EP2193228A2 - Filament containing hyaluronic acid in free acidic form and method for making same - Google Patents

Abstract

The invention relates to a method for producing by wet spinning a continuous filament containing hyaluronic acid in the free acidic and particularly water-soluble form. The preparation method of the invention comprises the following steps: a) preparing an aqueous solution suitable for spinning of hyaluronic acid or a salt thereof, preferably a sodium hyaluronate solution; b) extruding said solution through an extrusion die; c) forming the filament by passing the extruded solution in a bath of concentrated acetic acid, particularly at more than 80%, followed by stretching and drying. The invention also relates to a filament containing hyaluronic acid in the free acidic form, wherein said filament has swelling properties in water and physiological liquids and can be solubilised in water under certain conditions.

Description

 FILAMENT BASED ON HYALURONIC ACID IN FREE ACIDIC FORM

AND ITS PROCESS OF OBTAINING

The present invention relates to the field of biomaterials and relates to a method of preparation by wet spinning a hyaluronic acid-based filament, especially soluble in water. The invention also relates to a filament based on hyaluronic acid, said filament having swelling properties in water and physiological fluids and being otherwise able to solubilize in water under certain conditions. The invention also relates to the use of the hyaluronic acid-based filament thus obtained for the production of various biomaterials.

Hyaluronic acid is a major constituent of connective tissue. It was discovered in bovine vitreous humor in 1934 by Karl Meyer [2] but its chemical structure could only be determined in the 1950s [3, 4] (numerals in square brackets refer to bibliographical references at the end of the description).

It is found mainly in the epidermis (2-4 mg / ml), dermis (-0.5 mg / ml), umbilical cord (~ 4 mg / ml), vitreous humor and synovial fluid.

Hyaluronic acid is a polyelectrolyte, that is to say a polymer bearing ionizable groups capable of dissociating in polar solvents, to form a charged polymer chain surrounded by more or less mobile counter ions. The presence of charges gives the polyelectrolyte solutions remarkable physicochemical properties corresponding to many applications.

Polyelectrolytes are used for their rheological properties as thickeners or gelling agents, especially in cosmetics [1]. They are also used for their adsorption properties at the interfaces.

In addition, they are widely present in biological media, which recommends their use in the context of biomedical devices.

Because of its viscoelastic properties giving it a great lubricating power, hyaluronic acid is used for viscosupplementation [5], the injection of hyaluronic acid into the joint with the aim of restoring the synovial fluid homeostasis by improving its rheological properties and promoting an endogenous production of hyaluronic acid. The properties of hyaluronic acid have also been used in ophthalmology [6] It is used in gel form, as a protective agent for eye cells when in contact with surgical instruments and implants, during surgery. Interventions in microsurgery of the eye. The main HA formulations marketed for this type of application are Healon® (Advanced Medical Optics, USA), Opegan® and OpeganHi® (Santen Pharmaceuticals, Japan)

Thanks to its great power of water retention, hyaluronic acid plays a key role in the hydration of the skin. It interacts with the collagen to give the cells a certain rigidity contributing to the suppleness of the skin and, by associating with the proteoglycans of the skin, the hyaluronic acid forms a network able to prevent the passage of the macromolecules (often toxic ) and to facilitate that of small electrolytes and water.

For all these reasons, hyaluronic acid is used in the cosmetic field for the formulation of creams or gels.

A wet-spinning process of hyaluronic acid was developed in the 1960s and 1970s to allow the preparation of hyaluronic acid films with an oriented structure [7,8,9]. The method is the adaptation of a protocol and a device developed for the preparation of DNA samples. In the wet spinning process described in document 9, a solution of potassium hyaluronate (2.5 to 3 mg / ml in a solution of 0.1M KCl) is extruded continuously through a die having 720 channels. cylindrical cylinders each having a diameter of 70 μm and a length of 1.5 mm. The potassium hyaluronate solution is extruded into a bath containing 75-80% ethyl alcohol in 0.1M KCl. The potassium hyaluronate fibers precipitate and are then bundled and wound on a rotating cylinder. Said fibers are then dried which leads to the formation of a film by coalescence.

Short fibers (nanofibres) of hyaluronic acid, grouped in membranes, have also been obtained by the technique of "electro-spinning" and "blowing-assisted electro-spinning" [10,11], The processes mentioned do not make it possible to obtain hyaluronic acid filaments but only membranes made up of networks of nanofibers, that is to say fibers of very short length.

Moreover, materials based on hyaluronic acid fibers have been obtained by crosslinking hyaluronic acid in the presence of crosslinking agents of the carbodiimide or epoxide type (crosslinking agents, a non-exhaustive list of which is for example cited in the document US 2007066816). These materials, however, have the significant disadvantage of being toxic to humans, which severely limits their interest in all uses in vivo. In addition, these materials are insoluble in water.

The present invention proposes to overcome the aforementioned disadvantages presented by the known materials based on hyaluronic acid.

Its primary objective is to provide a new process for producing a hyaluronic acid material in the form of a continuous filament which is free of a chemical crosslinking agent.

The term "filament" is defined in the sense of the invention as a continuous unit fiber of very great length, being not normally interrupted during its manufacturing process, the length of said filament being measured in meters or at least tens of centimeters.

According to a first aspect, the invention relates to a method of preparation by wet-spinning a filament based on hyaluronic acid in free (or protonated) acid form, said process being characterized in that it comprises the following steps: a) preparing a spinnable aqueous solution of hyaluronic acid or a salt of hyaluronic acid, preferably a solution of sodium hyaluronate; b) extruding said solution through an extrusion die; c) forming the filament by passing the extruded solution in a bath of concentrated acetic acid, the concentration of which is sufficiently high to coagulate the extruded solution in the form of a coagulated filament, stretching and drying.

By the term "spinnable solution" is meant a solution whose characteristics, in particular rheological, make it capable of being extruded continuously. The formation of the filament is done by coagulation. The hyaluronic acid solution or the hyaluronic acid salt, which is extruded through the die, is progressively set in mass as it passes through the coagulation bath, the coagulating agent of which is the concentrated solution of acetic acid. For example, acetic acid is concentrated to more than 80%, preferably more than 90%. At this concentration, hyaluronic acid is no longer soluble. Thus, the diffusion of the acetic acid solution in the extruded solution makes it possible to pass the hyaluronic acid from the liquid state to a state of physical hydrogel, in the form of a hyaluronic acid / acetic acid complex, up to obtaining a continuous filament whose section is completely coagulated, stretching and drying giving said filament its mechanical characteristics making it easily manipulated. In the case where the extruded solution is a solution of a hyaluronic acid salt, acetic acid causes its hydrolysis on the one hand hyaluronic acid and on the other hand an acetate which is soluble in the acid solution concentrated acetic acid; in this way, a physical hydrogel in the form of a hyaluronic acid complex in free acid / acid form is obtained in the same manner, starting from a solution of hyaluronic acid or a hyaluronic acid salt. acetic.

Preferably the spinnable solution is at a concentration of at least 0.8% by weight of hyaluronic acid or the equivalent weight of the hyaluronic acid salt. Below this concentration, the viscosity of the solution is insufficient to allow continuous extrusion. Advantageously, it is at a concentration of the order of 1 to 2% by weight of hyaluronic acid. Of course the concentration may vary depending on the diameter of the die used.

Preferably, the preparation of the spinnable solution consists in dissolving in water a given quantity of sodium hyaluronate in order to obtain the desired concentration, then in degassing the solution obtained, in order to eliminate the dissolved gases which can generate bubbles during the formation of the filament.

The draw ratio may be low, for example from 1.05 to 1.10, being in this case carried out on the coagulated filament, during drying, in order to exert a sufficient tensioning of said filament to maintain its geometry. and avoid any deformation during travel until winding. However, it is preferable to draw at a higher rate, for example 2 or more, under conditions that allow this stretching to occur primarily on the spinnable solution extruded between the die outlet and the coagulation bath. This stretching, possible because of the excellent viscoelastic properties of the solutions, in particular of sodium hyaluronate, makes it possible to adjust the diameter of the filament and its mechanical properties.

The drying is generally complete, so as to obtain a filament which is exclusively composed of hyaluronic acid in free acid form and a small proportion of water, of the order of 8 to 12% by weight when it is conditioned in a normal atmosphere. More intensive drying would destroy the hyaluronic acid / acetic acid / water complex by removing acetic acid and water. Nevertheless, the drying may possibly be partial either to obtain a filament free of acetic acid but having a greater proportion of water or to obtain a filament retaining a certain proportion of acetic acid in the context of applications in which the presence of this component is not crippling.

It should be noted that the water content of 8 to 12% is the usual water content of a polysaccharide such as hyaluronic acid in the dry state when it is in the non-ionized form, and in this case in free acid form. Conversely, any polysaccharide in ionized form, especially in saline form, contains under the same conditions from 16 to 18% water. The low water content of the hyaluronic acid filament in free acid form has important consequences on its properties, especially mechanical properties.

The filament obtained directly by the aforementioned method can hydrate and therefore swell in contact with water or physiological media; it remains soluble in water, under conditions which are in particular a function of its crystallinity and therefore of the stretching rate to which it has been subjected.

However, it may be desirable to reduce its reactivity to water. To do this, according to an alternative embodiment, the step of forming the hyaluronic acid filament in free acid form is followed by a step of coating said filament with a compound capable of slowing down its hydration, and therefore the swelling and solubilization of the filament, in water or in a physiological medium. The coating compound remains on the surface and does not diffuse deeply into the filament. The coating is carried out by passing the filament through a coating bath, for example containing from 0.10 to 10% by weight of the coating compound.

Said coating compound is, in one embodiment, a natural macromolecule such as chitosan or collagen. Since these natural polymers carry ionic sites, an electrostatic interaction occurs with polyanion / polycation-type hyaluronic acid, which increases the interaction between the coating compound and the filament. Moreover, in this case, as indeed in that of a polymer-based coating having no ionic charges, hydrogen bonds and hydrophobic interactions develop.

In another embodiment, the filament coating compound is a fatty compound of vegetable or animal origin, for example a vegetable wax deposited in the molten state.

It may also be advantageous to include in the inter-fibrillar spaces of the filament active principles, which are then able to be salted out when the filament is in contact with a liquid medium, in particular a physiological medium. This inclusion is obtained by dipping the filament in an impregnating bath containing said active ingredients, the porosity of the filament allowing them to diffuse inwardly of the filament in the inter-fibrillar spaces, and then by drying the filament thus impregnated. The expression "active principles" denotes all the molecules of biological interest in the field of biotechnologies, among them hormones, growth factors, interleukins, interferons, antibacterials, antifungals and biostimulants. The release rate of the active ingredient is a function of the interactions between said principle and the filament. The release is relatively fast if there is no interaction. On the other hand, if there are interactions, the release can be controlled either physico-chemically or by progressive biodegradation of the filament, especially in the latter case if the interactions are very strong.

The subject of the invention is also a filament based on hyaluronic acid in free acid form obtainable by the aforementioned method. Typically, the base filament, which is free of crosslinking chemicals, contains only hyaluronic acid in free acid form and water. It should be noted that the residual salt, in particular the sodium acetate formed during of the aforementioned process, is solubilized in the concentrated acetic acid solution and is therefore not in the filament. The absence of crosslinking agents guarantees a better biocompatibility since said agents have a certain toxicity and thus induce inflammatory reactions in vivo.

The filaments of the invention have, in the dry state, a water content of 8 to 12%. They have interesting swelling properties in physiological conditions. This makes them very good candidates for filling wrinkles.

The subject of the invention is also a coated filament based on hyaluronic acid which superficially comprises a coating of a compound capable of reducing its hydration, for example chitosan or collagen, or a fatty substance of animal or vegetable origin, in particular a wax of vegetable origin.

The subject of the invention is also a filament based on hyaluronic acid in free acid form which comprises active principles included in its inter-fibrillar spaces.

The invention also relates to the use of the hyaluronic acid-based filament in free acid form in cosmetics, in particular for filling wrinkles.

Another aspect relates to the use of the hyaluronic acid filament in free acid form of the invention for developing textile materials, for example nonwovens, for the manufacture of healing dressings.

Other features and advantages of the invention will appear on reading the detailed description and the following exemplary embodiments, as well as the appended figures in which:

FIG. 1 represents the hyaluronic acid filament in free acid form according to the invention, seen with the aid of an optical microscope; FIG. 2 shows the structure of the filament according to the invention, as observed with the aid of a scanning electron microscope; FIG. 3 represents the strain / stress curve obtained after a tensile test at 2 mm / min on a filament according to the invention, which is not very stretched; 4 represents the images obtained by X-ray diffraction for a macrofilament: unstretched (FIG 4a), stretched (FIG 4b) and very stretched (FIG 4c). The present invention relates to a method of preparation by wet spinning of a continuous filament based on hyaluronic acid in free or protonated acid form, from a spunable aqueous solution of hyaluronic acid or a sodium salt. hyaluronic acid, preferably sodium hyaluronate.

In the present description, the term "hyaluronic acid" should be understood as hyaluronic acid in free or protonated acid form.

To prepare the spinnable solution, a quantity of sodium hyaluronate is dissolved in water to obtain a viscous solution capable of spinning on its own, without dripping. It is noted that below 0.8% by weight, the solutions are not sufficiently viscous. For concentrations of the order of 1 to 2%, the solutions have a viscoelasticity such that they spin with a spatula. Of course, the concentration of sodium hyaluronate is chosen according to the diameter of the extrusion die. In an experimental setting, when the extrusion die is a fine needle whose diameter is of the order of 0.8 mm, excessive viscosities prevent the passage of the solution through the die, in which case in the examples below. Below are preferred 1% by weight solutions of sodium hyaluronate.

To avoid problems of rupture and that it is spinnable under good conditions, the solution used is subjected to a degassing step; in fact, the gases dissolved in the solutions form small bubbles which can be at the origin of embrittlement of the filament. Degassing is all the more important and difficult as the solutions are viscous.

The spinnable solution thus prepared is extruded through an extrusion die, immersed in a coagulation bath containing a coagulating agent, stretched and dried and the obtained filament is received. The extrusion conditions, in particular the extrusion rate, must allow the formation of a coagulated filament of constant diameter. The coagulation bath is a concentrated solution of acetic acid. The concentration of this acetic acid is sufficiently high to obtain that its progressive diffusion in the extruded hyaluronic acid solution makes it possible to pass hyaluronic acid from the liquid state to the state of a physical hydrogel, probably in the form of hyaluronic acid / acetic acid / water complex, to obtain a filament whose cross-section transverse is completely coagulated. It is acetic acid concentrated to more than 80%, preferably more than 90%.

In an experimental setting, the extrusion was carried out using a syringe equipped with a RAZEL R-99E syringe pump and a needle acting as an extrusion die, having a diameter of the order of 0, 8 mm. The extrusion rate was between 1, 2 and 15 cm / min.

The duration of the coagulation is controlled in three ways: by the speed of travel of the filament in the bath on the one hand, by the volume of solution contained in the reactor containing the coagulation bath on the other hand and finally by the path carried out by the filament inside this reactor. The residence time must be sufficient for the filament to be completely coagulated.

The drawing is carried out using a rotating motorized system, which pulls the filament at a speed above the extrusion speed and which optionally receives it. The draw ratio is the ratio between the speed in linear meter of said rotary system and the extrusion rate in linear meter. Stretching can occur on the coagulated filament; in this case, the draw ratio is small, of the order of a few percent, mainly for maintaining the geometry of the wire during its movement until it is received. Stretching can take place immediately at the die outlet on the extruded solution that has not yet been coagulated and possibly partly on the solution extruded during coagulation; in this case, the draw ratio is much greater, which may be of the order of 2 or even greater than 2, this drawing being mainly intended to adjust the diameter of the filament and its mechanical properties.

Drying, by any suitable means, is intended to remove excess coagulation bath and reduce the proportion of acetic acid and water in the filament. During total drying, the acetic acid is completely removed and the resulting filament contains only hyaluronic acid and water, with a water content of 8 to 12%.

The mechanical characteristics of the filament obtained are a function of the drying conditions and the degree of stretching. In particular the crystallinity rate of the filament evolves as the draw ratio.

The filament, which contains only hyaluronic acid and water, can hydrate on contact with water or physiological solutions, with subsequent swelling. It remains soluble in water, under varying conditions depending on its manufacturing parameters.

According to an alternative embodiment, in order to make it less sensitive to hydration, the hyaluronic acid filament undergoes a complementary coating step with a compound capable of slowing the hydration, and therefore the swelling and the solubilization of the filament, in water or in a physiological medium such as: blood, lymphatic or lacrimal fluid, etc. Said coating compound is, in one embodiment, a natural macromolecule such as chitosan or collagen. In another embodiment, the coating compound of the hyaluronic acid filament is a fatty compound of vegetable or animal origin.

According to a second aspect, the invention relates to a continuous filament based on hyaluronic acid obtainable by the above method. The known hyaluronic acid fibers obtained by electro-spinning have, according to WEB observations, diameters of less than 50 nm. The filament according to the present invention, obtained by the aforementioned method, has a diameter which is generally from one hundred to several hundred micrometers. In addition, it has a length that is not limited, which may be several meters or at least several tens of centimeters.

Typically, the hyaluronic acid-based filament of the invention is free from any chemical crosslinking agent, which makes it particularly suitable for in vivo uses in humans. It contains hyaluronic acid, water and possibly acetic acid. Preferably it contains exclusively hyaluronic acid and water, especially in the dry state with a water content of 8 to 12%.

It swells in water and in a given physiological environment. It is also able to dissolve in water. This dissolution in the water of the hyaluronic acid filament is not immediate; it is obtained by increasing the pH. Indeed, hyaluronic acid in its free acid form or protonated, that is to say, non-ionized is not directly soluble in water. It is thus necessary to sufficiently ionize the carboxylic sites to obtain a perfect solubilization. The hyaluronic acid filament was characterized by optical microscopic observation and scanning electron microscopy, by tensile tests as well as by X-ray diffraction, as presented hereinafter.

1. Determination of the diameter of the filaments

The average filament diameter was determined for each spinning speed using an optical microscope from four measurements taken at different locations along the length of the same filament. It turns out that the spinning speed has no influence on the diameter of the filaments.

The diameters measured are between 120 and 170 μm. However, the diameter of the wires can vary by changing the drawing ratio and the diameter of the die.

The image of a filament according to the invention as visualized by means of an optical microscope is shown in FIG. 1. It is a hyaluronic acid filament obtained from a solution of 1% by weight of sodium hyaluronate for a spinning speed of 5.9 cm / min.

2. Scanning electron microscopy

The surface condition and fracture profile of the hyaluronic acid filaments were observed using a Hitachi S800 scanning electron microscope at 15 kV after metallization with gold-palladium. The filaments were ruptured by immersion in liquid nitrogen from a filament surrounded by a needle. The images obtained from a poorly drawn filament of hyaluronic acid obtained from a 1% by weight solution of sodium hyaluronate are shown in FIG.

The filament appears at first to consist of several assembled fibers (Figure 2a), but the fracture surface (Figure 2b) clearly shows that the surface relief is due to folds. These folds or villi appear most certainly during the drying step. In Figure 2b, we also observe within these villi smaller objects such as fibrils that seem to align along the axis of the filament. Such "nanofibrils" have already been observed for chitosan fibers [12].

3. Mechanical properties

Traction tests were carried out using an Adamel-Lhomargy DY22 traction machine fitted with a 1ON sensor and a specific system for the wires. Samples were prepared from a 1% by weight solution of sodium hyaluronate coagulated in 99% concentrated acetic acid. These filaments were slightly stretched.

These filaments were then subjected to tensile stress at a speed of 2 mm / min, the length initially subjected to the load being set at 30 mm. For each type of sample, four tests were performed.

Young's modulus, breaking strength, and elongation at break were then determined for each sample, i.e., for each spinning speed.

An example of a tensile curve is given in FIG. 3. It corresponds to the strain / stress curve obtained after a tensile test at 2 mm / min on a thread that is not very stretched, the spinning speed being 4.7 cm / cm 2. min. It is observed that after a linear elastic deformation domain, the filament undergoes a beginning of plastic deformation. 4. Crystallinity: X-ray diffraction (WAXS)

The images obtained by X-ray diffraction are given in FIG. 4 for a hyaluronic acid filament: unstretched (FIG. 4a), stretched (FIG. 4b) and very stretched (FIG. 4c). These results demonstrate the development of the crystallinity of the samples studied.

The intensity profile was studied according to the azimuthal angle which allowed the calculation of the orientation function of Hermans:

/ = -0.02 for the unstretched sample (Figure 4a) and f = -0.113 for the highly stretched sample (Figure 4c)

Knowing that :

-f = -0.5 for planes perfectly aligned with the axis of a fiber, - / = 1 for planes perpendicular to the axis of the fiber, and - ^ = O for isotropically oriented planes, the values obtained show that the crystalline portions of the unstretched filament are very weakly oriented in a preferred direction while for the highly stretched sample, the crystalline portions are preferably oriented along the axis of the filament.

The crystallinity induces in the hyaluronic acid filament a parallel arrangement of nano fibrils, with formation of fibrillary inter-spaces. Targeted applications:

These filaments have particularly interesting properties for the biomedical and surgical fields. A first intended application is the filling of wrinkles, because of their swelling capacity and their crystalline nature. The more the sample is crystalline and the more its resolubilization, especially at pH biological media such as blood (pH 7.2 to 7.4) or tears (pH 8) will be slowed down.

Another application is the use of hyaluronic acid-based filaments for making textiles, in the form of nonwovens, fabrics or knits, especially in order to form healing dressings.

It is also envisaged to insert active principles in the inter fibrillary spaces, then transforming the filaments into systems adapted to the controlled release of active principles.

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[10] Um, I. C .; Fang, D. F .; Hsiao, B. S .; et al. Electro-spinning and electro-blowing of hyaluronic acid. Biomacromolecules (2004), 4, 1428-1436.

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Claims

1. Process for the preparation by wet spinning of a filament based on hyaluronic acid in free acid form, characterized in that it comprises the following steps: a) preparation of a spinnable aqueous solution of hyaluronic acid or of a salt of hyaluronic acid; b) extruding said solution through an extrusion die; c) passing the extruded solution in an acetic acid solution whose concentration is sufficiently high to coagulate the extruded solution as a coagulated filament, stretching and drying. 2. Method according to one of claims 1 to 9 characterized in that the acetic acid solution is concentrated to more than 80%, preferably more than 90%, especially 99%. 3. Method according to one of claims 1 or 2 wherein the preparation of the spinnable solution consists in dissolving in water a specific amount of sodium hyaluronate and then degassing before extrusion. 4.Procédé according to any one of claims 1 to 3 wherein the spinnable solution has a concentration by weight of hyaluronic acid or hyaluronic acid salt greater than 0.8%, preferably of the order of 1 to 2%. 5. Method according to one of claims 1 to 4 characterized in that the stretching occurs on the coagulated filament with a low draw ratio of the order of 1, 05 to 1, 10. 6. Method according to one of claims 1 to 4 characterized in that the stretching occurs on the extruded solution before and optionally during coagulation with a high draw ratio, especially of the order or greater than 2. 7. Method according to one of claims 1 to 6 characterized in that the drying conditions are adjusted so as to completely eliminate the acetic acid contained in the coagulated filament. 8. Method according to claim 7 characterized in that the drying conditions are adjusted so as to remove most of the water contained in the coagulated filament. 9. Method according to one of claims 1 to 8 characterized in that it comprises a complementary step of coating the filament with a compound capable of slowing the hydration of said filament, especially chitosan or collagen or a fatty compound of vegetable or animal origin. 10. Method according to one of claims 1 to 8 characterized in that it comprises a complementary step of inclusion of active principles in the inter-fibrillar spaces of the filament. 11. Filament based on hyaluronic acid in free acid form, in particular obtained by the method according to one of claims 1 to 6, characterized in that it contains hyaluronic acid, acetic acid and water. 'water. 12. Hyaluronic acid filament, in particular obtained by the process according to claim 7, characterized in that it contains exclusively hyaluronic acid and water in a small proportion, in particular from 8 to 12% by weight of water. 13. Filament according to one of claims 11 or 12 having a diameter of the order of or greater than 100 microns. Coated filament in which the filament according to one of claims 1 to 13 comprises a coating of a compound capable of slowing down its hydration, especially chitosan or collagen or fatty compound of plant or animal origin, especially vegetable wax. 15. Filament with inclusion in which the filament according to one of the claims 11 to 13 comprises active principles included in its inter-fibrillar spaces. 16. Inclusive filament according to claim 15 characterized in that the active ingredients are selected from hormones, growth factors, interleukins, interferons, antibacterials, antifungals and biostimulants. 17. Use of the filament according to one of claims 11 to 16 for developing textile materials, including non-woven, such as healing dressings. 18. Use of the filament according to one of claims 11 to 14 in cosmetics for filling wrinkles. 19. Use of the filament according to one of claims 15 or 16 to develop systems adapted to the controlled release of active ingredients.