BIOABSORBABLE MEMBRANE FOR TISSUE REGENERATION AND PREPARATION PROCEDURE

Abstract

Bioabsorbable membrane for tissue regeneration comprising: poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), polyvinylpyrrolidone-vinyl acetate (PVP), and poly(lactide-co-glycolide) (PLGA). The membrane comprises between 65 and 80% by weight of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), between 4 and 15% by weight of polyvinylpyrrolidone-vinyl acetate copolymer (PVP), between 0.5 and 10% by weight of poly(lactide-co-glycolide) (PLGA), and between 4 and 10% of a surfactant.

Description

Qualification Bioabsorbable membrane for tissue regeneration and preparation procedure The present invention relates to a bioabsorbable membrane for tissue regeneration and a preparation method, wherein the membrane comprises: poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), polyvinylpyrrolidone-vinyl acetate (PVP), and poly(lactide-co-glycolide) (PLGA). More specifically, the membrane comprises between 65 and 80 wt% of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), between 4 and 15 wt% of polyvinylpyrrolidone-vinyl acetate (PVP) copolymer, between 0.5 and 10 wt% of poly(lactide-co-glycolide) (PLGA), and between 4 and 10 wt% of a surface-facing agent. BACKGROUND Currently, the traditional treatment for severe burns and chronic wounds involves skin grafting using skin taken from an uninjured area of ​​the patient's own body. This procedure, called autografting, allows for permanent skin regeneration (Pham et al., 2007; Gómez et al., 2011). However, this traditional treatment is slow, painful, and complex when the affected areas are extensive. For example, in patients with diabetic foot ulcers, the affected area is usually amputated to prevent infection. The urgent need for permanent wound coverage in these patients has driven the development of tissue engineering for skin regeneration. Different types of bioengineered skin have been developed. Initially, skin substitutes consisted only of sheets of keratinocytes. Subsequently, it was shown that including a substrate or scaffold for cell growth could improve the skin regeneration process. For example, the following have been developed: a three-dimensional, acellular nylon matrix with porcine collagen peptides coated with a silicone sheet; a permanent, acellular allogeneic membrane generated from cadaveric skin; an acellular bilaminar product (with an outer silicone sheet and an inner sheet with a type I bovine collagen matrix); and a type I bovine collagen membrane with neonatal fibroblasts, onto which keratinocytes are seeded. IF-2019-425 9313 0-APN-ANP#INPI Page 1 of 22 allogeneic; a three-dimensional nylon matrix with porcine collagen peptides and neonatal human fibroblasts, and others. Each of these products has unique characteristics, but not all have achieved the expected results, and these biomaterials are scarce. The key to greater effectiveness in skin regeneration depends on the material used to develop the membranes and its interaction with the type of cell being cultured. Previous art requires the application of two membranes and grafts in successive procedures: one procedure for the regeneration of the dermal layer and another for the regeneration of the epidermal layer. BRIEF DESCRIPTION OF THE INVENTION A degradable membrane for tissue regeneration is provided, comprising poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), polyvinylpyrrolidone-vinyl acetate (PVP), and poly(lactide-co-glycolide) (PLGA). The membrane comprises between 65 and 80 wt% of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), between 4 and 15 wt% of polyvinylpyrrolidone-vinyl acetate (PVP) copolymer, between 0.5 and 10 wt% of poly(lactide-co-glycolide) (PLGA), and between 4 and 10 wt% of a surfactant. In a preferred embodiment, the membrane may further comprise between 0 and 10 wt% of an essential oil or a glycolic extract of plant origin. The membrane has a thickness between 50 and 500 µm; in a preferred embodiment, the membrane has a thickness between 90 and 110 µm. The membrane has a one side with a pore size between 20 and 44 pm and the other side with a pore size between 5 and 15 pm.The membrane comprises a breaking strength of between 4 and 15 MPa, a tensile modulus between 0.3 and 1.5 GPa and a strain at break greater than 1.3%. A procedure is provided for preparing the membrane described in the preceding paragraph, comprising the following steps: a. Dissolve PHBV in chloroform under agitation; b. add PLGA while maintaining agitation; c. add PVP while maintaining agitation; IF-2019-425 9313 0-APN-ANP#INPI 2 Page 2 of 22 d. When the polymers from the previous steps have dissolved, add a non-ionic surfactant under stirring. e. add toluene under agitation; f. add water while stirring, g. Spread the mixture obtained in the previous step on a surface; and h. Dry the spread mixture until a membrane between 90 and 110 µm thick is obtained. In a preferred embodiment, the stirring speed of step a is between 450 and 550 rpm and the heating temperature is between 35 and 45°C; the non-ionic surfactant of step d. is from the polysorbate group, the surface of step g. is made of polyester or a material compatible with the chlorinated solvent, and the drying step h. is carried out by evaporation. DESCRIPTION OF THE FIGURES Figure 1 shows a stress-strain curve for two membranes: prototype and final. They were developed from the following formulations: Final Prototype Formulation % weight PHBV 15.3 18.2 PVP VA 64 2.6 2.6 PLGA - 2.0 Tween 80 5.1 1.8 h2o 5.1 0.9 CHCh 60.9 68.0 Toluene 11.0 6.5 Figure 2 shows the representative stress-strain curve of the membrane of the invention without irradiation vs the membrane of the invention irradiated under gamma radiation sterilization conditions. Figure 3 shows in a curve the temporal evolution of the contact angle of the membrane of the invention. IF-2019-4259313 0-APN-ANP#INPI Page 3 of 22 Figure 4 shows SEM micrographs of the membrane faces; A fibroblast face and B epithelial cell face; the differences in porosity of both faces can be seen. Figure 5 shows fluorescence micrographs of HaCaT cells stained with DAPI and DiO 3 days after being seeded on the membrane of the invention compared with the control condition consisting of the culture plate. Total magnification 100X. Figure 6 shows the results of the cytotoxicity assay of the bioabsorbable membranes of the invention in NIH / 3T3 cells in direct contact with the material. Phase contrast, total magnification of 100X. Null control (complete culture medium); negative control (Teflon®); positive control (Latex); PHBV is the membrane of the invention. Figure 7 shows the results of the cytotoxicity assay of the bioabsorbable membranes of the invention in NIH / 3T3 cells in direct contact with the material. Phase contrast, total magnification 100X. Null control (complete culture medium); negative control (Teflon®); positive control (Latex); PHBV is the membrane of the invention. One-way analysis of variance (ANOVA), Dunnett's post-hoc test (p<0.0001) Figure 8 shows the results of the indirect cytotoxicity assay of the bioabsorbable membranes of the invention in NIH / 3T3 cells. Phase contrast, total magnification 100X. Null control (complete culture medium); negative control (Teflon®); positive control (Latex); PHBV is the membrane of the invention. Cells cultured in the presence of the pure extract (top panels) or the extract diluted 1 / 16 in complete medium (bottom panels). Figure 9 shows the cytotoxicity results of the bioabsorbable membranes of the invention in NIH / 3T3 cells with the pure material extracts. XTT assay. Null control (complete culture medium); negative control (Teflon®); positive control IF-2019-4259313 O-APN-ANP#INPI Page 4 of 22 (Latex); PHBV is the membrane of the invention. One-way analysis of variance (ANOVA), Dunnett post-hoc test (p<0.0001) Figure 10 shows the results of the cytotoxicity assay of the bioabsorbable membranes of the invention in NIH / 3T3 cells with the material extracts diluted 1 / 16. XTT assay. Null control (complete culture medium); negative control (Teflon®); positive control (Latex); PHBV is the membrane of the invention. One-way analysis of variance (ANOVA), Dunnett's post-hoc test (p=0.0538) Figure 11 shows the proliferation curves of HaCaT cells on membranes of the invention. Quantitative evaluation with XTT assay. PHBV is the membrane of the invention. Figure 12 shows the proliferation of NIH / 3T3 cells on the membranes of the invention; PHBV is the membrane of the invention. Quantitative evaluation with XTT assay. Figure 13 shows NIH / 3T3 fibroblasts on the upper surface of the membranes. DAPI stained. Total magnification 100X. Figure 14 shows the underside of the membranes. DAPI stained. Total magnification 100X Figure 15 shows HaCaT keratinocytes on the membrane of the invention. 25-day culture with Hematoxylin and Eosin staining. Scale bar 100 µm Figure 16 shows NIH / 3T3 fibroblasts on membranes. 25-day culture with Hematoxylin and Eosin staining. Scale bar 100 µm Figure 17 shows HaCaT keratinocytes on membranes. 7-day culture with DiO staining. Total magnification 100X. The dotted line on the right indicates the IF-2019-4259313 0-APN-ANP#INPI Page 5 of 22. Cell colony boundary on the membrane. The dotted line on the left indicates the boundary of the membrane of the invention. Figure 18 shows HaCaT keratinocytes on culture plates (top panel) and membranes of the invention (PHBV) (bottom panel). 7-day culture with DiO staining (left panels) and DAPI staining (right panels). Total magnification 100X. Figure 19 shows NIH / 3T3 fibroblasts on culture plates (top panel) and PHBV membranes of the invention (bottom panel). 7-day culture with DiO staining (left panel) and DAPI staining (right panel). Total magnification 100X. Figure 20 shows photographs of pig No. 1, Lesion No. 1 (left craniolateral) treated with the membrane of the invention; Lesion No. 2 (right craniolateral) treated with the membrane of the invention; Lesion No. 3 (left caudolateral) treated with Jelonet® petrolatum gauze; and Lesion No. 4 (right caudolateral) treated with Integra® membrane. A: Day 6 post-surgery, B: Day 9 post-surgery, C: Day 12 post-surgery, D: Day 18 post-surgery, E: Day 21 post-surgery, F: Day 24 post-surgery, G: Day 30 post-surgery, H: Day 33 post-surgery - Biopsy sampling Figure 21 (ac) shows the histological sections of Lesion 2 (right craniolateral) treated with the membrane of the invention, a) General appearance of the section; b) Detail of epidermis and c) dermis. Detail of dermis. E: epidermis, D: dermis. Figure 22(ab) shows the histological sections of Lesion 3 (left caudolateral) treated with Jelonet® petrolatum gauze. a) General appearance of the section, b) Detail of the dermis. E: epidermis, D: dermis. H&E Figure 23 illustrates the proliferation of HaCaT keratinocytes on membranes without (a) and with volatile plant extract (b). Culture time: 3 days. Top: DAPI staining (identifying nuclei). Bottom: DiO staining (identifying cell membranes). Observed with a Nikon TE2000 microscope, 60X objective. IF-2019-4259313 0-APN-ANP#INPI Page 6 of 22 DESCRIPTION OF THE INVENTION For the purposes of this application, “formulation” means the preparation of the emulsion or mixture of compounds that is spread over a surface and then dried by evaporation, ultimately resulting in the bioabsorbable membrane of the invention. For the purposes of this application, “composition” is understood to mean the qualitative and quantitative composition of the membrane of the invention completed after the evaporation and drying stage. The membrane of the invention allows the growth of dermal and epidermal tissue in a single step, meaning that the regeneration of both layers of the skin is carried out in an integrated and joint manner using a single membrane and performing a single surgical intervention. In order to improve the mechanical properties, a membrane comprising PHBV, PVP, and PLGA was developed. Table 1 presents some of the preferred compositions of the membrane of the invention. Table 1 Composition % weight PHBV 65-80 PVP VA 64 4-15 PLGA 0.5-10 Surfactant 4-10 The membrane composition may also include between 0 and 10% by weight of a plant extract. Furthermore, when it includes plant extract, it may not contain PLGA. The membrane comprises a polyester from the polylactic family. In particular, it comprises the poly(lactic-co-glycolic acid) (PLGA) copolymer, for example with an LA:GA fraction of 85:15. IF-2019-425 9313 O-APN-ANP#INPI Page 7 of 22 It was observed that the membrane resulting from a formulation with 3% by weight of PLGA relative to PHBV achieved the appropriate mechanical properties. That is, PLGA should be approximately 3% by weight relative to PHBV. The addition of toluene in the preparation of the membrane allows control of the solvent evaporation rate and consequently the stability of the emulsion during the evaporation stage. The mechanical behavior of the membrane of the invention was tested against the mechanical behavior of a membrane of the prior art. As can be seen in Figure 1 and Table 2, the mechanical properties of the membrane of the invention have greater deformation at break, less rigidity since the tensile modulus was reduced, and lower breaking stress. It was analyzed whether the application of gamma rays for membrane sterilization produced physical changes, for example modifications in tension and deformation. As can be seen in Figure 2, there is no significant variation in the irradiated membranes compared to the non-irradiated membranes. Only a reduction in maximum stress and maximum strain is observed. Table 2: Mechanical properties of membranes Tensile Modulus (GPa) Tensile Strength (MPa) Strain at Break (%) Prior Art Membrane 1.31 13.0 1.2 Membrane of the Invention 1.04 11.6 1.5 The minimum effective sterilization dose was validated by the Microbiology Laboratory of the Ezeiza Atomic Center of the National Atomic Energy Commission (CNEA) using standardized methodologies (ISO 13004:2013 (VDmax20 Method)). Sterilization was performed at the Semi-Industrial Irradiation Plant of the CNEA's Ezeiza Atomic Center. IF-2019-425 9313 0-APN-ANP#INPI Page 8 of 22 The membrane surface is strongly hydrophilic, exhibiting a reduction in the water contact angle over time as the substrate becomes hydrated. Figure 3 illustrates this reduction in the contact angle over time. As can be seen in Figure 4, there are differences in pore morphology and size between the membrane faces. The face in contact with the substrate onto which the emulsion is spread has smaller pores and is the face where epithelial cells (future epidermis) are seeded. On the other hand, the surface in contact with air during the manufacturing process results in larger pore sizes and is the face where fibroblasts (future neodermis) are seeded. The average pore sizes measured from the micrographs are shown in Table 3. Table 3 Pore sizes measured from SEM micrographs Pore ​​size (pm) Fibroblast face 32±12 Epidermal cell face 10 ±5 Figure 5 shows the results of the HaCaT cell proliferation assays on the membranes compared to the control sample. Qualitative analysis of cells in culture in direct contact with the membrane (Figure 6) shows no alterations in the normal cell morphology observed in the null and negative control cells. On the other hand, the positive control cells show a clear deviation from normal morphology (vacuolation, nuclear disintegration, decreased density of viable cells, cytolysis). Quantitative analysis of cells in culture in direct contact with the membrane (Figure 7) shows no cytotoxic effect of the bioabsorbable membrane of the invention. Cell viability quantified by the XTT assay shows no significant differences between the null and negative controls with respect to the membrane sample. The results of the indirect cytotoxicity assay show that cells cultured with the membrane of the invention in complete culture medium (Figure 8) do not show IF-2019-4259313 O-APN-ANP#INPI Page 9 of 22 alterations of normal cell morphology observed in null control and negative control cells. Quantitative analysis of the cultured cells (Figures 9 and 10) shows no cytotoxic effect of the bioabsorbable membrane of the invention. Cell viability, quantified by the XTT assay, shows no significant differences between the null and negative controls with respect to the membrane sample. In cell growth experiments, HaCaT cells showed sustained growth on the membranes of the invention for 4 weeks (Figure 11). In cell growth experiments, fibroblasts showed sustained growth in the membranes of the invention (Figure 12), although slower than the HaCaT keratinocyte line. Fibroblasts were observed adhering to the upper surface of the membranes (Figure 13). No cells were observed on the opposite surface of the membranes. No cells were observed on the insert membrane either (Figure 14). The NIH / 3T3 fibroblasts were unable to penetrate the membrane of the invention. Culture of immortalized human keratinocyte and immortalized murine fibroblast cell lines on the bioabsorbable membrane of the invention: After 25 days of culture, analysis of histological sections revealed the growth of HaCaT cells in multilayers on the surface of the membranes (Figure 15). No cell invasion into the membrane was observed in any of the analyzed sections or membranes. The tissue formed a stratified epithelium of four or more cell layers on the membrane. No development of a stratum corneum was observed in any of the analyzed membranes. After 25 days of culture, analysis of the histological sections revealed monolayer growth of NIH / 3T3 fibroblast cells on the surface of the membranes. No cell invasion into the membrane was observed in any of the sections or membranes analyzed (Figure 16). IF-2019-4259313 0-APN-ANP#INPI Page 10 of 22 In Figures 17 and 18, it is possible to appreciate the HaCaT keratinocytes on the membrane of the invention, without alterations in their morphology compared to the control cells that were cultured on the culture plate. NIH / 3T3 fibroblasts also do not show morphological alterations compared to the cells cultured in the culture plate when stained with DiO and DAPI (Figure 19). The results of the in vivo trials are shown in Figure 20. The healing of wounds treated with the membrane of the invention and autologous cells can be observed; this healing occurred more rapidly compared to the controls. The two wounds treated with the bioabsorbable membrane showed faster healing over time. In pigs, the epithelialized area was significantly larger in the bioabsorbable membranes of the invention compared to the positive control group (Jelonet and Integra). This is because the four epidermal cell strains extracted from a biopsy and separated were seeded in the pig. No remnants of the membranes were visualized in the biopsies (hematoxylin-eosin), indicating that the membrane was metabolized by day 33 of treatment, even before epithelialization was complete. After one month of follow-up, no differences were observed in either the dermis or the epidermis (Figures 21 and 22). The membrane containing either glycolic acid or essential oil extracts proved to be non-cytotoxic (3T3 fibroblast evaluation), and HaCat keratinocytes adhered to and proliferated on it, as illustrated in Figure 23, after 3 days in an incubator. For the purposes of this application, "vegetable extract" refers to essential oils derived from plants and glycolic acid extracts also obtained from plants, such as Aloe Vera. It is also noted that the porous structure of the membrane allows the simultaneous growth of both cell types (epithelial cells and fibroblasts) while preventing the invasion of one cell type into the niche of the other, thus favoring IF-2019-4259313 0-APN-ANP#INPI 11 Page 11 of 22 Communication between both cell types through chemical signals capable of passing through the pores. The membrane allows for the growth of dermal and epidermal tissue in a single step; that is, the regeneration of both skin layers is performed in an integrated and simultaneous manner using a single membrane and a single surgical procedure. The membrane of the invention has the necessary flexibility for the manipulation required by the surgeon. One of the distinguishing features of the membrane of the invention lies in the material used for its construction: a selection of natural polymers from the polyhydroxyalkanoate (PHA) group. PHAs are used in biomedicine, nanomedicine, and innovative medicines. The biological and mechanical properties of PHAs give the biomaterial excellent adhesion to the affected area and create a surface highly suitable for the simultaneous autologous growth of dermal and epidermal cells. However, the most significant innovation of the membrane of the invention is that it allows for the growth of dermal and epidermal tissue in a single step. This means that the regeneration of both skin layers will be performed in an integrated manner, within a single membrane, with a single surgical procedure. In contrast, scaffolds and matrices currently available on the market require the application of two grafts in two successive procedures: one for the regeneration of the dermal layer and another for the epidermal layer. This latter innovation is made possible by the interconnected network of pores present in the membrane of the invention. The pores in the lower region of the membrane have an optimal diameter for the growth of dermal fibroblasts. The pores in the upper region of the membrane have a smaller diameter, creating an optimal environment for cell development. In this way, both skin layers can develop simultaneously. The introduction of the skin regeneration membrane in the healthcare field aims to facilitate surgical and cell culture techniques, while simultaneously improving patients' quality of life. In this case, surgery times are reduced for both the doctor and the patient, thanks to the membrane design of IF-2019-4259313 O-APN-ANP#INPI 12 Page 12 of 22 of the invention has the advantage of eliminating the in vitro cell culture stage (which usually takes 15 days). Furthermore, the product's compatibility with the patient is greater, since it is made using the patient's own cells, obtained through a biopsy. This, in turn, provides significant aesthetic advantages to the patient, as the regenerated skin will have the same properties as the rest of the skin on their body. Likewise, the process is less invasive and traumatic, with shorter healing and scarring times, thus providing better functional results for the skin. This invention is best illustrated by the following examples, which should not be interpreted as imposing a limitation on its scope. On the contrary, it should be clearly understood that, after reading this description, other embodiments, modifications, and equivalents may be suggested to those skilled in the art, without departing from the spirit of the present invention and / or the scope of the appended claims. Examples Example 1: Procedure for preparing membranes The following materials were used in the procedure: a) Poly(3-hydroxybutyric acid -co-3-hydroxyvaleric acid) (PHBV) 12 mol% HV, plasticized with 10 wt% citric acid esters. CAS No. 80181-31-3 Mw: 201600, Mn: 84000, PDI: 2.4 b) PVP-VAc-Copolymer (PVP VA64) Synonyms: vinylpyrrolidone-vinyl acetate copolymer, Copovidone, Copovidonum, Copolyvidone, Copovidone. Mw: 45,000-70,000 CAS No. 25086-89-9 c) Poly(lactide-co-glycolide) 85 / 15 (LA / GA) (PLGA) Intrinsic Viscosity (IV) (dL / g): 0.55-0.75 d) Tween 80. Synonyms: Polyoxyethylene 20 Sorbitan Mono-Oleate, Polysorbate 80. CAS No. 9005-65-6 e) Chloroform (CHC13) Grade Pro-Analysis CAS N° 67-66-3 (f) Toluene, Pro-Analysis Grade No. 108-88-3 (g) Distilled water PHBV is added to one volume of chloroform, at a 1:3.7 ratio of PHBV:chloroform. The polymer mass is dissolved by stirring with a diver IF-2019-4259313 0-APN-ANP#INPI Page 13 of 22. Magnetic heating at a speed of between 450 and 550 rpm and heating between 35 and 45°C. Ensure that the heating is not excessive to avoid solvent loss. If solvent evaporates, replenish it immediately. Note: boiling point of chloroform 60°C. Once a solution free of suspended pellet residue is obtained, PLGA is added at a preferred ratio of 10:1 PHBV:PLGA. Stirring continues until the PLGA is completely dissolved. The PVP is weighed and added in a 14:1 ratio of PHBV:PVP to the stirring polymer solution. During polymer dissolution, partial evaporation of chloroform is observed due to heating and stirring. The polymer concentration must be checked by weighing to ensure it does not exceed the final concentration specified in the formulation. Once the polymers are completely dissolved, stirring is continued at a speed of 400 to 500 rpm. Tween 80 or any other surfactant is then added in a quantity or ratio of 10:1 PHBV:Tween 80. Stirring is then increased to between 600 and 700 rpm and maintained for approximately 30 to 40 minutes. Toluene is added in a 1:10 toluene:chloroform ratio and stirring is continued for about 20 to 30 minutes more. Finally, water is added in a quantity or ratio of 1:100 water:chloroform, measured with a micropipette. The entire mixture is stirred for at least 30 minutes at a speed of between 1500 and 1700 rpm. The final chloroform concentration is checked by weighing to ensure it matches the formulation concentration. Excess chloroform can be evaporated by gentle heating until the formulation concentration is reached. It is also checked that no polymers are adhering to the walls of the container, as these could alter the formulation. Any such solids should be incorporated into the mixture, and stirring should continue. IF-2019-4259313 0-APN-ANP#INPI Page 14 of 22 Table 4 Emulsion formulation Formulation % weight PHBV 13-23 PVP VA 64 1-3 PLGA 1-3 Tween 80 1-4 H2O 0.5-2 CHCh 65-75 Toluene 5-8 The formulation may also incorporate between 0 and 2% by weight of a plant extract, as a preliminary step to the addition of water. Once the formulation shown in Table 4 above is achieved, the emulsion is spread onto a substrate, such as a polyester substrate, using a blade with a 400 µm gap between the blade and the polyester surface. The formulation is at a temperature of between 25 and 35 °C during spreading. It evaporates at room temperature until the membrane reaches a thickness between 50 and 500 µm. The thickness depends on the body site where the membrane will be applied; for example, greater thicknesses are required in areas such as the heel, and minimal or lesser thicknesses on the inner part of the elbow. In a preferred embodiment, the membrane thickness is between 90 and 110 µm. Example 2: Characterization of membranes Mechanical properties: A stress-strain analysis was performed on the membranes obtained in Example 1. A Q-800 Dynamic Mechanical Analyzer (DMA) from TA Instruments was used. The experimental procedure suggested by the company for membranes under isothermal conditions was followed. Contact angle: The contact angles of water on the membranes were determined, following the procedure used in our laboratory (Ruiz, É. B. Hermida, and A. IF-2019-4259313 0-APN-ANP#INPI Page 15 of 22 Baldessari, Fabrication and characterization of porous PHBV scaffolds for tissue engineering, Journal of Physics: Conference Series, vol. 332, p. 012028, Dec. 2011) Porosity and morphology: The membrane surfaces were analyzed using scanning electron microscopy (SEM). The membranes were coated with gold and observed under high vacuum in an electron microscopy system. Pore size was determined by measuring the micrographs using ImageJ, an open-source image analysis software. Example 3: Cytotoxicity assays: Direct cytotoxicity: Direct cytotoxicity assays of the bioabsorbable membranes were performed according to an international standard method, in accordance with ISO 10993-5. NIH / 3T3 cells were cultured in direct contact with the bioabsorbable membrane. The cells were incubated at an appropriate density in a 24-well plate, with the sample under study placed in each well to occupy 10% of the well surface area, along with positive and negative controls. After 24 hours of incubation, qualitative and quantitative cytotoxicity assessments were performed. The qualitative assessment consisted of direct microscopic observation of the cells using vital stains and the determination of deviations from normal morphology (vacuolation, nuclear disintegration, and membrane integrity).The quantitative evaluation was performed by assessing the reducing potential of the cells using the XTT assay (Sigma-Aldrich (St. Louis, MO) according to the supplier's instructions. Indirect cytotoxicity: The analysis was performed according to an international standard method, in accordance with ISO 10993-5. An extract was prepared from the material to be analyzed, as well as from the positive and negative controls. The material was placed in culture medium at a material area (cm²) / culture medium (mL) ratio of 6:1 and incubated at 37°C with 5% CO₂ for 72 hours. NIH / 3T3 cells were cultured from these extracts in a 24-well plate for 24 hours. Qualitative evaluation consisted of direct observation of the cells under a microscope using vital stains. Quantitative evaluation was performed IF-2019-425 9313 0-APN-ANP#INPI Page 16 of 22 evaluating the reducing potential of the cells by the XTT assay (SigmaAldrich (St. Louis, MO) according to the supplier's instructions. Example 4: Cell proliferation assay The HaCaT keratinocyte cell line was cultured in DMEM medium with high glucose and L-glutamine (Gibco, Thermo Fischer Scientific, MA), supplemented with 10% fetal bovine serum (Gibco, Thermo Fischer Scientific, MA), penicillin, and streptomycin (Invitrogen, Thermo Fischer Scientific, MA). Cell cultures were maintained at 37°C in a humidified atmosphere with 5% CO2. 100 HaCaT cells were seeded onto membranes of the invention. Cultures were maintained in complete medium, as previously described, at 37°C in a humidified atmosphere with 5% CO2. Cell-free membranes were used as a null control, and cells were cultured on the plastic culture plate as a positive control. The cultures were analyzed at regular time intervals: 1 to 4 weeks, using the XTT-based in vitro viability assessment kit (Sigma-Aldrich (St. Louis, MO) according to the supplier's instructions. The NIH / 3T3 mouse fibroblast cell line was cultured in DMEM medium with high glucose and L-glutamine (Gibco, Thermo Fischer Scientific, MA), supplemented with 5% fetal bovine serum (Gibco, Thermo Fischer Scientific, MA), penicillin, and streptomycin (Invitrogen, Thermo Fischer Scientific, MA). Cell cultures were maintained at 37°C in a humidified atmosphere with 5% CO2. One × 10⁵ NIH / 3T3 cells were seeded onto membranes of the invention. Cultures were maintained in complete medium, as previously described, at 37°C in a humidified atmosphere with 5% CO2. Cell-free membranes were used as a null control, and cells were cultured on the plastic culture plate as a positive control. The cultures were analyzed at regular time intervals: 1 to 4 weeks, using the XTT-based in vitro viability assessment kit (Sigma-Aldrich (St. Louis, MO) according to the supplier's instructions. Example 5: Migration assay of immortalized murine fibroblasts on the bioabsorbable membrane of the invention IF-2019-4259313 0-APN-ANP#INPI Page 17 of 22 The fibroblast migration assay through membranes of the invention was performed in transwell plates. NIH / 3T3 fibroblasts were starved in antibiotic-containing culture medium for 16–18 hours. The cells were harvested and resuspended in medium without fetal bovine serum. A membrane with an 8 µm pore size insert was placed over it, and a known number of cells were seeded onto the membrane. As a positive control, a membrane with a pore size of 8 µm or larger was used. Complete medium (10% fetal bovine serum) was placed in the lower chamber. The cultures were incubated for 24 hours. The membranes and inserts were fixed with 4% paraformaldehyde (Sigma-Aldrich) and subsequently labeled with DAPI (Sigma-Aldrich) to visualize nuclei. The membrane face where the cells were seeded, the opposite face of the membrane and the insert were analyzed using a NIKON TE2000-U reverse optics epifluorescence microscope (Melville, New York, USA).The images were taken with a cooled Orca-AG digital CCD camera (Hamamatsu, Japan). Example 6: Culture of immortalized human keratinocyte and immortalized murine fibroblast cell lines on the bioabsorbable membrane of the invention: The HaCat immortalized keratinocyte cell line was cultured on the bioabsorbable membrane to evaluate its colonization and differentiation capacity. The NIH-3T3 immortalized murine fibroblast cell line was also cultured to evaluate its colonization capacity. The morpho-functionality of the generated cultures was evaluated through histological analysis using optical microscopy. The morphology and organization of the epidermal and dermal tissue generated in vitro on the membrane of the invention were assessed. Additionally, the cultures were evaluated using fluorescent dyes by epifluorescence microscopy. The HaCaT keratinocyte cell line was cultured in DMEM medium with high glucose and L-glutamine (Gibco, Thermo Fischer Scientific, MA), supplemented with 10% fetal bovine serum (Gibco, Thermo Fischer Scientific, MA), penicillin, and streptomycin (Invitrogen, Thermo Fischer Scientific, MA). Cell cultures were maintained at 37°C in a humidified atmosphere with 5% CO2. 5 x 10⁵ HaCaT cells were seeded onto membranes. Cultures were maintained in complete medium, IF-2019-425 9313 0-APN-ANP#INPI 18 Page 18 of 22 as previously described, at 37°C in a humid atmosphere with 5% CO2 for 25 days, with regular medium changes. Subsequently, the cultures were fixed in Bouin's solution (0.9% picric acid, 5% acetic acid, pure formaldehyde) for 24 hours and dehydrated in ascending series of ethyl alcohol. They were cleared with butyl xylene, embedded in paraffin, and sectioned in cross-section using a rotary microtome. The sections were mounted on positive slides. For further analysis, the sections were stained with hematoxylin and eosin (H&E). A bright-field microscope with an integrated digital camera was used for observation, analysis, and obtaining photomicrographs. The NIH / 3T3 fibroblast cell line was cultured in DMEM medium with high glucose and L-glutamine (Gibco, Thermo Fischer Scientific, MA), supplemented with 5% fetal bovine serum (Gibco, Thermo Fischer Scientific, MA), penicillin, and streptomycin (Invitrogen, Thermo Fischer Scientific, MA). Cell cultures were maintained at 37°C in a humidified atmosphere with 5% CO2. Five × 10⁵ NIH / 3T3 cells were seeded onto membranes. Cultures were maintained in complete medium, as previously described, at 37°C in a humidified atmosphere with 5% CO2 for 25 days, with regular medium changes. The cultures were fixed in Bouin's solution (0.9% picric acid, 5% acetic acid, pure formaldehyde) for 24 hours and dehydrated in ascending series of ethyl alcohol. They were cleared with butyl xylene, embedded in paraffin, and sectioned in cross-section using a rotary microtome. The sections were mounted on glass slides. For subsequent analysis, the sections were stained with hematoxylin and eosin. A bright-field microscope with an integrated digital camera was used for observation, analysis, and obtaining photomicrographs. The HaCaT and NIH / 3T3 cell lines were cultured under the same conditions described previously in this example. After 7 days of culture, the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 20 minutes at room temperature. The cultures were labeled with DAPI (Sigma-Aldrich) to visualize the nuclei; and with DiO (Molecular Probes, Invitrogen), a lipophilic dye IF-2019-4259313 0-APN-ANP#INPI 19 Page 19 of 22, which allows observation of the plasma membranes of the cells. The slides were mounted in PBS:glycerol for observation with a NIKON TE2000-U inverted optics epifluorescence microscope (Melville, New York, USA). The images were taken with a cooled Orca-AG digital CCD camera (Hamamatsu, Japan). Example 7: In vivo studies and trials in pigs: The experiment was conducted at the Center for Research and Development in Experimental Medicine (CIDME) at Maimonides University. This specialized animal facility uses farm animals as laboratory models. It has the infrastructure, equipment, and trained personnel to conduct preclinical trials of biomedical devices, biomaterials, and drugs. To evaluate the developed membranes for the treatment of deep burns, pigs were used in an excision wound model. 15 kg Yorkshire pigs were used. The animals were anesthetized and placed in a recumbent position. Four 10 cm x 10 cm full-thickness excision wounds were made on the animal's back. Adrenaline-soaked gauze was used to maintain hemostasis. The resorbable membranes of the invention and controls (an open wound treated with Integra® and a wound treated with Jelonet® petrolatum gauze) were placed in the wound beds. Each membrane was appropriately seeded with the autologous cell suspension, and the wounds were covered. To obtain the autologous epithelial cells, the biopsy was first washed with trypsin / EDTA (T / E) until the epidermis separated. The epidermis was then enzymatically digested by immersing it in T / E at approximately 37°C for 15 minutes.The T / E was then inactivated with culture medium and filtered using a 100 µm filter. The filtered epithelial cells, suspended in culture medium, were collected with a syringe and then spread onto the upper surface of the membrane. Finally, the membrane was placed over the wound. The wound healing processes were monitored until epithelialization. During this time, the evolution of the wounds was recorded photographically. Measurements were taken to assess wound contraction and any signs of inflammation and infection. Once the experiment was completed, the animals were sacrificed and biopsies were taken to evaluate wound epithelialization. Page 20 of 22 cellular, vascularization of the wound and biodegradation of the material, by immunohistochemistry. Example 8: Membrane with plant-based extract of essential oil type with antibacterial properties: Table 5 details the preferred composition for the formulation and for the finished membrane “preferred composition” as listed for the original membrane (column % dry weight). Table 5: - ! ... and < Emulsion formulation % weight Membrane composition % dry weight PHBV 18.0 71.2 PLGA 1.86 7.36 PVP VA 64 2.52 9.98 Surfactant 1.80 7.13 Plant extract 1.08 4.27 Water 0.77 — CHCh 67.2 — Toluene 6.78 — The procedure was carried out in a similar manner to how it is detailed in Example 1, with the only variation being the addition of an extract, also under agitation at 700-800 rpm. Optional: It was found that it is also possible to incorporate the plant extract in a single step along with the surfactant, in the proportions used. Different quantities of the membrane components shown in Table 6 were tested. Table 6 Emulsion formulation % weight Membrane composition % weight IF-2019-425 9313 0-APN-ANP#INPI Page 21 of 22 PHBV 13-23 65-80 PLGA 0-3 0-10 PVP VA 64 1-3 4-15 Surfactant 1-4 4-10 Plant Extract 0.5-2 1-10 Water 0.5-2 CHC13 65-75 Toluene 5-8 Example 9: Preparation of the membrane with glycolic extract of plant origin; from a plant with anti-inflammatory and healing properties. Membranes were obtained with 10% dry mass extract, assisting the emulsion with gentle sonication. The preferred emulsion formulation and membrane composition are detailed in Table 7. Table 7: Preferred emulsion formulation and composition MEMBRANE WITH PLANT EXTRACT Emulsion formulation % weight Membrane composition % weight PHBV 18.1 70.9 PVP VA 64 2.40 9.92 Surfactant 2.11 8.27 Glycolic plant extract 2.78 10.8 CHCl3 67.7 — Toluene 6.65 — The step of adding the glycolic extract was performed at the end of the procedure shown in Example 1, stirring at 1500 rpm, replacing the step of adding HjO. Finally, the preparation can be lightly sonicated for up to 7 minutes. Optionally, it can be prepared without PLGA, as shown in Table 5, while still exhibiting sufficient mechanical properties. IF-2019-425 9313 0-APN-ANP#INPI Page 22 of 22 Argentine Republic - National Executive Branch 2019 - Year of Exports Additional Signature Sheet Graphic Report Number: IF-2019-42593130-APN-ANP#INPI CITY OF BUENOS AIRES Wednesday, May 8, 2019 Reference: 20190100898 The document was imported by the GEDO system with a total of 22 page(s). Digitally signed by GESTION DOCUMENTAL ELECTRONICA - GDE DN: cn=ELECTRONIC DOCUMENTARY MANAGEMENT - GDE, c=AR, o=GOVERNMENT SECRETARY OF MODERNIZATION, ou=ADMINISTRATIVE MODERNIZATION SECRETARY, serialNumber=CUIT 30715117564 Date: 2019.05.08 19:18:45-03'00' Darío Julio Martin Mayares Administrative Advisor National Patent Administration National Institute of Industrial Property Digitally signed by GESTION DOCUMENTAL ELECTRONICA GDE DN: cn=GEST10N ELECTRONIC DOCUMENTARY - GDE, c=AR, o=GOVERNMENT SECRETARY OF MODERNIZATION, ou=ADMINISTRATIVE MODERNIZATION SECRETARY, serialNumber=CUIT 30715117564 Date: 2019.05.08 19:18:46 -03'00'

Claims

1. A bioabsorbable membrane for the regeneration of mammalian epithelial tissue, characterized in that it comprises between 65 and 80% by weight of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), between 4 and 15% by weight of polyvinylpyrrolidone-vinyl acetate (PVP) copolymer, between 0.5 and 10% by weight of poly(lactide-co-glycolide) (PLGA), and between 4 and 10% of a surfactant, wherein one side has a pore size between 20 and 44 µm and the other side has a pore size between 5 and 15 µm. Nine claims follow.