008.0473. Tillman et al, The in vivo stability of electrospun polycaprolactone-collagen scaffolds in vascular reconstruction. Biomaterials, 30 (4), 583-588 (2009). https://www.sciencedirect.com/science / article / pii / S0142961208007813? via% 3Dihub. It has been observed that these variations in the concentration of collagen are not significant in the diameter of the fibr as obtained, while they are expensive due to the high proportion of this protein. In the present invention, the polymers are mixed in different ratios, for example PCL / COL between 19: 1 and 9: 1. The membranes obtained with collagen concentrations lower than 1% not only allow cell growth and a decrease in fiber diameter but also obtain a less expensive product that exhibits improved biological properties. In particular, said membranes comprise low concentrations of collagen with respect to PCL, while they significantly reduce the diameter of the fiber, with respect to the precursor polymer. In a particular embodiment of the present invention, the membranes are obtained by means of the electrospinning technique, in order to obtain a polymeric scaffold matrix. Since the protein used contains amide groups that make the conductivity of the solution increases, the elongation of the fiber is favored during the electrospinning process, resulting in fibers of smaller diameter. The PCL / COL membranes of the present invention are obtained by dissolving the polymers in a suitable solvent, selected from TFE, HFP, dimethylformamide and chloroform. In a particular embodiment of the present invention, the polymeric matrix is obtained by solubilizing the polymers in 2,2,2 trifluoroethanol with a total polymer concentration (PCL / COL) of 9% by weight. The PCL is mixed with collagen in a 19: 1 ratio, obtaining a particular concentration of 85.5 mg / mL of and 4.5 mg / mL of the polymers in the solvent, respectively. The PCL / COL membranes of the present invention are characterized by physical parameters such as a fiber diameter of 200 to 400 nm, porosity between 50 and 75% and hydrophobicity at a contact angle of 60 to 80 ° at 0 seconds and angle of contact from 5 to 15 ° to 5 seconds. The fiber diameter can be between about 235 and 355 nm. In one embodiment, the PCL / COL membranes of the scaffold are characterized by a fiber diameter of 295.5 nm and a porosity of 57.57%. Hydrophobicity is defined by a contact angle in 0 seconds of 71.45 ± 5.87 ° and a contact angle in 5 seconds: 10.13 ± 2.59 °. The mechanical and thermal properties exhibited by the PCL / COL membranes correspond to a Young's modulus of 6.986 ± 2.398 MPa, strain percentage of 114.08%, enthalpy of fusion (∆H_m) of 63.35 J / g, melting temperature (T_m) of 57.31 ° C and percentage of crystallinity (Xc) of 45.41%. Young's modulus measurement corresponds to a value close to that reported in the literature for human body tissues, particularly the parameters ∆H_m, T_m and X_c they are similar in the thermal characterization of PCL due to the high proportion of this polymer in the solution. Transforming growth factor β3 (TGF-β3) In the scaffold of the present invention, the intermediate layer is made up of transforming growth factor (TGF-β3). It is known that TGF-β3 injected into skin wounds reduces the inflammatory response and can limit the production of fibrotic tissue. However, this factor is unstable at room temperature, which limits its use for administration and its preservation since it is a protein susceptible to losing its activity with slight changes in pH or temperature (A. Roberts et al. The transforming growth factor-βs , Peptide growth factors and their receptors I, 419-472 (1991). Https://doi.org/10.1007/978-1-4612-3210-0_8, GM Saed et al. Transforming growth factor beta isoforms production by human peritoneal mesothelial cells after exposure to hypoxia, American Journal of Reproductive Immunology, 43, 285-291, (2000). https://doi.org/10.1111/j.8755-8920.2000.430507.x). Different factor stabilization mechanisms include chemical immobilization within or on a matrix and physical encapsulation in the delivery system. Immobilization involves chemical binding or affinity interaction between the polymer and the growth factor, while the physical method relies on encapsulation, diffusion, and controlled release of the growth factor from the substrate. In the present invention, TGF-β3 is encapsulated and immobilized in a matrix. Encapsulation is beneficial because it minimizes the factor's exposure to harsh conditions during processing while protecting its activity. In addition, the materials used in encapsulation and immobilization confer mechanical resistance, porosity and relevant degradation rates that allow them to be implanted. Encapsulation can be carried out with materials such as synthetic polymers, including poly (α-hydroxy acids), poly (orthoesters), poly (anhydrides), poly (amino acids), dextrin, poly (glucoside) (PGA), polyacid (l- lactic acid) (PLA), its copolymers and natural polymers such as fibroin, keratin, collagen, gelatin, fibrinogen, elastin, chitosan, hyaluronic acid, starch, carrageenan, cellulose, and alginate. In a particular embodiment of the present invention, TGF-β3 is encapsulated in calcium alginate, which exhibits suitable encapsulating properties to maintain the bioactivity of TGF-β3 and guarantee its bioavailability over time. Cells of the outer layer of the scaffold According to the present invention, the PCL / COL and TGF-β3 scaffold may comprise an outer layer, which is made up of cells that are selected from mesenchymal, epithelial progenitors (keratinocytes and stem cells multipotent epithelial cells) and / or chondrogenic (chondrocytes or chondroblasts). The cells of the outer layer are characterized by being cells capable of producing factors selected from angiogenic, epithelial, and immunomodulatory factors. Particularly and without being limiting, said factors are selected from among hepatocyte growth factor (HGF), basic fibroblast growth factor (FGFb), vascular endothelial growth factor (VEGF), platelet-derived growth factor AA (PDGF- AA), transforming growth factor beta 2 (TGF-β2), granulocyte colony stimulating factor (G-CSF), transforming growth factor beta 1 (TGF-β1), neurotrophic factors selected from nerve growth factor (NGF) , neurotrophin-3 (NT3), neurotrophin-4 (NT4), glial-derived neurotrophic factor (GDNF), chemokines selected from CCL5 chemokines (RANTES), monocyte chemoattractant protein 1 (MCP-1,) eotaxin, interferon gamma- inducible protein 10 (IP-10), CXCL1, CXCL, CXCL5, CXCL6, CXCL8, cytokines, hematopoietic growth factors selected from granulocyte macrophage colony stimulating factor (GM-CSF), leukemia inhibiting factor (LIF), interleukins IL-1α, IL-6, IL-8, IL-10, IL-11 and angiogenic factors selected from angiopoietin 1, angiogenin, placental growth factor (PLGF), thrombospondin 2 and endostatin. In one embodiment, the mesenchymal progenitor cells are mesenchymal stromal cells (CEM), particularly Wharton's gelatin mesenchymal stromal cells (CEM-GW), structurally located on the upper or lower PCL / COL layer. Structure and Function of Scaffolds for Tissue Regeneration or Repair The PCL / COL-TGF-β3 scaffold of the present invention has a multilayer structure, wherein a layer consisting of encapsulated transforming growth factor (TGF-β3) is disposes and immobilizes in the middle of an upper and a lower layer of PCL / COL membrane. Optionally, an outer layer of cells is disposed on one of the more superficial upper or lower layers of the scaffold. With this structural arrangement (called sandwich type), the PCL / COL membrane allows the encapsulated growth factor to migrate towards the skin lesion without losing physicochemical and biological properties, serving as a support that directs the action of TGF-β3 through a sustained release of the same. Additionally, the addition of CEM on the surface of the scaffold allows TGF-β3 to stimulate the production of various cellular factors that act as immune modulators that, together with TGF-β3, exert a synergistic effect on the skin regeneration and strengthening process. the immunity of the treated tissue. The PCL / COL-TGF-β3 scaffold can also have an arrangement comprising as many layers of PCL / COL and TGF-β3 as required according to the thickness of the skin lesion and the underlying diseases of the patient. This means that the composition of the scaffold can favor the adhesion, maintenance and proliferation of cells. In a particular embodiment, the cells are CEM-GW as a potential strategy for the regeneration of skin tissue. Manufacturing method The manufacturing method of the PCL / COL scaffolds of the present invention comprises the preparation of the polymeric solutions of polycaprolactone and collagen (PCL / COL) in a suitable solvent, selected from TFE, HFP, dimethylformamide, chloroform, acetone , in quantities between 300 and 500 µL, obtaining a concentration of the polymers in solution of between 9 and 12% w / v, in a PCL: COL ratio of between 19: 1 and 9: 1. In a particular embodiment, the solvent used is 2,2,2-trifluoroethanol (TFE), due to the fact that it has a high value of dielectric constant that favors the reduction of the diameter and the defects of the resulting fibers during the electrospinning process. In a particular embodiment, the PCL and COL polymers are found in a concentration of 85.5 mg / mL and 4.5 mg / mL, respectively, for a concentration of 9% w / v of total polymers in the solvent. Subsequently, the solutions of the polymers are processed using techniques such as lyophilization of the polymers in solution (Chang et al. The application of type II collagen and chondroitin sulfate grafted PCL porous scaffold in cartilage tissue engineering. Journal of Biomedical Materials Research Part A: An Official Journal of The Society for Biomaterials, The Japanese Society for Biomaterials, and The Australian Society for Biomaterials and the Korean Society for Biomaterials, 92 (2) 712-723 (2010). Https://onlinelibrary.wiley.com/doi/ abs / 10.1002 / jbm.a.32198), 3D printing of PCL with collagen coating (Yeo et al. Preparation and characterization of 3D composite scaffolds based on rapid-prototyped PCL / β-TCP struts and electrospun PCL coated with collagen and HA for bone regeneration. Chemistry of materials, 24 (5), 903-913, (2012). https://pubs.acs.org/doi/full/10.1021/cm201119q) and foundry (Ahn et al. A new hybrid scaffold constructed of solid freeform-fabricated PCL stru ts and collagen struts for bone tissue regeneration: fabrication, mechanical properties, and cellular activity. Journal of Materials Chemistry,, 22 (31), 15901-15909 (2012). https://doi.org/10.1039/C2JM33310D) to obtain the PCL / COL membrane. In the present invention, the PCL / COL membranes are obtained by the electrospinning technique, according to the protocol described in Example 13. In a particular embodiment, the volume of the electrospun solution for obtaining the membranes corresponds to 300 µL. In a particular embodiment, the electrospinning process is carried out at room temperature with humidity values between 50 and 55%. After electrospinning, the scaffolds are vacuum dried for 24 hours to remove any remaining solvent. The encapsulation of the transforming growth factor TGF-β3 is carried out by generating three solutions 1) sodium alginate solution (2% w / v), 2) TGF-β3 solution and 3) calcium chloride solution (3% w / v) and the capsules are obtained by the extrusion method. The encapsulation of TGF-β3 in calcium alginate for subsequent immobilization in a polymeric membrane, allows the design of delivery systems in a controlled manner, with a potential direct application on the lesion, which favors the formation of blood vessels, regulates chronic inflammation, minimize scar formation, speeding up repair processes. Once the growth factor has been encapsulated, a vertical electrospinning assembly is used, where a layer of PCL / COL polymer solution is electrospun, the capsules are placed on the membrane and a new layer of 300 µL of polymeric solution is electrospun, using the protocol method previously described. The capsules are thus immobilized on the scaffold in the form of a sandwich. The scaffolds are stored in a humid chamber at 4 ° C. Optionally, on one of the outermost membranes of the scaffold it is possible to grow an additional outer layer of cells, as for example selected from mesenchymal, epithelial and / or chondrogenic progenitors. In one embodiment, said outer layer cells correspond to Wharton's gelatin mesenchymal stromal cells (CEM-GW). For the cultivation of the cells, the procedure of Example 13 was followed. By means of the developed method, a scaffold is obtained that exhibits adequate physicochemical properties as a biological matrix, which guarantees the availability of growth factor TGF-β3 over time while preserving its bioactivity. Applications of PCL / COL-TGF-β3 scaffolds The PCL / COL-TGF-β3 scaffolds of the present invention can be used as skin substitutes that achieve complete tissue repair, preservation of skin elasticity, decreased formation of the scar, controlled delivery of growth factor that allows a sustained release over time, allowing TGFβ-3 to play a key role in tissue repair, reducing healing times, improving the appearance and functionality of the tissue . Therefore, the scaffolds developed in the present invention provide various benefits for tissue repair or regeneration, including tissue repair or regeneration in the event of any loss occurring for reasons such as genetic disorders, thermal trauma, chronic or acute wounds and even surgical interventions. that can generate deep lesions that cannot be successfully treated by conventional treatments and put patients' lives at risk. Additionally, and depending on the cell type that is grown on the scaffold, it can be used in the treatment of tissue lesions in the skin or in cartilage. If the seeded cells are of epithelial or mesenchymal origin, it is used in the treatment of burns, vascular and diabetic ulcers. When cells of chondrogenic or mesenchymal origin are incorporated, use in the treatment of circumscribed cartilage lesions (dissecting osteochondritis, patellar chondropathy, ankle chondromalacia, osteochondral lesions). The present invention will be presented in detail through the following examples, which are supplied for illustrative purposes only and not with the aim of limiting its scope. EXAMPLES Example 1. Preparation of poly ℇ-caprolactone (PCL) collagen (COL) / transforming growth factor (TGF-β3) scaffolds For the manufacture of the scaffold, pellets of poly ℇ-caprolactone (MW) ~ 80,000 g / mole (PCL, Sigma Aldrich) and calf skin type I collagen (Sigma-Aldrich) in solution with the organic solvent 2,2,2-trifluoroethanol ≥99% (TFE, Merck), where the ratio of the polycaprolactone (PCL) polymers and collagen in solution at 19: 1 and 9: 1 w / w, their concentration at 9 and 12% w / v and the volume of the electrospinning solution at 300 µL and 500 µL. From the 300 µL solution an 8x8 cm and 40 µm thick membrane was obtained and from the 500 µL solution a 8x8 cm and 70 µm thick membrane. Figure 1 schematically illustrates the structure of the scaffolds of the invention. The compositions developed for the manufacture of the PCL / COL scaffold are shown below:

Table 1. PCL / COL formulations developed for the production of scaffolds To evaluate the influence of the addition of collagen in the PCL membranes, in addition to the combinations shown in Table 1, a reference solution of PCL without collagen was made, in concentrations of 90mg / mL (9% w / v) and 120mg / mL (12% w / v) in TFE, the solutions were kept under magnetic stirring at 500 rpm for six hours at room temperature. To identify the properties of the scaffolds obtained from the formulations exemplified in Table 1, the PCL / COL membranes obtained were characterized by means of techniques such as (SEM), contact angle, (FT-IR), (XPS), (TGA), (DSC). The evaluation of the physicochemical properties is shown in the following examples. The characterization of the scaffolds was carried out based on four general aspects, in order to identify and establish the influence of collagen on the PCL polymeric support. The first was to analyze the structural and morphological properties of the scaffolds, using techniques such as Fourier transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), contact angle, X-ray emitted photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and mechanical properties. The second was to evaluate the release of the growth factor from the microcapsules by means of the enzyme-linked immunosorbent assay (ELISA). The third aspect was to determine the viability of the scaffolds with a cytotoxicity test by means of resazurin test and in vitro cell proliferation by monitoring by fluorescence microscopy and fourth, the functionality of the construct was evaluated by the production of growth factors (transformant β, b-FGF, VEGF-A, HGF) and epithelial differentiation markers involved in the skin tissue regeneration processes of Wharton's gelatin mesenchymal stromal cells (CEM-GW) stimulated by scaffolds, using Luminex® technology. Example 2: Evaluation of fiber morphology The morphology of PCL / COL membranes and control PCL membranes was determined by observation by scanning electron microscopy (SEM) (JEOL-JSM-7600F). Figure 2 shows the morphology of the PCL membranes, while Figures 3A to 3H illustrate the PCL / COL membranes. Samples were viewed using magnification between 5000x and 10000x to determine fiber diameters and stimulate pore size using image analysis software. By means of the images obtained by scanning electron microscopy, the average fiber diameter and the percentage of porosity were defined for each formulation of the PCL / COL scaffold design, the results are summarized in the following table:

Table 2. Results of fiber diameter and porosity for the PCL / COL formulations The use of different proportions of collagen with respect to PCL and different percentages of total weight of the sample, indicated low variations between the averages of fiber diameter, finding that low concentrations of collagen can be used at low volumes with a similar result. The reduction of the fiber diameter evidenced in the fibers of the PCL / COL scaffold compared to that of PCL without collagen is attributed to the use of a protein that contains amide groups throughout its structural conformation, which increases the conductivity of the solution. . By increasing the conductivity of the solution, the bending instability during electrospinning can be increased, therefore, the jet path becomes longer and a greater stretching of the solution is induced, causing the reduction of the fiber diameter. . The increase in elongation forces and the greater flexural instability resulted in fibers of smaller diameter with respect to the fibers obtained with the precursor polymer (PCL). Porosity showed an inverse trend to fiber diameter, therefore, it can be inferred that, by decreasing the size of the fiber, the percentage of porosity of the scaffold increases and also its surface area. The scaffolds with less electrospun volume presented a higher percentage of porosity, due to the fact that when electrospinning a greater quantity of solution, a shielding effect was presented on the fibers, limiting the pores of the scaffold. Decreasing the volume of electrospun solution increases the wettability of the scaffolds, because the culture medium can better penetrate the fibers, this statement is supported by the contact angle analysis. Porosity is a crucial factor in the manufacture of scaffolds with potential application in tissue repair, while the porous nature is essential to facilitate cell infiltration and proliferation. A high degree of porosity guarantees a sufficient exchange of gas and nutrients, for cellular maintenance and subsequent wound healing. Formulations with an electrospinning volume of 0.3 mL will show better cell proliferation and maintenance characteristics than formulations with higher electrospinning. It is possible to show that the electrospinning technique provided a mechanism for the production of fibrous scaffolds, with high porosity, a wide distribution of pore diameters, a high ratio of volume per surface area and morphological similarities with respect to collagen fibers. that make up the native extracellular matrix (ECM). Particularly, in the human body, the basement membrane is a fibrous structure of 40 to 220 nm, made up of type IV collagen and laminin, where the fiber diameters of the PCL / COL scaffolds show a considerable approach to this value with an average diameter of 299.8 nm ± 69.96 nm. Example 3. Hydrophobicity tests This was performed by establishing the tangent angle of a drop of liquid with a solid surface at the base. The contact angle of the liquid fall on a solid surface is defined by the mechanical equilibrium of the fall under the action of three interfacial tensions: solid-vapor γsv, solid-liquid γsl and liquid-vapor γlv. This equilibrium is known as Young's equation. The interaction between the drop and the PCL / COL and PCL scaffolds was performed using Pinacle Studio software and a goniometer (Ramehart inc Model 100-07-00). A 2 µL drop of distilled water was deposited on the surface of the scaffolds and the angles were calculated from the drop contact with the support in the software (ImageJ). Surface wettability is an important property of biomaterials and can affect cell adhesion, maintenance, and proliferation. The contact angle that is generated from contact with water in the scaffold can be used to evaluate the wettability, so to get an idea of the behavior that the scaffolds will have in a physiological environment it is important to know the wetting characteristics of the membranes , in order to guarantee cell adhesion and proliferation. Below are the contact angle values for PCL AND PCL / COL scaffolds:

Table 3. Contact angle results for PCL / COL formulations Most synthetic polymers, including PCL, are hydrophobic due to the low presence of carbonyl groups in their chain, while natural polymers such as proteins (collagen) they are hydrophilic. After combining the collagen with PCL, it was observed that the water drop was absorbed within the first 5 seconds of contact with the scaffold fibers, resulting in a contact angle of less than 60 ° at 5 seconds, that is, The addition of collagen to the PCL polymeric matrix provides the scaffold with carboxylic groups and amide groups that reduce the hydrophobicity of the scaffold by up to 50% at the moment of contact of the drop of water with the scaffold, making the scaffolds highly Wettable, parameter of great importance for cell culture. It is concluded then that the addition of collagen to the PCL modified the hydrophobicity favoring cell culture because the scaffolds presented contact angles less than 90 °, considering them as wettable surfaces. According to the above, the scaffolds that present a higher volume of electrospun solution present a greater angle, due to the fact that the permeability of is reduced by the greater number of fibers, evidencing their resistance to being wetted, a result that is confirmed with porosity percentages calculated by scanning electron microscopy. Regarding the amount of collagen present in the different formulations, no evidence was found that in the scaffolds with a higher proportion; the drop will be absorbed faster, which implies that collagen in small proportions can change the polarity of the scaffold. Example 4. Loss of mass analysis by TGA This analysis allows measurements to be carried out to determine the composition of the materials and to predict their stability in a defined temperature range for each type of material, being useful to characterize materials that present loss or gain in weight. due to decomposition, oxidation or dehydration. The thermal transitions of the scaffolds were determined in a TGA Q5000 equipment from TA Instruments, approximately 5 mg of sample was placed and it was subjected in a range of temperatures, ranging from room temperature (Dear inventors, specify in degrees centigrade to which it is equivalent this value) up to 250ºC with a heating rate of 10ºC / min under the N ₂ atmosphere. The determination of the transition energies and temperatures was carried out using the TA Universal Analysis software. The results were analyzed for each mass loss in each material, where the TGA curve of the PCL control matrix shows that the decomposition temperature of the PCL was evident at 346.02 ° C with a mass loss of 93.54%, showing the thermal degradation of a single stage. The collagen thermogram shows a weight loss in several stages, the first of 42.71 ° C which corresponds to the loss of water acquired by hydration of the collagen fibers, whose mass represents 3.38%, and the second to 137.63 ° C corresponding to the loss of water linked to the collagen molecule and the breakdown of polypeptide chains with a loss of mass of 12.26%, the degradation of type I collagen is approximately from 210 ° C. For the collagen used, the decomposition occurred at around 246.85 ° C, corresponding to the degradation of the organic groups of the molecule, around 42.25% of the total mass of the sample. The remaining mass is finished degrading near 520 ° C leaving a residue of 17.26%.

Table 4. Results of the thermal stability analysis for the PCL / COL formulations The TGA thermograms of the PCL scaffold showed a continuous line until reaching decomposition temperature. The PCL / COL fibers, on the other hand, showed a progressive loss of weight before the decomposition temperature and a decrease in the degradation temperature in the PCL / COL scaffolds, attributed to the release of water molecules from the fibers and the breakdown of collagen. The PCL / COL scaffolds 6 and 8 present a higher denaturation temperature due to the fact that they comprise a higher concentration of synthetic polymer, resembling the degradation temperature of PCL. In the case of the PCL / COL 4 and 6 scaffolds that present a higher concentration of collagen, the reduction in the loss of mass over the decomposition temperature is evidenced, due to the elimination of volatile substances contributed by the collagen in the scaffolds. In the decomposition stages in the TGA analysis, it was observed that the signals for PCL and collagen were not constant for all mixtures, but they vary to a small degree and suggest the interaction between collagen and PCL, so the thermal property of a it could be affected by the other. Suggesting that the reason for the variations in thermal properties is due to a possible interaction of amide groups of collagen with carboxyl groups of PCL. Example 5. Analysis of functional groups by FT-IR In order to confirm a possible interaction of PCL with collagen, electrospun matrices obtained from various combinations of collagen and PCL were subjected to FT-IR analysis. The characterization of the functional groups of the scaffolds was determined in the Nicolet 6700 Thermo Scientific equipment using infrared spectroscopy with a Fourier transform with attenuated total reflection, where the sample was characterized using a measurement range of 4000 to 500 cm ^-1 at room temperature. For type I collagen used in scaffolding, NH stretch vibrations, CH2 vibrations, C = O vibrations of amide bonds, the bending vibrations of -NH, the bending vibrations of CH2 and the vibrations of C- N can be observed respectively at 3309, 2938, 1724, 1548, 1375 and 1292 cm ^-1 . The peaks observed for PCL only at 2940 and 2865 cm ^-1 are due to vibrations of CH ₂ , the intense signal at 1728 cm ^-1 corresponds to the vibrations of C = O, at 1452 cm ^-1 bending vibrations of CH ₂ and 1240 COC asymmetric tension vibration, suggesting that the electrospinning process does not modify the composition. With respect to the FT-IR spectra of the different PCL / COL formulations made, the signal of decreased intensity with respect to the FT-IR of the PCL, observed at 1724 cm ^-1, is attributed to the reduced availability of PCL C = O In the interaction with collagen, the C = O vibrations observed at 1630 cm ^-1 suggest that the collagen fibers are well dispersed and as a result the amide bonds may be highly delocalized in the adjacent C = O group. The slight changes in the signals of the FT-IR spectra with respect to the PCL, show the interactions that occur between collagen and PCL during the electrospinning process as follows:

Table 5. FT-IR signals for PCL / COL and PCL formulations The state of the art suggests the use of high concentration of collagen to identify the presence of nitrogen groups in the scaffold, however, in the present invention It was possible to determine that the low natural polymer versus synthetic polymer ratios can achieve similar results, reducing scaffold production costs. Example 6. Analysis of transitions by DSC For the determination of thermal transitions of the scaffolds, the samples were dried in a desiccator with a VacuuBrand model RZ 2.5 vacuum pump. Then about 10mg of sample was placed in a TA Instruments DSC Q100 equipment. For the PCL sample and one of PCL / COL 19: 1 at 12% in THF solution, a cycle was made from -50 ° C to 150 ° C, returning to -50 ° C with a heating rate of 20 ° C / min. under an atmosphere of N ₂ , for the other samples a heating branch from 25 ° C to 150 ° C was used. For the analysis of the transitions of the PCL, a heating cycle of the PCL was carried out, ranging from -60 ° C to 150 ° C and a cooling cycle from 150 ° C again to -60 ° C, yielding values of crystallization temperature (Tc) of 32.39 ° C, melting temperature (T _m ) of 57.42 ° C and glass transition temperature (T _g ) of 59.53 ° C. For collagen, since it can dehydrate or denature when heated, the characteristic endothermic peaks in the DSC thermogram are evident at 79.50 ° C due to the elimination of free water, with a high enthalpy value 119, 7 J / g and a second signal near 186.51 ° C with an enthalpy of 85.14 J / g, these signals have often been referred to as the collagen dehydration temperature (TDcol). PCL / COL scaffolds showed higher stability compared to natural collagen. In the DSC analysis of the different scaffold mixtures, the peaks around 57.42 ° C correspond to the melting point of PCL and the peaks around 186.51 ° C correspond to the melting (denaturation) of collagen. Masking of collagen denaturation was observed in the curves corresponding to different PCL / COL ratios, which may be due to the high proportion of PCL in the scaffolds. These results confirm those found in the TGA analysis, because there was no evident weight loss, up to 155 ° C, since the denaturation of collagen does not produce volatile substances, which could affect the mass of the residue. Additionally, the percentage of crystallinity (Xc) of the PCL / COL and PCL scaffolds was calculated using the following equation:

The percentage of crystallinity of PCL / COL scaffolds is affected by the concentration of collagen present in the fibers, thus, the percentage of crystallinity of PCL / COL is lower than that of PCL scaffolds and for PCL / COL scaffolds 19: 1 crystallinity was higher than 9: 1 scaffolds, as evidenced below:

Table 6. Melting point, enthalpy and percentage of crystallinity for the PCL / COL and PCL formulations The percentage of crystallinity (Xc) of the scaffolds is related to the rapid solidification process of electrospinning, since this process hinders the development of crystallinity, because the chains do not have enough time to form crystals. A high percentage of crystallinity affects cell adhesion because cells have preferences over amorphous surfaces. The low variability between the crystallization percentages between the pure polymer and the polymer in combination with collagen is due to the difference in the injection speed in obtaining the fibers, in the case of the PCL solution a higher flow was used, This flow reduces the re-christianization time of the polymer before the formation of the fibers. For example, in one modality the enthalpy values (∆Hm) were 63.35 J / g, melting temperature (Tm) of 57.31 ° C and percentage of crystallinity (Xc): 45.41%. Example 7. Analysis of the scaffold surface by XPS The measurements of X-ray photoelectron spectroscopy (XPS) constitute a tool to monitor the changes in the surface that appear after several physical-chemical treatments, therefore, it was used as a technique for the superficial chemical characterization of scaffolds from 2 to 3 nm in depth. They were performed on a VG-ESCALAB photoelectron microscope using a monochromatic Mg X-ray source (h = 1253.6 eV). The spectra were obtained with a constant energy step E0 = 50 eV with an electron detachment angle of 45 ° with respect to the normal surface. Spectral deconvolution analysis was performed with Origin 9® software. The surface of the scaffolds was analyzed in order to know the molecules present on the surface that make the cells adhere to the scaffolds. For the pure PCL spectrum, the signals of C1s and O1s were detected, in the case of C1s there were four signals (CC / CH at 284.6 eV, CO at 285.7 eV, OCO at 287.5 eV O-C = O at 288.7) and for O1s two signals (* OC = O at 533.1 eV and OC = O * at 531.7 eV) which correspond to the XPS analysis reported in the literature for PCL. Thus, according to the results obtained by XPS, it is confirmed that the scaffolding manufacturing process does not generate any change in the surface of the material, with which we can guarantee that cell adhesion is occurring in the polymeric material used. As shown below, the XPS results of PCL / COL scaffolds showed a signal attributed to N 1s, for the scaffold with the highest proportion of collagen (PCL / COL 4), which confirms the presence of collagen on the surface of the scaffold, for the other combinations collagen is undetectable due to the high proportion of PCL with respect to the protein, inferring that the protein is not located on the surface of the scaffolds.

Table 7. C 1s and O 1s signals for PCL / COL formulations Example 8. Material mechanical properties The tensile strength of electrospun nanofibers was determined using an INSTRON 5500R universal tensile testing machine (Instron Inc., MA, USA), using a 500 N load cell at a 10 mm / min [0.4 in / min] crosshead speed over the sample. The scaffolds were evaluated by uniaxial tension according to the ASTM D1708 standard, where the Young's modulus of the samples was estimated from the slope of the discharge curve in the region of maximum load. The experiments were carried out in triplicate and the results were reproducible in each specimen. PCL and PCL / COL scaffolds were evaluated by uniaxial stress according to ASTM D1708. Next, the values for Young's modulus and the percentage of deformation obtained for the different scaffolds are shown:

Table 8. Results of Young's modulus and percentage of deformation for the PCL / COL and PCL formulations Due to the formation of porous structures (an important parameter in the exchange of nutrients in biological studies) the electrospinning technique significantly reduces the mechanical properties of the materials, for example, an 80kD PCL scaffold obtained by extrusion can have a Young's modulus of 190 ± 6 MPa compared to the 22.634 ± 2.484 found for the electrospun PCL membrane. For the formulations developed in the present invention, it was found that those scaffolds that contain collagen present a decrease in Young's modulus compared to the PCL scaffolds, this is due to the fact that the PCL scaffolds present thicker fibers and less pore area, that directly affects this property, in addition to the possible electrostatic interactions that are generated between the protein and the polymeric chains of the polyester that decrease the resistance to deformation, since the proteins do not have great mechanical properties. The PCL / COL scaffolds with a higher electrospun volume presented Young's moduli with a slight increase due to the fact that having a greater volume of solution the number of fibers present is greater and therefore the porosity is reduced, generating scaffolds with a higher degree of tenacity. Regarding the amount of collagen, no significant differences were evidenced in Young's modulus, inferring that the lowest concentration of collagen can be used without representing a significant change with the membranes of higher concentration. Regarding the concentration by weight of the scaffold, those of 12% by total weight presented higher Young's modulus due to the increase in PCL in the membrane. The elastic behavior with subsequent deformation without neck formation was an evident behavior in the stress vs. deformation test, since no reduction in the cross-sectional area was observed before failure. The pure polymer PCL and the scaffolds containing collagen present Young's modulus and final stress significantly lower in some cases up to one third of the Young's modulus of the PCL scaffold, this can also be the product of the possible electrostatic interactions generated between the protein and the polymeric chains of the polyester that make the scaffolds less tenacious, since the proteins do not have great mechanical properties. The percentages of deformation before failure exceed 100% deformation, values that reach up to 171% deformation, attributed to the fiber diameters of the scaffolds since, being in submicron sizes and presenting pore area greater than 50%, they are It can present a rearrangement of the fibers due to the tension exerted that gives the material the possibility of elongating without generating breaks. Natural polymer scaffolds often have poor mechanical properties and although synthetic polymer scaffolds have improved mechanical properties, they do not possess inherent biological affinity. Therefore, membranes composed of synthetic and natural polymers generate a scaffold with an optimal combination of mechanical integrity and biological function. The measurement of the Young's modulus of scaffolds is a vital parameter to estimate the similarity to the characteristics of the skin, therefore, to establish a comparison with the properties of the skin, the following table reports the values as Young's modulus of skin found in literature:

Table 9. Young's modulus of the skin According to the Young's modulus or elasticity values obtained for the PCL / COL scaffolds, it can be inferred that the scaffolds present elastic properties with values similar to those reported for different areas of the skin, therefore which PCL / COL scaffolds could be classified as a good candidate to be used as a dermal replacement. Example 9. Cell Adhesion Assay Scaffold cytocompatibility was evaluated by measuring cell adhesion of outer layer cells seeded on one of the scaffold PCL / COL membranes, eg, human Wharton GFP gelatin mesenchymal stromal cells (CEM-GW), for which, CEM-GW transduced with green-fluorescent protein (CEM-GW-GFP) and wild-type (CEM-GW) were seeded. The evolution of the cell adhesion of CEM-GW-GFP on the PCL / COL scaffolds with pure PCL scaffolds was performed by inverted fluorescent microscopy at 24 h, 48 h, 15 days and 22 days for the first test and 24 h, 48h, 72h, 96h and 120h for the second trial. Proliferation of CEM-GW cells seeded into scaffolds was assessed using a resazurin cell proliferation and viability assay (Sigma-Aldrich, St Louis, MO, USA). USA). The CEM-GWs were seeded at a density of 5x10 ⁴ cells on the 1 cm diameter scaffolds in a 24-well plate. Cells were allowed to adhere for 24 hours, after which the medium was exchanged for 500 µl of a 10% solution of resazurin in fresh culture medium. After incubating the cells for 3 hours, 100 µL aliquots were measured using a microplate reader (Synergy; BioTek, USA) at 450 and 570 nm and the background absorbance was measured at 450 nm. Data were represented as the mean ± standard deviation. Statistical significance was tested using a Student's t test, and differences were considered significant if p <0.05. After 1, 3, 6, 9, 12 days of culture, the redox activity in the cytosol of the viable cells was evaluated by means of the reduction of resazurin, as evidenced in Figure 5. The determination of the viability of cells maintained in in vitro culture is a necessary requirement if these scaffolds are to be carried to implantation in vivo. In order to select the scaffold with the best formulation for the adhesion, maintenance and proliferation of the CEM-GW, a monitoring of the culture of CEM-GW GFP on the scaffolds was carried out, in vitro. Fluorescence images of the scaffolds were analyzed at four times (24h, 48h, 15 days and 22 days) and cell morphology and confluence in the PCL and PCL / COL scaffolds were compared. The formulations containing collagen (particularly PCL / COL 1 and PCL / COL 2) showed a positive response to cell adhesion, the cells at 24 h presented a fibroblast morphology typical of CEM-GW, also throughout the culture period. cells gradually proliferated on the scaffold. For the other PCL / COL 3 to PCL / COL 8 formulations, the cells at 24h had circular morphology, which indicates that the cells had problems for adhesion to the scaffold, after 48h the cells take on the fibroblast morphology and their proliferation over time. In the case of the PCL scaffold, the cells at 24h had circular morphology, which indicates that the cells had problems for adhesion to the scaffold, attributed to the hydrophobicity of the material. Example 10. Statistical analysis The statistical design 2 ³ was analyzed, in order to determine if the association between each of the experimentally obtained response variables (degree of cell adhesion by means of monitoring by fluorescence microscopy of the CEM-GW, diameter of fiber and percentage of porosity obtained by means of the analysis of the images of scanning electron microscopy SEM, and determined contact angle in a goniometer) and each variable included in the model (electrospun volume, proportion and concentration) are statistically significant. The p-value of each of the response variables was compared with the significance level to evaluate the null hypothesis. The null hypothesis corresponds to the fact that the coefficient of the term is equal to zero, which implies that there is no association between the variables and the response. Using the R statistical software, the statistical model contemplated at the beginning of the study was analyzed. The following table shows the design variables versus the response variables obtained experimentally.

Table 10. Relationship between the study variables and the response variables of the statistical design for the PCL / COL formulations. For the values of degree of adhesion, the coefficient of determination of the model was R ² 0.8452, this value infers that the Response variable is adjusted to the study model, indicating the degree of relationship that exists between the variance of the regression of the model and the variance of the response variable. With a significance level of 0.01541, the null hypothesis is rejected and it is affirmed that there is an association between the variables and the response of the study, in this case the variable that presents the most significance in the response is the total concentration of the scaffold, indicating that scaffolds with a lower total concentration favor cell adhesion in scaffolds. For the fiber diameter values, the coefficient of determination of the model was R ² 0.6399, a value that is far from the linearity of the model, for which it is inferred that the response variable did not have a good effect on the study model. With a significance level of 0.2216, the null hypothesis is accepted, which assumes that there is no association between the variables and the study response, inferring that the fiber diameter variable is independent of the parameters of the study model. Regarding the percentage of porosity, the coefficient of determination of the model was R ² 0.9171, this value indicates that the response variable fits the study model. The study variable that most affects the response variable is electrospun solution volume, indicating that lower solution volumes allow a higher percentage of porosity in the scaffold. With a significance level of 0.01253, the null hypothesis is rejected and it is affirmed that there is an association between the variables and the study response. For the contact angle value, the coefficient of determination of the model was R ² 0.8112, this value suggests that the response variable fits the study model. At the maximum levels of electrospun solution volume and total scaffold concentration, there is an increase in the contact angle, it presented a significance level of 0.04245, the null hypothesis was rejected and it was affirmed that there is an association between the variables and the response. of the study. Example 11. Evaluation of the encapsulation efficiency of the capsules loaded with TGF-β3 TGF-β3 is a protein susceptible to losing its activity with slight changes in pH or temperature. Looking for a release strategy that minimizes the risk of protein denaturation and guarantees the presence of the protein for a longer time, the obtention of capsules by ionic gelation by extrusion from sodium alginate was developed due to its biosafety and inertia towards encapsulated proteins. The calcium alginate capsules obtained by the extrusion method had an average size of 1.56 mm ± 0.66 mm, the size of the capsules is proportional to the caliber of the syringe used, the capsules showed a smooth surface, uniform with a 0.03 ± 0.002 faith that classifies them as capsules with a high sphericity factor. The distribution of Uniform size and high sphericity of the capsules can be attributed to the control of the variables of the ionic gelling process: concentration of the alginate solution, distance of the drop of the drop and immersion time in CaCl ₂ . Alginate is an anionic polysaccharide, while transforming growth factor β3 is positively charged, which can interact electrostatically with the polymer chain, allowing the factor to be physically immobilized in the polysaccharide capsules. This chemical interaction guarantees the preservation of the biological properties of the factor by subjecting the capsules to environmental agents or changes in temperature. The permeable membrane of the capsules and the degradation of the polymer allowed the immobilized growth factor to be solubilized in polar media and released in time. The total protein in the system was analyzed to infer the amount that should be present in each calcium alginate capsule, for which 500 ng of TGF-β3 were suspended in 200 µL of sodium alginate solution, after gelling. Ionic it was calculated that for every 200 µL of solution 40 capsules of calcium alginate are obtained containing 12.5 ng of TGF-β3 for each capsule. 5 capsules were added per cm ² of scaffold, obtaining a theoretical concentration of 62.5 ng of TGF-β3 per scaffold. The encapsulated amount of TGF-β3 in the five capsules was quantified by means of ELISA quantification, adding citric acid to a well, generating the rupture of the capsules and quantifying the total amount of the factor in the solution, obtaining this experimental value and knowing the theoric value. The encapsulation efficiency (% EE) using the following equation:

Example 12. Addition of the TGF-β3 loaded capsules in the PCL / COL scaffolds After evaluating the characteristics described in Example 10 and the cell viability, the PCL / COL 1 formulation was chosen to make the addition of the loaded microcapsules of the transforming growth factor TGF-β3 and evaluation of the addition of the capsules loaded with TGF-β3 in the scaffold. Example 13. Method of obtaining the PCL-COL / TGF-β3 scaffold For the purposes of the present invention, the scaffold exemplified below is found with the encapsulated transforming growth factor (TGF-β3). a- Electrospinning of the membrane: The polymers were dissolved in trifluoroethanol at a concentration of 9% of the total weight of the polymers in the solution, where 19 parts correspond to PCL and 1 part to COL. The solution was stirred magnetically at 500 rpm for 6 hours at room temperature. Each solution was loaded into 10 mL syringes with a 0.8 mm internal diameter blunt needle and incorporated into the injection system (NE injector model 4000 Syringe Pump Company) of the electrospinning equipment (power supply HV 350R Unlimited Company Amherst). For the poly ℇ-caprolactone / Collagen (PCL / COL) solutions, a voltage of 19 Kv was applied, a distance from the needle to the collector of 15 cm and a flow of 0.6 mL / h. A control scaffold of only polycaprolactone was also manufactured, for this to the 9% PCL solution, a voltage of 19 KV was applied, a distance from the needle to the collector of 15 cm and the flow was increased 1 mL / h due to the change in viscosity exhibited by the solution. A 10x10 cm copper collector covered with aluminum was used. homogenization of the polymers in the solution, this was loaded into a 10 ml syringe and incorporated into the injection system of the electrospinning equipment. The polymeric matrices were obtained at 19 Kv, 15 cm from the needle to the collector and a constant flow of the solution of 0.6 mL / h. The electrospinning process was carried out at room temperature with humidity values between 50 and 55%. The scaffolds were dried under vacuum for 24 hours to remove possible solvent remnants on the fibers. b- Encapsulation: For encapsulation, three solutions were generated: - Sodium alginate solution (sodium salt of alginic acid from Sigma-Aldrich brown algae) (2% w / v): 0.20 g sodium alginate was dissolved in 10 mL of ultra-pure water, this solution was left under magnetic stirring at 500 rpm for 3 hours at 30 ° C, then the homogeneous solution was sterilized by filtration on a 0.45 µm filter. - TGF-β3 solution (Sigma-Aldrich): 50 µL of PBS containing 1% BSA as carrier protein at 500 ng were added to reconstitute the growth factor that is lyophilized, this solution was stirred at 100 rpm for 10 min . - Calcium chloride solution (Sigma-Aldrich) (3% w / v): 6 g of CaCl ₂ were dissolved in 200 mL of ultra-pure water, this solution was left under magnetic stirring at 500 rpm for 30 minutes at room temperature. The previous solutions were stored at 4 ° C prior to use, in order to avoid denaturation of the growth factor in the manufacture of the capsules. The calcium alginate capsules loaded with TGF-β3 were obtained by the extrusion method as described below: 50 µL of TGF-β3 solution were mixed with 200 µL of sodium alginate solution at 100 rpm for 30 min. Solution was resuspended several times to ensure complete homogenization. The mixed slurry was dropped through a 30G needle into a beaker containing 100 mL of CaCl2 solution that was under constant magnetic stirring. The capsules formed instantly when the alginate solution entered the CaCl ₂ solution, stirring was maintained for 30 min. Finally, the excess CaCl ₂ solution was removed, washing the capsules with 0.9% saline three times and stored in 1X PBS until use. The encapsulation efficiency (% EE) of TGF-β3 in the calcium alginate capsules was calculated using the following equation:

Where m _e corresponds to the amount of encapsulated TGF-β3 (data obtained by generating the rupture of the capsules in suspension and quantifying the amount of factor released), and mT is the amount of TGF-β3 added to the sodium alginate solution. Data shown in Example 11. c- Immobilization of factor capsules on PCL / COL membranes: For the introduction of the calcium alginate capsules loaded with TGF-β3 in the scaffolds, a vertical electrospinning assembly was used, where 250 µL of a layer of polymeric solution is first electrospun, the capsules with a diameter of 1.5 mm are placed on said membrane and a new layer of 50 µL of polymeric solution is electrospun, where the capsules are immobilized on the scaffold , in the form of a sandwich. The scaffolds were stored in a humid chamber at 4 ° C. d- Arrangement of the outer layer of cells: Finally, using the following methodology, CEM-GW were cultivated on the scaffold: The scaffolds were irradiated with gamma rays in order to eliminate possible pathogens present in the fibers. Simultaneously, the CEM-GW in passage 4 were seeded in culture flasks T75 until reaching 70-80% confluence. Detachment of adherent cells was achieved by a 5 minute incubation in 0.05% trypsin - solution of 0.25% EDTA, 37 ° C. The cells were then centrifuged and the pellet was suspended in fresh culture medium. The cells were then seeded on the scaffolds at a density of 5x10 ⁴ cells / cm ² scaffold. Cell adhesion and morphology were observed during the experiment, using cells transduced with green-fluorescent protein (CEM-GW-GFP). The evolution of the cell adhesion of CEM-GW-GFP on the scaffolds was carried out by inverted fluorescent microscopy at 24 h, 48 h, 15 days and 22 days and for the factor release assay 24 h, 48 h, 72 h, 96 h and 120h. Example 14. Release profile of TGF-β3 in the alginate capsules and in the PCL / COL scaffold The amount of TGF-β3 released over time was quantified by means of the enzyme-linked ELISA method, in order to estimate the Percentage of TGF-β3 released for each time with the experimental data obtained from the quantification. Once the amount of protein was obtained in each case, the amount of this released at each time was determined. The calculations were carried out using the following expression: Where:

m (L): mass of TGF-β3 released by the capsules. m (C): mass of TGF-β3 in the capsules. % Protein released: TGF-β3 released by the capsules.

Table 11. Release profile of TGF-β3 on the PCAT scaffold vs TGF-β3 capsules The release in time of the scaffold infiltrated capsules (PCAT) and the capsules directly into the delivery system (capsules + TGF -β3). As evidenced in Figure 4, the TGF-β3 encapsulated and incorporated in the scaffold (PCAT) had a duration of its release of 8 days, while the capsules in suspension showed a release of the factor in 5 days, so that said Results suggest an extension in the availability time of the factor incorporated into the scaffold. These differences in release time can be attributed to the fact that the beads in the PCAT system present an additional reinforcement, provided by the PCL / COL fibers that cover the bead, while, for the system of capsules in suspension, the agitation constant release system and regular changes of medium cause the capsules to weaken and present possible physical damage. It is well known that controlled release systems must have a maximum dose appropriate for the tissue where the implant is to be made. Studies around the subject suggest that for in vivo therapies in connective tissue repair, the amounts of TGF-β3 it can be within a wide dose range from 50-250 ng or 100 ng / mL to 5 µg / m. However, the application of high doses (> 900 ng / mL TGF-β3) has been associated with side effects such as inflammation and tissue erosion. Therefore, the maximum dose of TGF-β3 released for the PCAT scaffold was evaluated, which was 42745.13 ± 2525.90 pg / mL, which compared to the capsules was 35924.34 ± 3108.19 pg / mL, the two systems presented the same number of capsules, the difference between the concentrations is mainly attributed to the variability of the factor contained in each capsule. After carrying out the three consolidated linear adjustments in the following table, it is evident that the experimental points that show a greater adjustment to a straight line, and that the value of the correlation factor R ² close to corresponds to n = 0.

Table 12. TGF-β3 release kinetics Therefore, the release kinetics is of order 0 and the kinetic constant is the following: / 7í5 ^ For the scaffold with capsules infiltrated in the scaffold and / 7í5 For the capsules in suspension .

This suggests that for the PCAT system in one day 11.37% of the total TGF-β3 is released and for the capsules loaded with TGF-β3, 20.11% of the total encapsulated TGF-β3 is released daily. Zero order release kinetics refers to the possibility of prolonging the presence of the protein without increasing the dose over time. In a traditional dosage, after administration of a dose of protein, the concentration reaches a maximum from which it decreases until the protein has been fully used. In view of the above, the encapsulation in calcium alginate turned out to be an adequate methodology in the controlled delivery of the growth factor over time, the use of a polymeric support to add the capsules helps to preserve the release of the factor for a longer time, mainly due to the possible electrostatic interactions that may occur due to the anionic nature of the alginate and the cationic nature of the protein, this added to the additional support provided by the fibers that cover the capsules, acting as an additional protective barrier. Thus, for the PCAT scaffold, the release was in eight days with a reaction order of 0 and a release constant of 226.73 ^ ^{^} was compared with the release of the

suspension capsules, for which there was a release time of 5 days with a release constant of 301.04 ^ ^{^}

Example 15. Monitoring of the PCL / COL 1 scaffold with the addition of calcium alginate capsules loaded with TGF-β3 After immobilization of the capsules loaded with TGF-β3 in the PCL / COL scaffold, a microscopic monitoring of fluorescence with CEM-GW-GFP cells where shorter times (24h, 48h, 72h, 96h and 120h) than those taken into account for the cell adhesion assays developed on the membranes were evaluated. The evaluation of cell proliferation was carried out as shown in Figure 5, under three different conditions 1) for the PCL / COL 1 scaffold (PC), 2) for the PCL / COL 1 scaffold with alginate capsules calcium cells immobilized on the fibers (PCA), and 3) for the PCL / COL 1 scaffold with calcium alginate capsules loaded with TGF-β3 immobilized on the fibers (PCAT). There are no significant differences in terms of proliferation of CEM-GW-GFP for PCA scaffolds, inferring that calcium alginate does not generate cytotoxicity, therefore it presents a positive proliferation response. In the case of PCAT, this scaffold presents a release in time of TGF-β3, which is evident by the progressive increase in proliferation of CEM-GW-GFP, because TGF-β3 plays an essential role in the processes repair and regeneration of tissues, since they trigger biological effects such as proliferation, cell, the generation of blood vessels (angiogenesis) and cell migration (chemotaxis). Example 16. Cell proliferation analysis of CEM-GW cells by resazurin assay Cell proliferation was evaluated in vitro by fluorescence microscopy monitoring and the resazurin reduction method, in order to establish the relative cytotoxicity of the materials used. for shaping scaffolding. Due to the property of living cells to maintain a reducing environment within their cytoplasm and mitochondria, it is possible to use a redox agent such as the indicator resazurin that changes from the oxidized form (blue) to the reduced form (red). The absorbance values were treated with the following equation to obtain the% reduction of resazurin.

With the data obtained from the ELISA plate reader and applying the described equation, the analysis of cell proliferation was performed on the PC, PCA and PCAT scaffolds, where as specified in Example 15 and Figure 5, PC refers to PCL / COL 1, PCA to PCL / COL 1 with immobilization of calcium alginate capsules without growth factor and PCAT to PCL / COL 1 with immobilization of calcium alginate capsules with growth factor.

Table 13. Viability of CEM-GW cells on PC, PCA and PCAT scaffolds The proliferation analysis shows that the PCAT scaffolds with encapsulated TGF-β3 present a significant difference in cell proliferation with respect to the other scaffolds (Fig. 5 ), this because human transforming growth factor β3 (TGF-β3) is a multifunctional bio-active protein generally involved in the regulation, proliferation, differentiation, migration and survival of many different types of cells. It is also a strong stimulator of the synthesis and deposition of extracellular matrix proteins (ECM) by fibroblasts, osteoblasts and endothelial cells; Furthermore, it induces the expression of integrins and receptors that mediate cellular interactions with extracellular matrix proteins. Fluorescence imaging and reduction analysis indicate that calcium alginate encapsulated transforming growth factor β3 did not lose its bioactivity during capsule preparation or in the scaffold sterilization process. Example 17. Evaluation of the secretory profile of factors in the CEM-GW on the scaffolds The CEM-GW were cultured on the scaffolds of i) polycaprolactone / collagen (PCL / COL), ii) PCL / COL with alginate capsules without TGF- β3 (PCA) and iii) PCL / COL with alginate capsules are TGF-β3 (PCAT). Once the CEM-GW had been cultured on the PC, PCA and PCAT scaffolds, the supernatant was collected until 12, 24, 48, 72 and 96 of culture, it was clarified by means of a 6-minute centrifugation at 1200 rpm, and it was stored at -20 ° C until use. Finally, the production of TGF-β1, TGF-β2, TGF-β3, PDGFaa, FGF-b, HDF was evaluated. EGF, VEGF-AA and angiopoietin I by means of an immunoassay based on magnetic microspheres. Luminex LXSAHM R&D Systems Minneapolis, Canada, according to manufacturer's instructions. Briefly, for the activation of TGF-β, aliquots of 100 µL of collected sample were taken for each time and each donor, to this medium 25 µL of 1N HCL was added, then 25 µL. NaOH / HEPES, each solution was dissolved in 200 µL of conditioned medium and 100 µL of treated sample was added to the 96-well lacquer. However, for the detection of the other factors, 50 µl aliquots were taken and placed directly in the 96-well plate. After placing the samples, 50 µL of magnetic macroparticles are added, incubated for 2 hours at room temperature, with constant stirring. At the end of the incubation time, three washes were carried out on the plate and then a mixture of biotin antibodies was added and it was incubated for one hour at room temperature, after washing, streptavidin-PE was added to the microparticles for 30 minutes and incubated again at room temperature. The microparticles were washed and resuspended before reading on the Luminex / 100. The data were processed with the GraphPad prism 7 software, to observe the behavior of the cytokines over time for each scaffold. The secretion profile for TGF-β1 (Fig. 6A) shows that there is a considerable concentration, approximately 9213.20 µg / mL of this factor. In the different scaffolds there is a trend of consumption of this factor present in the culture medium. TGF-β3 is only present in scaffolds with encapsulated factor, because CEM-GW do not produce this factor (Fig. 6C). TGF-β1 has a very broad spectrum of functions, which depend on the state of cellular activation, its concentration, the balance of expression of other cytokines and the physiological conditions of the cells on which it acts. Regarding the secretion profiles for TGF-β2 (Fig. 6B) in time, it showed significant differences in secretion according to the culture support, a marked increase in the secretion of this factor in the PCAT scaffolds is notable. The PCAT scaffold showed significant differences with respect to the other scaffolds, because the encapsulated TGF-β3 induces other intracellular events, such as the regulation of growth factors that intervene in cell differentiation. In Figure 7E and B it can be seen that the scaffolds grown with cells consume the factors, generating a tendency to decrease them over time. The cells in the different matrices follow a similar utilization behavior, which is directly related to cell proliferation. In agreement with the data obtained for the percentage of relative viability of the polymeric matrices by the resazurin test (Fig. 5), where the increase in cellular metabolic activity on the scaffolds is observed over time, with a significant difference for TGF β3 membrane after 72 h (p-value 0.009), attributed to the presence of human transforming growth factor β3 (TGF-β3) which is a multifunctional bio-active protein involved in proliferation, migration processes and cell survival (Tang, QO, et al. TGF-β3: a potential biological therapy for enhancing chondrogenesis. Expert Opinion on Biological Therapy, 2009.9 (6), 689-701), (Lebrin, F., et to the. TGF-β receptor function in the endothelium. Cardiovascular research, 2005. 65 (3), 599-608). In Figure 7F, a production of VEGFaa is observed in the scaffolds without TGF-β3 although lower. The most relevant is the reduction of the factor in the presence of TGF-β3, mainly due to the fact that VEGFaa, in addition to being an angiogenic factor, stimulates epiltelization and collagen production (Zografou, A., et al. Improvement of skin- graft survival after autologous transplantation of adipose-derived stem cells in rats. Journal of plastic, reconstructive & aesthetic surgery, 2011.64 (12), 1647-1656), (Capla, JM, et al. Skin graft vascularization involves precisely regulated regression and replacement of endothelial cells through both angiogenesis and vasculogenesis. Plastic and reconstructive surgery, 2006. 117 (3) .836-844), (Bao, P., et al. The role of vascular endothelial growth factor in wound healing. Journal of Surgical Research , 2009. 153 (2), 347-358). The hyperactivity of this factor causes an excess in its accumulation on the extracellular matrix and fibrosis, for which an apparent inhibition of VEGFaa production occurs when there is an exogenous application of TGF-β3, mainly because this is an antifibrotic factor. that modulates the production and deposition of collagen mainly type I associated with better wound healing and scar reduction results (Tang, QO, et al. TGF-β3: a potential biological therapy for enhancing chondrogenesis. Expert Opinion on Biological Therapy, 2009 . 9 (6), 689-701). Figure 7C shows that the CEM-GW grown in the polystyrene plate (control) present a constant production of FGF-b, with a maximum of 60.79 pg / mL. Particularly, in the scaffolds there is an induction to the production of the factor, four times greater than that of the control, a behavior that is evidenced in all the polymeric matrices, reaching values of 272.74 pg / mL, this notable increase is attributed to the surface conformation of the electrospun polymer and the microporous structure that favors cell adhesion, nutrient diffusion, the expressions of factors related to re-epithelialization and blood vessel formation (Turnbull, G., et al. 3D bioactive composite scaffolds for bone tissue engineering. Bioactive Materials, 2018. 3 (3), 278-314), (Gregor, A., et al. Designing of PLA scaffolds for bone tissue replacement fabricated by ordinary commercial 3D printer. Journal of biological engineering, 2017. 11 (1) 3). The presence of this factor is very important during wound repair as it promotes cell migration through surface receptors such as integrins, during the formation of granulation tissue. Previous research has shown that FGF-b participates in the inhibition of the transient phenotypic change of fibroblasts from granulation tissue into myofibroblasts and regulates their apoptosis process, promoting tissue repair without scar formation (Shi, H., et al. The anti-scar effects of basic fibroblast growth factor on the wound repair in vitro and in vivo. PloS one, 2013. 8 (4)), (Spyrou, GE, et al. The effect of basic fibroblast growth factor on scarring. British journal of plastic surgery, 2002.55 (4), 275-282), (Akasaka, Y., et al. Basic fibroblast growth factor promotes apoptosis and suppresses granulation tissue formation in acute incisional wounds. The Journal of Pathology: A Journal of the Pathological Society of Great Britain and Ireland, 2004. 203 (2), 710-720). In addition, it modulates the production of type I and III collagen and intervenes in its deposition, accelerating the healing processes (Shi, H., et al. The anti-scar effects of basic fibroblast growth factor on the wound repair in vitro and in I live. PloS one, 2013. 8 (4)). These characteristics classify FGF-b as a promising therapeutic agent for skin tissue regeneration processes. In Figure 7A it is observed that, although there is angiopoietin in the medium, at 24 hours the control cells begin to produce it, while the cells grown on the scaffolds begin to produce it after 96 hours. Angiopoietin I stimulates the formation of granulation tissue by acting as chemoattractants for neutrophils, macrophages and fibroblasts, therefore, they are important modulators of angiogenesis during wound healing through cellular regulation. This factor is taken advantage of by the average content of angiopoietin I present in the medium, which corresponds to 7272.96 pg / mL during the first 72 h of culture. In Figure 7D, a high production of HGF is observed in the control with a maximum production value of 15754.414 pg / mL at 96 h, as in the cells grown in the scaffolds and there are no significant differences (p-value = 0.7045), which indicates that the scaffolds do not inhibit factor production with respect to cells grown on the culture plate. HGF plays a promoting role in tissue regeneration, protection and homeostasis (Matsumoto, K., et al. HGF – Met pathway in regeneration and drug discovery. Biomedicines, 2014. 2 (4), 275-300). In addition, it has an inhibitory role in the progression of chronic inflammation and is an antifibrotic factor involved in the dedifferentiation processes of epidermal cells (Li, J., et al. HGF accelerates wound healing by promoting the dedifferentiation of epidermal cells through- integrin / ILK pathway. BioMed research international, 2013). From the foregoing, it can be inferred that the secretion of factors by CEM-GW can vary substantially depending on the composition of the material with which the scaffold is manufactured and the addition of conjugated proteins or peptides. Previous studies affirm that the biomaterials used to make scaffolds can modify the behavior of EMF in culture, due to the changes in the microenvironment provided by the scaffold (Lane, SWDA Williams et al., Modulating the stem cell niche for tissue regeneration. Nature biotechnology, 2014. 32 (8), 795), (Anderson, H., et al. Mesenchymal stem cell fate: applying biomaterials for control of stem cell behavior. Frontiers in bioengineering and biotechnology, 2016. 4, 38). Certain materials are used in the stimulation of these cells to control the differentiation and / or the production of factors for a particular cell lineage (Qazi, T., et al. Biomaterials that promote cell-cell interactions enhance the paracrine function of MSCs. Biomaterials , (2017) 140, 103-114). Next, the quantification of the factor production of the CEM-GW on the scaffolds is shown: The production of factors in (pg / mL) at 72 hours of cell culture on the scaffolds, the control is the cells on the culture plate

Table 14. Quantification of the production of factors produced by the CEM-GW on the scaffolds With the results shown in the examples it is shown that the PCAT scaffolds are viable for the generation of a scaffold with CEM and the results obtained suggest that the generated scaffolds they could be a possible skin substitute, which in the long term could be used in the treatment of skin lesions caused by burns, diabetic and / or vascular ulcers.

Claims

CLAIMS 1. A poly (ℇ-caprolactone) -collagen / TGF-β3 scaffold, comprising at least: a lower layer, an intermediate layer and an upper layer; wherein the upper and lower layers are a polycaprolactone (PCL) and collagen membrane; and the intermediate layer is made up of transforming growth factor (TGF-β3).

2. The scaffold according to Claim 1, wherein the PCL and collagen in the membrane are in a ratio between 9: 1 and 19: 1.

3. The scaffold according to Claim 1, wherein the transforming growth factor (TGF-β3) is encapsulated and immobilized in a matrix.

The scaffold according to Claim 1, further comprising an outer layer of cells selected from mesenchymal, epithelial and / or chondrogenic progenitors located on one of the upper or lower layers.

The scaffold according to Claim 4 wherein said outer layer cells are mesenchymal stromal cells (CEM).

6. The scaffold according to Claim 4 wherein said outer layer cells produce factors selected from angiogenic, epithelial, and immunomodulatory factors.

7. The scaffold according to Claim 6, wherein said cells of the outer layer produce growth factors selected from HGF, FGFb, VEGF, PDGF-AA, TGF-β2, G-CSF and / or TGF-β1, chemokines selected from RANTES , MCP-1, Eotaxin, IP-10, CXCL1, CXCL, CXCL5, CXCL6 and / or CXCL8, neurotrophic factors selected from NGF, NT3, NT4 and / or GDNF, cytokines and hematopoietic growth factors selected from G-CSF, GM -CSF, LIF, IL-1α, IL-6, IL-8, IL-10 and / or IL-11 and factors angiogenic agents selected from angiopoietin 1, angiogenin, PLGF, thrombospondin 2 and / or endostatin.

8. The scaffold according to Claim 1, wherein the membrane of the upper and lower layers is characterized by the following parameters: diameter of fibers from 200 to 400 nm; porosity between 55 and 75%; hydrophobicity contact angle from 60 to 80 ° at 0 seconds and contact angle from 5 to 15 ° at 5 seconds.

9. Use of a scaffold according to Claim 1 for tissue repair or regeneration.

10. A method for the manufacture of a poly (ℇ-caprolactone) -collagen / TGF-β3 scaffold according to Claim 1, comprising: a) preparing a polymeric solution of polycaprolactone and collagen (PCL / COL) in a suitable solvent ; b) electrospinning a layer of the polymeric solution obtained in a); c) placing on the electrospun layer of step b) capsules of transforming growth factor (TGF-β3); d) electrospinning a new layer of polymer solution from step a) over the capsule layer from step c); e) optionally, arranging a layer of cells on one of the electrospun layers of steps b) or d).

The method of Claim 10 wherein the polymeric solution of step a) comprises PCL / COL, in a ratio between 9: 1 and 19: 1, respectively, in a concentration between 9 and 12% w / v in the solvent.

12. The method of Claim 10 wherein the suitable solvent is selected from TFE, HFP, dimethylformamide, chloroform, acetone.

13. The method of Claim 12 wherein the suitable solvent is 2,2,2-trifluoroethanol (TFE).