MATRICES FOR TISSUE ENGINEERING IN THE FORM OF FOAMS, FIBRES AND/OR MEMBRANES FORMED OF POLYMERS, CERAMICS, POLYMERIC COMPOSITES AND/OR CERAMIC COMPOSITES CONTAINING BIXA ORELLANA L. EXTRACT AND METHOD OF PRODUCTION

Abstract

The present invention relates to matrices for tissue engineering in the form of foams, fibres and/or membranes formed of polymers, ceramics, polymeric composites and/or ceramic composites containing Bixa orellana L. extract capable of inducing tissue regeneration in vivo and in vitro, preventing inflammatory processes and fungal and bacterial contamination during processes of regeneration. The matrices can be two-dimensional or three-dimensional, and have the morphology, porosity and pore size required for tissue growth and regeneration, said structure being particularly suitable for tissue growth in vitro or tissue regeneration in vivo, and the regeneration of hard or soft tissue. Methods for producing said matrices are also described, which include impregnating the materials with the extract and subsequently processing the scaffolds in the form of foams, fibres or membranes, which can be carried out by electro spinning, producing foams by leaching particles, the method of foaming, lyophilization or casting.

Claims

1. Matrices for Tissue Engineering in the form of foams, fibers and/or membranes consisting of polymers, ceramics, polymeric composites and/or ceramic composites characterized by containing an active substance, the extract of Bixa orellana L., with concentration in the range of 0.01% to 50% w w; and the method of obtaining it.

2. Matrices for Tissue Engineering in the form of foams, fibers and/or membranes consisting of polymers, ceramics, polymeric composites and/or ceramic composites as described in claim 1. is characterized by inducing tissue regeneration in vivo, tissue growth in vitro, and avoiding inflammatory processes and contamination with fungi and bacteria during the processes of regeneration and growth of new tissues.

3. The Matrices for Tissue Engineering according to claim 1 characterized by a material in the form of foams consisting of structures containing with open and interconnected pores with size in the range of 40-900 μm and porosity in the range of 30-90%.

4. The Matrices for Tissue Engineering according to claim 1 characterized by a material in the form of lined or randomly oriented fiber mats consisting of filaments with average diameters in the range of 1 nm and 100 μm, with smooth, rough and/or porous surface.

5. The Matrices for Tissue Engineering according to claim 1 characterized by a material in the form of membranes containing pores or not, with an average thickness in the range of 0.1 μm and 10 μm, with smooth and/or rough surface.

6. The Matrices for Tissue Engineering according to claims 1 to 5, characterized by a biocompatible material from the group of polymeric, ceramic and/or composite materials containing the active substance.

7. The Matrices for Tissue Engineering, according to claim 6, characterized by a biocompatible material from the group of natural and/or synthetic biodegradable polymeric materials, as well as the combination of these in the form of copolymers and/or blends, containing the active substance.

8. The Matrices for tissue engineering, according to claim 6, characterized by a material from the group of non-degradable polymeric materials, containing the active substance.

9. The Matrices for Tissue Engineering, according to claim 6, characterized by a biocompatible material of the class of ceramic materials containing the active substance.

10. The Matrices for Tissue Engineering, according to claim 6, characterized by a biocompatible material from the group of composite materials consisting of polymeric materials, according to claims 7 and 8, and/or ceramic materials, according to claim 9, reinforced with particles and/or fibers, according to claims 7-9, added in a concentration ranging from 0.01% to 70% w/w.

11. Method of obtaining biocompatible material from the group of polymeric, ceramic and composite materials containing the active substance as defined in claims 6 to 10, characterized by the following steps: a. Impregnation of polymeric, ceramic and composite materials with the active substance; b. Drying of the material containing the active substance;

12. Method for obtaining biocompatible material from the group of polymeric, ceramic and composite materials containing the active substance, according to claim 11 (a), characterized by adding the solution in organic solvent of the active substance, with a concentration in the range of 0.005% to 50% w/w, over a period of time from 5 minutes to 48 hours to materials in the polymer class, according to claims 7 and 8 and/or materials in the ceramic class, according to claim 9, in powder, extract or grains.

13. Method for obtaining biocompatible material from the group of polymeric, ceramic and composite materials containing the active substance, according to claim 11 (b), characterized by drying the material incorporated with the substance by increasing the temperature, in the range of 30-200° C., pressure reduction and lowering of temperature, use of supercritical fluid and/or application of vacuum with or without temperature change.

14. Method of obtaining the biocompatible polymeric material as defined in claims 1 and 6 characterized by: a. processing the matrices in the form of foams; b. processing the matrices in the form of fibers; c. processing the matrices in the form of membranes;

15. Method of biocompatible material from the group of polymeric, ceramic and composite materials in the form of foams, according to claim 14 (a), characterized by (i) Addition of the porogenic agent to a polymeric mass and/or ceramic mass in suspension or not, in proportions from 1:1 to 20:1 w/w material/porogenic agent, the porogenic agent being selected from the group of: particles soluble in aqueous solution, frozen or effervescent particles and/or droplets of solvents insoluble in the dispersion of the polymeric material or ceramic material, particles obtained from the gas product formation reaction, or gas foams; (ii) Transfer of the polymeric mass and/or ceramic mass in suspension or not containing the porogenic agent to mold and promote drying, temperature increase, in the range of 30-200° C., pressure reduction and lowering of temperature or application of vacuum with or without temperature change. (iii) removal of the porosity-forming agent through leaching, chemical reaction, porogenic fusion, sintering, pressure reduction and porogenic dissolution.

16. Method of biocompatible material from the group of polymeric materials and composites in the form of fibers, according to claim 14 (b), characterized by including the methods for producing non-woven mats of aligned or with random orientation that includes the following steps: (i) dissolving the polymer impregnated with the active substance in the corresponding solvent, obtaining a solution with a concentration between 5 and 35% w/w, temperatures from 25 to 150° C.; (ii) electrospinning of the solutions, c)nsisting of a mono or coaxial electrospinning process, characterized by the electrospinning conditions of the nanofibers: voltage in the range of 5 to 40 kV, temperature in the range of 5 to 60° C., using a distance between the tip of the needle and the surface of the collector in the range of 3 cm and 30 cm, with flow in the pump in the range of 0.005 to 10 mL/h and rotation in the range of 1 to 6000 rpm in the collection cylinder.

17. Method of biocompatible material from the group of polymeric and composite materials in the form of membranes, according to claim 14 (c), characterized by obtaining the membranes by the solvent casting process that includes the preparation of the solutions of the polymeric materials and/or composites, according to claims 7, 8 and 10, with concentration in the range of 5 to 90% w/w, temperature in the range of 25 to 150° C., followed by spreading the solution on a flat surface and drying in temperature in the range from 25 to 200° C.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1—are SEM images of cellulose acetate nanofibers containing Bixa orellana L. extract.

[0024] FIG. 2—are SEM images of PVA nanofibers/Bixa orellana L. extract.

[0025] FIG. 3—is an SEM image of the interior of the PVA foam/extract of Bixa orellana L. reticulated with chemical cross-linking agent.

[0026] FIG. 4—is an Optical microscopy image of the surface of PVA foams/extract of Bixa orellana L. Reticulated with chemical cross-linking agent.

[0027] FIG. 5—is an SEM image of PVA foams/extract of Bixa orellana L. reticulated with chemical cross-linking agent.

[0028] FIG. 6—is Poly butylene succinate (PBS) foams/Bixa orellana L. extract.

[0029] FIG. 7—is an SEM image of the composite PVA/extract of Bixa orellana L. reinforced with magnetic nanoparticle.

[0030] FIG. 8—is an SEM image of the PVA composite/annatto extract Bixa orellana L. reinforced with bioglass.

[0031] FIG. 9—is an SEM image of the PVA composite/Bixa orellana L. extract reinforced with bioglass with its respective chemical analysis by EDS.

DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention relates to a new matrix (scaffold) in the form of foams, fibers and/or membranes consisting of polymers and/or polymer composites reinforced with ceramics containing Bixa orellana L. extract as a bioactive molecule capable to induce tissue regeneration in vivo, tissue growth in vitro and also avoid inflammatory processes and contamination with fungi and bacteria during the regeneration and creation processes of the new tissue. The matrices can be two-dimensional or three-dimensional in the form of foams, fibers and/or membranes and present morphology, porosity and pore size required for tissue growth and regeneration.

[0033] In the present invention, biocompatible polymers are considered any material of natural or synthetic origin ordered in the form of chains (dimeric or polymeric) associated in the form of homopolymers, copolymers or polymeric blends, whether of synthetic or natural materials, which do not promote any harmful reaction to the organism or cells and that has degradation due to physiological processes (such as hydrolysis, enzymatic degradation, among others) without generating toxic products.

[0034] The natural polymers that can be used to produce scaffolds in the form of foams, fibers and/or membranes that include collagen, gelatin, fibrin, fibronectin, fibrinogen, laminin, proteoglycans, elastin, hyaluronic acid, chondroitin sulfate, polyamino acids, polysaccharides, chitosan , chitin, silk, alginate, modified cellulose, however, any other polymer of vegetable or animal origin can be used.

[0035] Biocompatible, biodegradable synthetic polymers that can be used to produce scaffolds in the form of foams, fibers and/or membranes include poly (lactic acid), poly (L-lactic acid) poly (D-Lactic acid), poly (glycolic acid), polycaprolactone, poly (epsilon-caprolactone), poly (lactic-co-glycolic acid), poly (epsilon-caprolactone-co-glycolic acid), poly (epsilon-caprolactone-co-L-lactic acid), polydioxanone, polygluconate , poly (lactic acid-ethylene co-oxide), poly (vinyl alcohol), poly (hydroxybutyrate), poly (hydroxypropionic acid), polyphosphoester, poly (alpha-hydroxy acid), polycarbonates, polyamides, polyanhydrides, polyamino acids, polyorthoesters , biodegradable polyurethanes, cellulose acetate, poly (butylene succinate) and poly (hydroxybutyrate).

[0036] The biocompatible polymers of the group of non-degradable polymeric materials that can be used for the production of scaffolds in the form of foams, fibers and/or membranes include aliphatic polyesters, polyacrylates, polymethacrylates, non-degradable polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl fluoride, polyvinyl fluoride imidazole, chlorosulfonated polyolefins, polyethylene oxide, polyvinyl alcohol, polytetrafluoroethylene, polyamides, silicone, poly (styrene-co-butadiene).

[0037] The polymer/ceramic composites that can be used to produce scaffolds in the form of foams, fibers and/or membranes include the biocompatible and biodegradable or non-degradable polymers mentioned in the previous paragraphs reinforced with particles or ceramic fibers. In the present invention, ceramic material is any inorganic and non-metallic material, biocompatible, whose synthesis can be carried out by heating at high temperatures or co-precipitation at low temperatures, resulting in a material in the form of crystalline fibers or particles (for example, triphosphate calcium, hydroxyapatite, magnetite, maghemite) or amorphous (for example, glass) that has the ability to induce tissue growth in vitro, tissue regeneration in vivo, direct growth or tissue regeneration and/or induce hyperthermia. Among the ceramic materials used, bioactive and biodegradable calcium phosphates, bioactive and biodegradable ceramics, bio glass, biocompatible iron oxides can be highlighted.

[0038] The polymer matrices and polymer/ceramic composites in the form of foams, fibers or membranes contain a concentration of Bixa orellana L. extract that ranges from 0.01% to 50%, but preferably from 0.1% to 25%, in particular from 0.5% to 15% (w/w).

[0039] The polymer/ceramic composites containing Bixa orellana L. extract can have a concentration of particles and/or reinforcement fibers in the proportion that varies from 0.01% to 90%, but preferably from 0.1% to 50%, especially of 0.5% to 10% (w/w). The variation in the concentration of reinforcements allows to control mechanical properties, bioactivity, rate of regeneration, growth and direction of growth/tissue regeneration.

[0040] The polymeric, ceramic and composites matrices containing Bixa orellana L. extract in the form of foams have different populations of interconnected pores, whose size range varies from 40-900 μm and porosity in the range of 30-90%.

[0041] The polymeric, ceramic and composite matrices containing Bixa orellana L. extract in the form of fibers are made up of filaments with an average diameter in the range of 1 nm and 100 μm, with a smooth and/or porous surface.

[0042] The polymeric, ceramic and composite matrices containing Bixa orellana L. extract in the form of membranes containing Bixa orellana L. extract with average thickness in the range of 0.1 μm and 10 μm, with smooth and/or rough surface.

[0043] The polymeric matrices, ceramics and composites containing Bixa orellana L. extract with concentration in the range of 0.01% to 50% w/w.

[0044] Preparation of polymeric matrices, ceramics and composites in the form of foams, fibers, membranes containing Bixa orellana L. extract The processing methods used to obtain the polymer matrix or polymer/ceramic composite containing Bixa orellana L. extract are described below.

[0045] Foams—The process of obtaining matrices in the form of foams consists of the combination of methods of particle leaching, foaming and lyophilization. The particle leaching technique involves molding the dissolved or fused polymer around the porogenic agent, drying or solidifying the polymer and leaching the porogenic agent to generate the polymers with an interconnected pore network. The technique produces scaffolds with controlled porosity (over 93%), pore size (over 500 mm) and crystallinity. By adjusting the manufacturing parameters such as type, quantity and size of the porogenic agent, the porous scaffolds can be adapted for a specific application. The Foaming technique uses a chemical reaction whose product is a gas that is trapped in the form of bubbles within the polymeric matrix. The lyophilization technique, on the other hand, consists of producing a polymer solution that is frozen and then subjected to cryogenic cooling, followed by the application of a low pressure to remove ice crystals via sublimation. Lyophilization removes water and solvent, producing the scaffold with interconnected pores and porosity above 90% and an average diameter of 15 to 35 μm.

[0046] Fibers—In the present invention, matrices with fibrous morphology are obtained using the electrospinning technique. From this technique, it is possible to form filaments, or fibers, on a micro and nanometric scale. The matrices are prepared by dissolving the polymer in an appropriate solvent. The process takes place through the application of a high voltage between a metallic capillary (needle), connected to a syringe, which contains the polymeric solution and an electrically grounded collector. The solution is ejected through the capillary at a constant rate controlled by an injection pump. When the electric field exceeds the surface tension of the solution, it forms a polymeric jet, which becomes thinner as a result of the evaporation of the solvent, occurring the formation of fibers that are attracted to the collector. The thickness and morphology of the fibers depend on the physical-chemical properties of the solutions, such as viscosity, surface tension, and also on process parameters such as the solution flow, dielectric potential and distance between the needle tip and the collector.

[0047] Membranes—The technique known as solvent casting consists of pouring a solution of the polymer into a mold and allowing the solvent to evaporate forming a solid film and film containing the polymeric material in the form of non-porous membranes.

[0048] In the present invention, the process of obtaining polymeric matrices or polymer/ceramic composites containing Bixa orellana L. extract in the form of fibers, foams or membranes comprises the following steps: [0049] 1—Obtaining Bixa orellana L. extract. This step consists in preparing suspensions of Bixa orellana L. extract in methanol by adding the seeds in organic solvent in the proportion (3:1) (organic solvent/seeds) (m/m) . The suspension is stirred at 55° C. for a period in the range of 10 minutes to 24 hours, then the suspension is filtered, obtaining a suspension of Bixa orellana L. extract in ethanol with a concentration in the range of 1% to 10% m/m. [0050] 2—Obtaining the impregnated polymers with Bixa orellana L. extract. The polymeric, ceramic and/or composite mass is mixed with a volume of Bixa orellana L. extract sufficient to obtain the desired proportion of active extract molecules. This mixture is left to stand for evaporation of the solvent. The obtained system is dried obtaining the polymer impregnated with extract of Bixa orellana L. In some variations of the method the extract is added during the processing of the polymeric matrix or polymer/ceramic composite. [0051] 3—Processing of polymers, ceramics or polymer/ceramic composites—In this stage, fibers, foams or membranes are produced, using the processing techniques corresponding to each matrix form described above.

EXAMPLE 1

Preparation of Cellulose Acetate Fibers Containing Bixa orellana L Extract

[0052] The cellulose acetate solution/Bixa orellana extract with a concentration in the range 1 to 50% w/w was obtained by adding the polymer impregnated with Bixa orellana L., to an acetone /dimethylformamide solution (3:1, v/v) with constant stirring followed until complete solubilization at 40° C. The fibers were obtained by electrospinning the suspension under tension in the range of 5 to 40 kV, in temperature in the range of 5 to 6° C., using a distance in the range of 3 cm to 30 cm between the tip of the needle and the surface of the collector, with flow in the range of 0.005 to 10 mL/h in the pump, and rotation in the range of 1 to 6000 rpm in the collecting cylinder. The SEM images obtained for the cellulose acetate fibers/Bixa orellana L. extract in the different electrospinning conditions are shown in FIGS. 1.

EXAMPLE 2

Preparation of Polyvinyl Alcohol Fibers (PVA) Containing Bixa orellana L Extract

[0053] The aqueous solution of PVA/Bixa orellana L. extract with concentration 1 to 50% w/w was obtained by adding the PVA impregnated with Bixa orellana L. in ultra pure water under constant agitation at a temperature in the range of 25 to 70° C. by electrospinning the suspension under tension in the range of 5 to 40 kV, in temperature in the range of 5 to 60° C., using a distance in the range of 3 cm to 30 cm between the tip of the needle and the surface of the collector, with flow in the range from 0.005 to 10 mL/h in the pump, and rotation in the range from 1 to 6000 rpm in the collecting cylinder. The SEM images obtained for the PVA fibers/Bixa orellana L. extract in the different electrospinning conditions are shown in FIGS. 2.

EXAMPLE 3

Preparation of Polyvinyl Alcohol Foams (PVA) Containing Bixa orellana L. Extract Crosslinked with Glutaraldehyde

[0054] Initially an aqueous suspension of PVA/Bixa orellana L. extract and the porogenic agent was obtained by adding the PVA impregnated with Bixa orellana extract L. and the porogenic agent in ultra pure water under vigorous stirring for 1 minute. This suspension was stirred at a temperature in the range of 50-90° C. The homogeneous solution obtained was cooled to room temperature and then hydrochloric acid was added under gentle stirring. The porous sample obtained was frozen and left to stand at that temperature for 3 days. After this period, the sample was cut into cubes that were dipped in sodium hydroxide solution containing crosslinking agent and kept under agitation. The samples obtained were cooled and left to rest. Then, they were washed to leach the residual salt. The samples were frozen in liquid nitrogen and lyophilized. The SEM images of the porous foams of PVA/extract of Bixa orellana L. obtained are shown in FIGS. 3, 4 and 5.

EXAMPLE 4

Preparation of Polyvinyl Alcohol Foams (PVA) Containing Physically Crosslinked Bixa orellana L.

[0055] Initially, an aqueous suspension of PVA/Bixa orellana L. extract and the porogenic agent was obtained by adding the PVA impregnated with Bixa orellana L. extract and the porogenic agent in ultra pure water under vigorous stirring for 1 minute. This suspension was stirred at a temperature in the range of 50-90° C. The homogeneous solution obtained was cooled to room temperature and then hydrochloric acid was added under gentle stirring. The porous sample obtained was frozen and left to stand for 3 days. After this period, the sample was cut into cubes that were subjected to 5 cycles of thawing at room temperature, followed by freezing. Then, they were washed to leach the residual salt. The samples were frozen in liquid nitrogen and lyophilized.

EXAMPLE 5

Preparation of Poly Butylene Succinate (PBS) Foams Containing Bixa orellana L. Extract

[0056] The PBS containing Bixa orellana L. extract was dissolved in organic solvent, the solution obtained was poured onto a flat surface containing a layer of porogen agent. The obtained system was left to stand at room temperature and after complete evaporation of the solvent the sample was washed several times until the agent was completely leached. The SEM images of the porous foams of PBS/Bixa orellana L. extract obtained are shown in FIG. 6.

EXAMPLE 6

Preparation of Polymeric Polyurethane (PU) Membranes Containing Bixa orellana L. Extract

[0057] The PU impregnated with Bixa orellana L. extract was dissolved in dimethylformamide (DMF) to obtain a concentration solution in the range of 1 to 30% m/v. The mixture was stirred for 24 hours at a temperature in the range of 30° C.-50° C., until complete PU solubilization. Subsequently, the solution was poured on a surface, the evaporation of the solvent occurred at a temperature in the range of 30° C.-50° C., for 24 hours.

EXAMPLE 7

Preparation of PVA Composite/Bixa orellana L. Extract Reinforced with Nanoparticulate Magnetite

[0058] The aqueous solution of PVA/Bixa orellana L. extract with concentration 1 to 50% w/w was obtained by adding the PVA impregnated with extract in a aqueous suspension of nanoparticulate magnetite with concentration in the range of 0.5 to 30% m/m under constant agitation at temperatures in the range of 30° C.-50° C. The composites in the form of fibers reinforced with magnetic nanoparticles were obtained by electrospinning the suspension under tension in the range of 5 to 40 kV, in temperature in the range of 5 to 60° C., using a distance in the range of 3 cm to 30 cm between the tip of the needle and the surface of the collector, with flow in the range of 0.005 at 10 mL/h in the pump, and rotation in the range of 1 to 6000 rpm in the collecting cylinder. The SEM images obtained for the composite are shown in FIGS. 7.

EXAMPLE 8

Preparation of PVA Composite/Bixa orellana L. Extract Reinforced with Bioactive Glass

[0059] The aqueous solution of PVA/Bixa orellana L. extract with concentration 1 to 50% w/w was obtained by adding the PVA impregnated with Bixa extract orellana L. in a bioactive glass suspension with a concentration of 0.5 to 30% w/w under constant agitation, temperatures in the range of 30° C.-50° C., followed by sonication. Composites in the form of fibers reinforced with bioglass particles were obtained by electrospinning the suspension under tension in the range of 5 to 40 kV, in temperature in the range of 5 to 60° C., using a distance in the range of 3 cm to 30 cm between the tip of the needle and the surface of the collector, with flow in the range of 0.005 to 10 mL/h in the pump, and rotation in the range of 1 to 6000 rpm in the collection cylinder. The SEM images obtained for the composite are shown in FIGS. 8 and 9.

[0060] Although the preferred version of the invention has been illustrated and described, it should be understood that it is not limited. Various modifications, changes, variations, substitutions and equivalents may occur, without departing from the scope of the present invention.