WO2020206067A1

WO2020206067A1 - Bioabsorbable membrane for tissue regeneration and process for preparing the same

Info

Publication number: WO2020206067A1
Application number: PCT/US2020/026312
Authority: WO
Inventors: Élida Beatriz HERMIDA; Ignacio Edgardo RUIZ ARIAS; Alberto Nazareno Bolgiani
Original assignee: Consejo Nacional De Investigaciones Científicas Y Técnicas; Universidad Nacional De San Martín; Inis Biotech Llc
Priority date: 2019-04-05
Filing date: 2020-04-02
Publication date: 2020-10-08
Also published as: AR115030A1

Abstract

A 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 from 65 to 80 wt% of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), from 4 to 15 wt% of polyvinylpyrrolidone-vinyl acetate (PVP) copolymer and from 0.5 to 10 wt% of poly(lactide-co-glycolide (PLGA) and from 4 to 10 wt% of a surfactant.

Description

BIOABSORBABLE MEMBRANE FOR TISSUE REGENERATION AND

PROCESS FOR PREPARING THE SAME

The present invention refers to a bioabsorbabie membrane for tissue regeneration and a process of preparation thereof, where the membrane comprises: po!y(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), polyvinylprrolidone-viny l acetate (PVP) and poly(lactide-co-glycoiide (PLGA). More specifically, the membrane comprises from 65 to 80 wt% of poly(3- hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), from 4 to 15 wt% of polyvinylpyrrolidone-vinyl acetate (PVP) copolymer and from 0,5 to 10 wt% of poly(lactide-co-giycolide (PLGA) and from 4 to 10 wt% of a surfactant.

BACKGROUND

Current state-of-the-art treatments of severe burns and chronic wounds consist in grafting skin from a non-injured area of the same patient. This process is designated auto-grafting or self-grafting and provides for a permanent cutaneous regeneration (Pham et al, 2007; Gomez et at, 2011). However, this traditional treatment is slow, painful and complex when the affected areas are extensive. For example, in the case of patients suffering from diabetic foot, the affected area is generally amputated in order to avoid infections.

The pressing need to permanently address the wounds of these patients has encouraged the development of tissue engineering for cutaneous regeneration. Different classes of bionengineered skin have been developed. Initially, cutaneous substitutes consisted only of keratinocyte sheets. Later, it was demonstrated that inclusion of a substrate or“scaffold” for cell growth could improve the process of skin regeneration. For example, the following approaches have been developed: a three-dimensional, acellular, nylon matrix with porcine collagen peptides covered by a silicone sheet; a permanent, acellular, allogenic membrane produced from cadaver skin; an acellular bilameiiar product (with an external silicone sheet and an internai sheet comprising a bovine type i collagen matrix); a bovine type l coiiagen membrane with neonate fibroblasts, on which allogenic keratinocytes are seeded; a three-dimensional nylon matrix with porcine collagen peptides and neonate human fibroblasts, and others.

Each of these products have distinguishing features, but not all of them have achieved the expected results and availability of these biomaterials is poor. The key to a higher efficacy of a skin regeneration process depends on the material used for developing membranes and its interaction with the cultured cell type. in the prior art, it was necessary to apply two membranes and grafts in subsequent interventions. The first procedure is meant to regenerate the dermic layer and the second is for regenerating the epidermic layer.

SUMMARY OF THE INVENTION

A degradable membrane for tissue regeneration comprising poly(3- hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), polyvinylpyrrolidone-vinyl acetate (PVP) and poly(lactide-co-glycolide) (PLGA) is provided. The membrane comprises from 65 to 80 wt% of po!y(3-hydroxybutyrate-co-3- hydroxyva!erate) (PHBV), from 4 to 15 wt% of polyvinylpyrrolidone-vinyl acetate (PVP) copolymer and from 0.5 to 10 wt% of poly(iactide-co-glycolide (PLGA) and from 4 to 10 wt% of a surfactant in a preferred embodiment, the membrane may further comprise from 0 to 10 wt% of an essential oil or a glycolic extract of plant origin. The membrane has a thickness from 50 to 500 pm. In a preferred embodiment the membrane has a thickness from 90 to 1 10 pm. One side of the membrane has a pore size from 20 to 44 mm and the other side has a pore size from 5 to 15 pm. The membrane has a tear strength from 4 to 15 MPa, a tensile modulus from 0,3 to 1 ,5 GPa and a strain at break of more than 1.3

%.

A process for preparing the membrane described in the above paragraph is also provided, which comprises the steps of: a. dissolving PHBV in chloroform under stirring; b. adding PLGA still under stirring; c. addding PVP still under stirring; d. once polymers of the above steps are dissolved, adding a non- ionic surfactant under stirring. e. adding toluene under stirring; f. adding water under stirring, g. extending the mixture obtained in the above step on a surface; and h. drying the extended mixture until a 90 to 1 10 pm thick membrane is obtained. In a preferred embodiment, stirring speed in step a is from 450 to 550 rpm and heating temperature is from 35 to 45 °C; the non-ionic surfactant of step d. belongs to the group of polysorbates, the surface of step g. is made of polyester or a material compatible with the chlorinated solvent and drying step h. is carried out by evaporation.

DESCRIPTION OF THE DRAWiNGS

Fig. 1 shows a stress-strain curve of two membranes: prototype and final. They were made from the following formulations:

Fig. 2 shows the representative stress-strain curve of the membrane of the invention without irradiation vs. the membrane of the invention irradiated under conditions of gamma radiation sterilization.

Fig. 3 shows a curve of time evolution of the contact angle of the membrane of the invention.

Fig. 4 shows SEM micrographs of the membrane sides; A: fibroblast side and B: epithelial cell side; note the differences in porosity between the two sides.

Fig. 5 shows fluorescence micrographs of HaCaT cells stained with DAPI and DiO 3 days after being deposited on the membrane of the invention compared to the control condition represented by the culture plate. 100X total magnification.

Fig. 6 shows cytotoxicity assay results of the bioabsorbable membranes of the invention on NIH/3T3 cells in direct contact with the material. Phase contrast, 100X total magnification. Null control (complete culture medium); negative control (Teflon®); positive control (Latex); PHBV is the membrane of the invention.

Fig. 7 shows the cytotoxicity assay results of the bioabsorbable membranes of the invention on NIH/3T3 cells in direct contact with the material. Phase contrast, 100X total magnification. Null control (complete culture medium); negative control (Teflon®); positive control (Latex); PHBV is the membrane of the invention. One-way analysis of variance (ANOVA), post-hoc Dunnett^’s test (p<0.0001)

Fig. 8 shows indirect cytotoxicity assay results of the bioabsorbable membranes of the invention on NIH/3T3 cells. Phase contrast, 100X total magnification. Null control (complete culture medium); negative control (Teflon®); positive control (Latex); PHBV is the membrane of the invention. Cells cultured in the presence of pure extract (upper panels) or extract diluted 1/16 in complete medium (lower panels).

Fig. 9 shows cytotoxicity results of the bioabsorbable membranes of the invention on NIH/3T3 cells with pure material extracts. 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 test post-hoc (p<0.0001)

Fig. 10 shows cytotoxicity assay results of the bioabsorbable membranes of the invention on NIH/3T3 cells with 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 test post-hoc (p=0.0538)

Fig. 1 1 shows proliferation curves of HaCaT cells on membranes of the invention. Quantitative assessment by XTT assay. PHBV is the membrane of the invention.

Fig. 12 shows NIH/3T3 cell proliferation on the membranes of the invention; PHBV is the membrane of the invention. Quantitative assessment by XTT assay.

Fig. 13 shows NIH/3T3 fibroblasts on the upper side of the membranes. DAPI staining. 100X total magnification.

Fig. 14 shows the lower side of the membranes. DAPI staining. 100X total magnification

Fig. 15 shows HaCaT keratinocytes on the membrane of the invention. Twenty-five days culture with Hematoxylin and Eosin staining. Scale bar 100 pm

Fig. 16 shows NIH/3T3 fibroblasts on the membranes. Twenty-five days culture with Hematoxylin and Eosin staining. Scale bar 100 pm

Fig. 17 shows HaCaT keratinocytes on the membranes. Seven days culture with DiO staining. 100X total magnification. The dotted line on the right indicates the cell colony boundary on the membrane. The dotted line on the left indicates the membrane of the invention boundary.

Fig. 18 shows HaCaT keratinocytes on culture plates (upper panel) and membranes of the invention (PHBV) (lower panel). Seven days culture with DiO staining (left panels) and DAPI (right panels). 100X total magnification.

Fig. 19 shows NIH/3T3 fibroblasts on culture plates (upper panel) and membranes of the invention PHBV (lower panel). Seven days culture with DiO staining (left panel) and DAPI (right panel). 100X total magnification.

Fig. 20 shows pictures of pig N°1 , Injury n°1 (cranial-left lateral) treated with the membrane of the invention; Injury n°2 (crania I- right lateral) treated with the membrane of the invention, injury n°3 (left caudolateral) treated with Jeionet® paraffin gauze and injury n°4 (right caudoiateral) 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 - Biopsies co flection

Fig. 21 (a-c) shows histology sections from Injury 2 (cranial-right lateral) treated with the membrane of the invention a) General appearance of the cut; b) Detailed view of epidermis and c) dermis. Detailed view of dermis. E: epidermis, D; dermis.

Fig. 22 (a-b) shows histology sections from Injury 3 (left caudoiateral) treated with Jeionet® paraffin gauze a) General appearance of the cut. b) Detailed view of dermis, E; epidermis, D: dermis. H&E

Fig. 23 illustrates HaCaT keratin ocyte proliferation on membranes without (a) and with volatile plant extract (b). Culture period; 3 days. Top: DAP I staining (showing nuclei). Bottom: DiO staining (showing cell membranes). Observation with Nikon TE2000 microscope, 60X objective.

DESCRIPTION OF THE INVENTION

For the purposes of the present application a“formulation” is understood as an emulsion or mixture of compounds which is spread on a surface and then dried by evaporation, finally resulting in the bioabsorbable membrane of the invention.

For the purposes of the present application, a“composition” is understood as a quali- and quantitative composition of the finished membrane of the invention upon completion of the steps of evaporation and drying.

The membrane of the invention makes it possible to grow dermic and epidermic tissue in a single step, i.e., regeneration of both skin layers is carried out in a joint and integrated manner using a single membrane and performing only one surgical intervention. in order to improve mechanical properties, a membrane comprising PHBV, PVR, PLGA was developed. Table 1 shows some of the preferred compositions for the membrane of the invention.

Table 1

The membrane composition may also comprise from 0 to 10 wt% of a plant extract. Indeed, if it comprises a plant extract it may not contain PLGA.

The membrane comprises a polyester of the polylactic family in particular, it comprises a po!y(iactic-co-giycolic acid) (PLGA) copolymer, for example with a fraction of 85:15 LA:GA. it is noted that the resulting membrane of a formulation comprising 3 wt% of PLGA relative to PHBV showed the appropriate mechanical properties. That is, PLGA should comprise about 3 wt% relative to PHBV.

Addition of toluene to the membrane preparation makes it possible to control solvent evaporation speed and consequently the stability of the emulsion during the evaporation step.

The mechanical behaviour of the membrane of the invention was tested against the mechanical behaviour of a prior art membrane.

As may be seen in Figure 1 and Table 2, the mechanical properties of the membrane of the invention provide a higher strain at break, less stiffness as the tensile modulus is lower and a lower tear strength. it was analyzed whether application of gamma rays for sterilizing the membrane produced physical changes, for example modifications of stress and strain.

As can be observed in Figure 2 there is no significant change between irradiated membranes and non-irradiated membranes. Only a reduction of maximum stress and maximum strain was noted.

TABLE 2: MECHANICAL PROPERTIES OF THE MEMBRANES

The minimum effective dosis of sterilization was validated at the Microbiological Laboratory [Laboratorio de Microbioiogia], Ezeiza Atomic Center [Centro Atomico Ezeiza], National Atomic Energy Commission [ Comision Nacional de Energia Atomica] (CNEA) using standarized methodologies (ISO Standard 13004: 2013 (Method VDmax20)). Sterilization was carried out at the Semi Industrial Irradiation Facility [Pianta de Irradiacion Semi Industrial] of the Ezeiza Atomic Center, CNEA.

The surface of the membrane is strongly hydrophilic, with a decrease of the water contact angle over time as substrate hydration occurs. Figure 3 shows a decrease of the contact angle over time.

As may be seen in Figure 4, there are differences in morphology and pore size between the two sides of the membrane. The side in contact with the support over which the emulsion is spread has pores of a smaller size, and it is the side on which epithelial cells are seeded (future epidermis). On the other hand, the surface in contact with air during the manufacturing process has larger pore sizes, and it is the side where fibroblasts are seeded (future neodermis). Table 3 shows mean pore sizes measured from micrographs.

Table 3: Pore sizes measured from SEM micrographs

Figure 5 shows the results of proliferation assays with HaCaT cells on the membranes as compared to a control sample.

A qualitative analysis of cells grown in direct contact with membranes (Figure 6) does not show any alteration of normal cell morphology, which is observed in null control and negative control cells in addition, positive control cells show a clear bias from normal morphology (vacuolation, nuclear disintegration, reduction of viable cell density, cytolysis).

A quantitative analysis of cultured cells in direct contact with the membrane (Figure 7) shows no cytotoxic effect of the bioabsorbable membrane of the invention. Cell viability quantified by an XTT assay shows no significant difference between null and negative controls relative to a membrane sample.

The results of an indirect cytotoxycity assay demonstrate that cells grown with the membrane of the invention in complete culture medium (Figure 8) show no alterations in normal cell morphology, which are observed in null control and negative control cells.

A quantitative analysis of the cells grown in culture (Figures 9 and 10) shows no cytotoxic effect of the bioabsorbable membrane of the invention. Cell viability quantified by an XTT assay shows no significant difference from null and negative controls relative to the membrane sample. in cell growth experiments, HaCaT cells show a sustained growth on the membranes of the invention for 4 weeks (Figure 11). in cell growth experiments, fibroblasts showed a sustained growth on the membranes of the invention (Figure 12), although slower when compared to the HaCaT keratinocyte line.

Adhered fibroblasts were observed on the upper side of the membranes (Figure 13). On the opposite side of the membranes no cells are observed. Also, no cells were observed on the membrane of the insert (Figure 14). NIH/3T3 fibroblasts were unable to cross the membrane of the invention.

Culture of immortalized human keratinocyte cell lines and immortalized murine fibroblasts on the bioabsorbable membrane of the invention: After 25 days of culture, an analysis of histological sections revealed growth of HaCaT cells in multiple layers on the surface of membranes of (Figure 15). None of the analyzed sections and membranes showed invasion of ceils within the membrane. The tissue formed a stratified epithelium, of 4 or more cell layers on the membrane. No development of stratum corneum was observed on any of the analyzed membranes.

After 25 days of culture, an analysis of histological sections revealed growth of NIH/3T3 fibroblast cells in monolayer on the surface of the membranes. None of the analyzed sections and membranes showed invasion of cells within the membrane (Figure 16). in Figures 17 and 18 HaCaT keratinocytes may be seen on a membrane of the invention, with no morphological alterations relative to control cells grown on a culture plate. Further, NIH/3T3 fibroblasts show no morphological alterations of cells grown on culture plates when stained with DiO and DAPI (Figure 19).

The results of in vivo assays are shown in Figure 20, where healing of wounds treated with the membrane of the invention and autologous celts may be observed, and this healing occurred faster as compared to controls. Both wounds treated with the bioabsorbable membrane have a faster evolution over time. in pigs, the epithelized area was significantly larger with the bioabsorbable membranes of the invention as compared to a positive control group (Jelonet e Integra.) This is because, in pigs, 4 epidermic ceil lines obtained from a biopsy were seeded, and separated.

No remaining membranes were visualized in the biopsies (hematoxylin- eosin), which means that the membrane is metabolized 33 days after treatment, even before epithelization is completed. After a month of evolution, no differences in dermis or epidermis were observed (Figures 21 and 22).

Membranes comprising a glycolic-type plant extract or an essential oil- type extract were not cytotoxic (assessed with 3T3 fibroblasts) and keratinocytes of the HaCat line adhered and proliferated on this membrane, as illustrated in Figure 23, after 3 days in a culture oven. For the purpose of the present application a plant extract is to be understood as essential oils of plant origin and a glycolic acid extract also of plant origin, for example from Aloe Vera.

It is further noted that the porous structure of the membrane makes it possible to grow simultaneously of both cell types (epithelial cells and fibroblasts) but prevents invasion of one cell type in the niche of the other, thus favouring cornunication between both cell types by means of chemical signals capable to traverse the pores. The membrane makes it possible to grow dermic and epidermic tissue in a single step, i.e., regeneration of both skin layers is carried out in a joint and integrated manner using a single membrane and performing only one surgical intervention.

The membrane of the invention has the necessary flexibility for the manipulation required by the surgeon.

One of the distinctive features of the membrane of the invention is based on the material used for its construction: a selection of natural polymers belonging to the group of polyhydroxyalkanoates (PHAs). PHAs are used in biomedicine, nanomedicine, and innovative medicines. The biological and mechanical features of PHAs impart an excellent adherence to the biomaterial in the affected area and generate a very suitable surface for simultaneous autologous growth of dermic and epidedermic cells.

However, the most important innovation of the membrane of the invention is that it makes it possible to grow dermic and epidermic tissue in a single step. This means that regeneration of both skin layers is carried out in an integrated manner, on a single membrane with only one surgical intervention. In contrast, scaffolds and matrixes currently available on the market require aplication of two grafts, in two succesive interventions: one for regenerating the dermic layer and the other for the epidermic layer. The latter innovation may be achieved as a result of an interconnected network of pores of the membrane of the invention. Pores of the lower region of the membrane have an optimal diameter for growth of dermic fibroblasts. Pores of the upper region of the membrane have a smaller diameter, thereby creating an optimal environment for cell development. Thus, both skin layers may develop simultaneously. introduction of membranes for skin regeneration into the healthcare field intend to facilitate surgical and cell culture techniques; and, at the same time, increase quality of life of the patients. In this case surgery times are reduced, both for the physician as for the patient, since the design of the membrane of the invention provides the advantage of removing the step of in vitro cell culture (which usually takes 15 days). Furthermore, compatibility of the product with the patient is higher, given that it is based on the patient’s own cells, obtained by biopsy in turn, this imparts important aesthetic advantages to the patient, as skin to be regenerated will have the same properties as the rest of the skin of his or her body. Also, the process is less invasive and traumatic, with shorter recovery and wound healing times, thus providing better functional results to the skin.

This invention is better illustrated in the following examples, which should not be construed as a limitation of the scope thereof. On the contrary, it should be clearly understood that after reading the present description other embodiments, modifications and equivalents thereof may be possible, which may be envisioned by a person of skill without departing from the spirit of the present invention and/or the scope of the appended claims.

EXAMPLES

EXAMPLE 1 : Membrane preparation procedure

The following materials were used in the procedure: a) Poly(3-hydroxybutyric acid-co-b-hydroxyvaleric acid) (PHBV) 12% molar of HV, plasticized with 10 % by weight citric acid esters. CAS N° 80181- 31-3

Mw: 201600, Mn: 84000, PDI: 2.4 b) PVP-VAc-Copolymer (PVP VA64) Synonyms: viylpyrrolidone-vinyl acetate copolymer, Copovidone, Copovidonum, Copoiyvidone, Copovidon. Mw: 45.000-70.000 CAS N° 25086-89-9 c) Poiy(lactide-co-glycolide) 85/15 (LA/GA) (PLGA) intrinsic Viscosity (IV) (dL/g): 0.55-0.75 d) Tween 80. Synonyms: Polyoxyethylene 20 Sorbitan monooieate, Poiysorbate 80. CAS N° 9005-65-6 e) Chloroform (CHCl₃) Pro-Analysis Grade CAS N° 67-66-3 f) Toluene, Pro-Analysis Grade N° 108-88-3 g) Distilled water

To a volume of chloroform, PHBV was added in a ratio of 1 :3.7 PHBVxhloroform. The polymer mass was dissolved by stirring with a magnetic bar at 450-550 rpm and heated to 35-45°C. Heating was controlled so as not to be excessive and avoid solvent loss. In case of evaporation, the solvent was replenished immediately. Note: chloroform boiling point is 60°C.

Once a solution was obtained without the presence of suspended pellets, the PLGA was added in a preferred ratio of 10:1 PHBV:PLGA. Stirring continued until complete dissolution of PLGA,

PVP was weighted and added at a ratio of 14:1 PHBV:PVP into the polymer solution with stirring.

During dissolution of the polymers, heating and stirring induced partial evaporation of chloroform. Weight of the polymer was controlled in order to avoid it to be higher than the final concentration according to the formulation.

Once complete dissolution of polymers was achieved, stirring continued at 400-500 rpm. Tween 80 or another surfactant were then added in an amount or ratio of 10:1 PHBV:Tween 80. Later, stirring was increased at 600-700 rpm, and maintained for about 30-40 min.

Toiuene was added at a ratio of 1 :10 toiueneichloroform and stirring continued for about 20-30 min more.

Finally, water was added an amount or ratio of 1 :100 watenchloroform by measuring with a micropipette. The mixture was stirred for at least 30 min at 1500-1700 rpm.

Weight must be controlled to ensure that the final chloroform concentration matches the concentration according to the formulation. Chloroform excess was evaporated by gentle heating to reach the formulation value. It was checked that there were no polymers adhered to the walls that could modify the formulation. If so, those solids were incorporated into the mixture and stirring continued.

An additional 0-2 % by weight of a plant extract may be further added into the formulation before water addition. Once the formulation shown in Table 4 above was achieved, the emulsion was spread on a substrate, for example a polyester substrate, and spread by means of a blade with a clearance of 400 pm between the blade and the polyester surface. During spreading, the formulation had a temperature of 25-35 °c

Evaporation took place at room temperature until a membrane thickness of 50-500 pm was achieved. Thickness depends on the part of the body where membrane is to be applied, for example more thickness is required for body sites like heels, and minimum or lower thickness values for the inside of elbows. In a preferred embodiment, membrane thickness is from 90 to 110 pm.

Example 2: Membrane characterization

Mechanical properties:

A stress-strain analysis was perfomed for membranes obtained in Example 1 using a Dynamic Mechanical Analyzer or DMA Q-800 brand from TA Instruments. The experimental procedure suggested by the manufacturer for membranes in endothermic conditions was followed.

Contact angle:

Water contact angles on the membranes were determined, according to the procedure utilized in our laboratory (Ruiz, E. B. Hermida, and A. Baldessari, Manufacturing and characterization of porous PHBV scaffolds for tissue engineering, Journal of Physics: Conference Series, voi. 332, p. 012028, Dec. 201 1 )

Porosity and morphology:

Membrane surfaces were analyzed by means of scanning electron microscopy (SEM). Membranes were coated with gold and observed using a high vacuum electron microscopy apparatus. Pore size was determined by measuring the micrographs using a public software for image analysis, imaged.

Example 3: Cytotoxicity assays:

Direct cytotoxicity: Bioabsorbabie membrane direct cytotoxicity assays were conducted according to an international standard method, as described in IS010993-5 standard.The NIH/3T3 ceils were cuitured in direct contact with the bioabsorbabie membrane. Cells were incubated in a 24-weil piate at an appropriate density, the test sample was then placed in each well to fill 10% of the well surface, and also positive and negative control samples. Qualitative and quantitative cytotoxicity assessments were made after 24 hours of incubation. The qualitative assessment consisted in microscope direct observation of cells using vital dyes and determination of normal morphology alterations (vacuolization, nuclear disintegration, and membrane integrity). Quantitative assessment included evaluation of the reduction potential of cells utilizing the XTT assay (Sigma-Aldrich (St. Louis, MO) according to the manufacturers specifications.

Indirect cytotoxicity: it was conducted according to an international standard method, the I S010993-5 standard. An extract of the material to be analyzed was obtained using positive and negative controls: the material was placed in culture medium in material area (cm²)/culture medium (mi) ratio of 6/1 and it was incubated at 37°C with 5% C02 for 72 h. These extracts were used for NIH/3T3 cell culture in a 24-well plate for 24 h. Qualitative evaluation consisted in microscope direct observation of cells using vital dyes. Quantitative assessment included evaluation of the reduction potential of ceils utilizing the XTT assay (Sigma- Aldrich (St. Louis, MO) according to the manufacturer specifications. Example 4: Cell proliferation assay

The HaCaT keratinocyte cell line was grown in DMEM medium with high glucose and L-giutamine (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 kept at 37 °C in a humidified atmosphere with 5% CO₂. 1x10⁵ HaCaT cells were seeded over the membranes of the invention. The culture was kept in complete medium, as described above, at 37°C in a humidified atmosphere with 5% CO₂. Cell-free membranes were used as null control while cells cultured over the plastic culture plate were used as the positive control.

The cultures were analyzed at regular intervals: 1 to 4 weeks, using the in vitro viability kit based on XTT (Sigma-Aldrich (St. Louis, MO) according to the manufacturer’s specifications.

The mouse fibroblast cell line, NIH/3T3, 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 kept at 37°C in a humidified atmosphere with 5% CO₂. 1x10⁵ NIH/3T3 cells were seeded over the membranes of the invention. The cultures were kept in complete medium, as described above, at 37°C in a humidified atmosphere with 5% CO₂. Cell-free membranes were used as null control while cells cultured over the plastic culture plate were used as positive control.

The cultures were analyzed at regular intervals: 1 to 4 weeks, using the in vitro viability kit based on XTT (Sigma-Aldrich (St. Louis, MO) according to the manufacturer’s specifications. Example 5: immortalized murine fibroblasts migration assay on the bioabsorbabie membrane of the invention

Fibroblast migration through the membranes of the invention was tested in transwell plates. NIH/3T3 fibroblasts were harvested in culture medium with antibiotics for 16 to 18 h. Cells were collected and resuspended in a medium without fetal bovine serum. A membrane was placed over an insert with an 8 pm pore size and a known number of cells were placed over it. A membrane with 8 pm pore size or more was used as positive control. Complete medium (10% fetal bovine serum) was placed in the lower chamber. The cultures were incubated for 24 h. The membranes and the inserts were fixed with 4% paraformaldehyde (Sigma-Aldrich) and then labeled with DAPI (Sigma-Aldrich) for visualization of nuclei. The membrane side where the cells were seeded, the opposite side of the membrane and the insert were analyzed under an inverted epifluorescence microscope NIKON TE2000-U (Melville, New York, USA). The images were taken with a cooled CCD digital camera Orca-AG (Hamamatsu, Japan).

Example 6: Culture of immortalized human keratinocyte and immortalized murine fibroblast cell lines on the bioabsorbabie membrane of the invention:

The immortalized keratinocyte Ha Cat cell line was cultured on the bioabsorbabie membrane in order to assess its colonization and differentiation capacity. In turn, the immortalized murine fibroblasts NIH-3T3 cell line was cultured, in order to assess its colonization capacity.

Evaluation of morphofunctionality was assessed for the generated cultures by means of histological analysis under light microscope. Morphology and organization of epidermic and dermic tissue generated in vitro on the membrane of the invention were assessed. Additionally, assessment of cultures using fluorescent dyes was performed under an epifluorescence microscope.

The keratinocyte HaCaT cell line was grown 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). The cell cultures were kept at 37°C in a humidified atmosphere with 5% CO₂. 5x10⁵ HaCaT cells were seeded on the membranes. The cultures were kept in complete medium, as described above, at 37°C in a humidified atmosphere with 5% CO₂ for 25 days, with regular medium changes.

Then, the cultures were fixed in Bouin solution (0.9% picric acid, 5% acetic acid, pure formaldehyde) for 24 h, dehydrated in ethylic alcohol upward dilution series. Then they were clarified with xylol-butyl, embedded in paraffin and cut cross-sectionally with a rotary microtome. The slices were mounted on positively charged slides. For further analysis, the sections were stained with hematoxylin and eosin (H&E). Observation, analysis, and microphotography imaging were performed with a light microscope equipped with a digital camera.

The NIH/3T3 fibroblasts 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). The cell cultures were kept at 37 °C in a humidified atmosphere with 5% CO₂. 5x10⁵ NIH/3T3 cells were seeded on the membranes. The cultures were kept in complete medium, as described above, at 37° C in a humidified atmosphere with 5% CO₂ for 25 days, with regular medium changes. The cultures were fixed in Bouin solution (0.9% picric acid, 5% acetic acid, pure formaldehyde) for 24 h, dehydrated in an ethyl ic alcohol upward dilution series. Then they were clarified with xylol-butyl, embedded in paraffin and cut cross-sectionally with a rotary microtome. The slices were mounted on positively charged slides. For further analysis, the sections were stained with hematoxylin and eosin. Observation, analysis, and microphotography imaging were carried out with a light microscope equipped with a digital camera.

HaCaT and NIH/3T3 cell lines were cultured in the same conditions previously described in this example. After 7 days of culture, the cells were fixed with 4% paraformaldehyde (Sigma-A!drich) for 20 minutes at room temperature. The cultures were labeled with DAPI (Sigma-Aldrich) for visualization of nuclei; and with DiO (Molecular Probes, invitrogen), which is a lipophilic dye that allows identification of plasmatic membranes of the cells. The samples were mounted in PBS:g!ycerol for observation under an inverted epifluorescence microscope NIKON TE2000-U (Melville, Nueva York, USA). The images were taken with a cooled CCD digital camera Orca-AG (Hamamatsu, Japan).

Example 7; in vivo studies and assays in pigs:

The experiment was carried out in the Research and Developmental Center for Experimental Medicine ( Centro de Investigation y Desarrolto en Medicina Experimental, CIDME) at the Maimonides University. It is a facility specialized in animals that houses farm animals as laboratory models. The facilities, the equipment, and skilled personnel are suitable for preclinic trials biomedical devices, biomaterials and drugs.

To evaluate the designed membranes for the treatment of deep burns, pigs were used as a model of excision wound. 15 kg Yorkshire pigs were used. The animals were anesthetized and disposed in reclined position. 4 total thickness wounds were performed by 10 cm x 10 cm excision on the back of the animal. An adrenaline embedded gauze was used for achieving the hemostasis. The resorbable membranes of the invention were placed on the wound beds, and also the controls (open wound and treated with Integra^®, and treated with paraffin gauze dressing Jelonet^®). Each of the membranes was suitably seeded with the autologous cell suspension and used for covering the open wounds. To obtain the autologous epithelial cells the biopsy was first washed with trypsin/EDTA (T/E) until the epidermis was separated. Then, the epidermis was enzymatically digested by soaking in T/E at 37°C for about 15 minutes. T/E was subsequently inactivated with culture medium and filtered with a 100 mm filter. The filtered epithelial cells suspended in culture medium were transferred with a syringe to the upper side of the membrane. Finally, the membrane was placed over the wound.

Wound healing processes were controlled until epithelialization. During this period, evolution of the wounds was photographically recorded. Measurements were taken to assess the contraction of the wounds, as well as inflammation and infection signs. After completion of the experiment, animals were sacrificed and biopsies were obtained in order to assess wound epithelialization, cellular density, vascularization and biodegradation of the material, by means of i m m unoh istochem istry .

Example 8: Membrane with plant-based essential oil type extract with antibacterial properties:

Table 5 depicts the composition of the preferred formulation and the “preferred composition” of the finished membrane as specified for the original membrane (column % dry weight). Table 5:

The procedure was carried out as described in Example 1, the only difference being the step of extract addition, also with stirring at 700-800 rpm.

Optional: It was also proved that it is possible to add the plant extract in a single step with the surfactant, with the same ratios.

Various amounts of the membrane components were assayed as shown in Table 6.

Table 6

Example 9: Membrane prepared with a plant-based glycolic extract; using a plant with anti-inflammatory and wound healing properties.

Membranes were obtain using 10 % dry weight extract, aiding emulsion formation with gentle sonication. The preferred emulsion formulation and membrane composition are described in Table 7.

TABLE 7 : PREFERRED EMULSION FORMULATION AND COMPOSITION OF THE

MEMBRANE WITH PLANT EXTRACT

The step of glycolic extract addition was performed at the end of the procedure shown in Example 1 , under stirring at 1500 rpm, instead of the water addition step.

Finally, the preparation can be gently sonicated for 7 minutes. Optionally, the membranes can be prepared without PLGA, as shown in Table 5, maintaining appropriate mechanical properties.

Claims

CLAIMS: Having thus specifically described and determined the nature and the best mode for carrying out the present invention, we claim ownership and exclusive rights to:

1. A bioabsorbabie membrane for tissue regeneration, characterized by comprising po ly (3-hydroxybuty rate-co-3- hyd roxy val erate ) (PHBV), polyvinylpyrrolidone-vinyl acetate (PVP) and poly(lactide-co-glycoiide (PLGA).

2. The membrane according to claim 1 , characterized by comprising from 65 to 80 wt% of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), from 4 to 15 wt% of polyvinylpyrrolidone-vinyl acetate (PVP) copolymer and from 0.5 to 10 wt% of poly(lactide-co-glycolide (PLGA) and from 4 to 10 wt% of a surfactant.

3. The membrane according to claim 1 , characterized by comprising from 0 to 10 wt% of a plant extract.

4. The membrane according to claim 3, characterized by comprising a plant extract selected from essential oils and glycolic extract.

5. The membrane according to claim 1 , characterized in that it has a thickness from 50 to 500 pm.

6. The membrane according to claim 5, characterized in that it has a thickness from 90 to 1 10 pm.

7. The membrane according to claim 1 , characterized in that one of its sides has a pore size from 20 to 44 pm and the other side has a pore size from 5 to 15 pm.

8. The membrane according to claim 1, characterized by comprising a tear strength from 4 to 15 MPa, a tensile modulus from 0,3 to 1 ,5 GPa and a strain at break higher than 1.3 %.

9. A process for preparing the membrane of claim 1 , characterized by comprising the steps of; a. dissolving PHBV in chloroform under stirring; b. adding PLGA still under stirring, until PLGA is dissolved; c. addding PVP still under stirring; d. once the polymers of the above steps are dissolved, adding a surfactant under stirring. e. adding toluene under stirring; f. adding water under stirring, g. extending the mixture obtained in the above step on a substrate; and h. drying the extended mixture until a 50 to 500 pm thick membrane is obtained.

10. The process according to claim 9, characterized in that in step a. stirring speed is from 450 to 550 rpm and heating temperature is from 35 to 45 °C.

1 1. The process according to claim 9, characterized in that the surfactant of step d. is a non-ionic surfactant.

12. The process according to claim 1 1 , characterized in that the non-ionic surfactant is a polysorbate.

13. The process according to claim 9, characterized in that the substrate of step g. is selected from the group consisting of polyester and poly-tetrafluorethylene

14. The process according to claim 9, characterized in that the drying step h. is carried out by evaporation.

15. The process according to claim 9, characterized in that, in step a., the PHBV:chloroform ratio is 1 :3.7.

16. The process according to claim 9, characterized in that, in step b., PLGA is added at a PHBV:PLGA ratio from 3:1 to 10:1.

17. The process according to claim 9, characterized in that, in step c., PVP is added at a PHBV:PVP ratio of 14:1.

18. The process according to claim 9, characterized in that, in step d., a surfactant is added at a PHBV:surfactant ratio of 10:1.

19. A method for treating wounds, characterized by comprising: a) seeding filtered autologous epithelial cells on a membrane of claim 1 ; and b) applying the seeded membrane of the previous step on a wound