Method for producing a hydrogel

Abstract

The present invention relates to a method for producing a hydrogel comprising a cross-linked polysaccharide, in particular, a method for producing an injectable hydrogel comprising cross-linked hyaluronic acid. The hydrogel has mechanical properties suitable for filling soft tissues. The present invention also relates to a hydrogel, preferably injectable, that can be obtained by the method and a composition containing the hydrogel.

Description

[0001] DESCRIPTION

[0002] TITLE: PROCESS FOR PREPARING A HYDROGEL

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to a method for preparing a hydrogel comprising a crosslinked polysaccharide, in particular, to a method for preparing an injectable hydrogel comprising crosslinked hyaluronic acid. The hydrogel has mechanical properties suitable for filling soft tissues. The present invention also relates to a hydrogel, preferably injectable, obtainable by the method and to a composition comprising the hydrogel.

[0005] TECHNOLOGICAL BACKGROUND

[0006] Polysaccharides, such as glycosaminoglycans, are widely used in the medical and aesthetic fields, particularly for soft tissue filling. In particular, the majority of products marketed for aesthetic applications are based on hyaluronic acid.

[0007] To improve skin quality, gels prepared from unmodified hyaluronic acid are interesting because they have the advantage of being perfectly biocompatible. However, after in vivo implantation, they degrade very quickly. Thus, the effects of such gels are only short-lived.

[0008] To increase the in vivo durability of hyaluronic acid gels and their resistance to degradation, hyaluronic acid is usually modified by crosslinking. Crosslinked hyaluronic acid gels can be obtained by different preparation methods.

[0009] In particular, polysaccharides, including hyaluronic acid, can be modified using crosslinking agents, such as epoxy agents, aldehydes like glutaraldehyde, divinyl sulfone (DVS), or polyamines. Currently, the most commonly used crosslinking agent is 1,4-butanediol diglycidyl ether (BDDE). Polysaccharides can be modified under different conditions of pH, time, pressure, and temperature.

[0010] For reasons of product safety and performance, the provision of sustainable in vivo compositions comprising a biocompatible polysaccharide, the most natural and therefore the least modified, in particular the least crosslinked, is sought. To reduce the amount of crosslinking agent required to obtain a polysaccharide-based gel with mechanical properties suitable for its use, various modifications to the parameters of the crosslinking process have already been proposed, such as the addition of different salts, increasing the concentration of hyaluronic acid and / or sodium hydroxide in the crosslinking medium, or adjusting the duration and temperature of the crosslinking reaction (WO2014 / 064633; WO2016 / 096920; WO2017 / 016917; Sukwha Kim et al., Carbohydrate Polymers, 2018, 202, 545-553).

[0011] However, below a certain threshold, the prepared gels no longer exhibit suitable mechanical properties. In particular, hyaluronic acid gels crosslinked with 1,4-butanediol diglycidyl ether (BDDE) with a degree of modification of approximately 1% are very poorly cohesive and / or do not withstand the heat sterilization conditions generally applied to this type of product (autoclave sterilization). These products, which are unstable during sterilization, ultimately exhibit a predominantly viscous component, which is not desirable for soft tissue filling applications.

[0012] Thus, a need remains for the provision of a process for preparing a gel based on polysaccharides, in particular hyaluronic acid, which makes it possible to further reduce the quantities used of conventional crosslinking agent while allowing the prepared gel to exhibit mechanical properties suitable for filling soft tissues.

[0013] BRIEF DESCRIPTION OF THE INVENTION

[0014] The subject of the invention is a process for preparing a polysaccharide-based hydrogel comprising the following steps: a) providing at least one polysaccharide; b) providing at least one crosslinking agent and / or at least one functionalizing agent, the functionalizing agent allowing crosslinking of the polysaccharide by sol-gel reaction; c) preparing a reaction medium comprising a solvent, the polysaccharide(s) and the crosslinking agent(s) and / or functionalizing agent(s); d) crosslinking of the polysaccharide: d 1 ) by reaction of the polysaccharide with the crosslinking agent(s); or d2) by sol-gel reaction of the functionalized polysaccharide, the functionalized polysaccharide being obtained by reaction of the polysaccharide with the functionalizing agent(s);wherein the crosslinking of the polysaccharide according to d1) or d2) is carried out under conditions which do not allow the sublimation of water, at a pressure P and at a temperature T higher than the temperature of the eutectic point of the reaction medium as measured at pressure P and lower than the temperature of the freezing point of the reaction medium as measured at pressure P.;

[0015] Advantageously, the crosslinking agent comprises at least two functional groups Z, identical or different, chosen from isocyanate, amino, epoxide, carboxyl, N-succinimidyloxycarbonyl, N-sulfosuccinimidyloxycarbonyl, halocarbonyl, isothiocyanate, vinyl, formyl, hydroxyl, sulfhydryl, hydrazino, acylhydrazino, aminoxy, carbodiimide groups, and an acid anhydride residue.

[0016] Advantageously, the functionalizing agent is a Chem molecule. It has the following formula: in which:

[0017] T represents an isocyanate, amino, epoxide, carboxyl, N-succinimidyloxycarbonyl, N-sulfosuccinimidyloxycarbonyl, halocarbonyl, isothiocyanate, vinyl, formyl, hydroxyl, sulfhydryl, hydrazino, acylhydrazino, aminoxy, carbodiimide group, or an acid anhydride residue;

[0018] A represents a chemical bond or a spacer group;

[0019] R5 and R6, which may be identical or different, represent a hydrogen atom; a halogen atom; a group -OR4 with R4 representing a hydrogen atom, an aryl group or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms; an aryl; or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms optionally substituted by one or more group(s) chosen from a halogen atom, an aryl and a hydroxyl;

[0020] R10 represents a hydrogen atom, an aryl group or an aliphatic hydrocarbon group containing from 1 to 6 carbon atoms.

[0021] The functional groups Z are advantageously identical and represent an epoxy or vinyl group, more preferably epoxy. The crosslinking agent is advantageously selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxy-octane, poly(ethylene glycol) diglycidyl ether (PEGDGE), 1,2-bis(2,3-epoxypropoxy)ethane (EGDGE), 1,3-bis(3-glycidyloxypropyl)tetramethyldisiloxane, poly(dimethylsiloxane) terminated at each end by a diglycidyl ether (CAS number: 130167-23-6), hydroxyapatite beads modified to carry epoxy groups and mixtures thereof.

[0022] The amount of crosslinking agent advantageously varies from 0.001 to 0.15 mole per 1 mole of repeating unit of the polysaccharide, preferably from 0.001 to 0.08 mole per 1 mole of repeating unit of the polysaccharide, even more preferably from 0.001 to 0.05 mole per 1 mole of repeating unit of the polysaccharide.

[0023] The crosslinking of the polysaccharide according to d1) or d2) is advantageously carried out for a period of at least 1 hour, preferably at least 3 hours, preferably at least 72 hours, preferably at most 27 weeks, under conditions which do not allow the sublimation of water at temperature T.

[0024] The crosslinking of the polysaccharide according to d1) or d2) is advantageously carried out for a period ranging from 2 to 25 weeks, preferably ranging from 2 to 20 weeks or 2 to 17 weeks, even more preferably from 3 to 8 weeks or from 4 to 7 weeks, under conditions which do not allow the sublimation of water at temperature T.

[0025] The pressure P is advantageously less than or equal to atmospheric pressure, advantageously between 0.7.10 5 Pa and 0.9.10 5 Pa or equal to atmospheric pressure. Step d) is advantageously carried out in a closed, airtight container which may be flexible or rigid, advantageously flexible.

[0026] In step d) the airtight container is advantageously placed at a temperature ranging from -35°C to -10°C at atmospheric pressure, preferably at a temperature of approximately -20°C at atmospheric pressure.

[0027] The invention also relates to a hydrogel capable of being obtained by the method according to the invention.

[0028] The invention also relates to a cosmetic or pharmaceutical composition comprising a hydrogel according to the invention.

[0029] The invention also relates to the hydrogel according to the invention or the composition according to the invention for its use in filling and / or replacing tissues.

[0030] The invention also relates to the cosmetic use of a hydrogel according to the invention or of a composition according to the invention for preventing and / or treating the alteration of the viscoelastic or biomechanical properties of the skin; for filling volume defects of the skin, in particular for filling wrinkles, fine lines and scars; for reducing nasolabial folds and bitterness folds; for increasing the volume of the cheekbones, chin or lips; for restoring the volumes of the face, in particular the cheeks, temples, the oval of the face, and the area around the eyes; for reducing the appearance of wrinkles and fine lines; or for stimulating, regenerating, moisturizing, firming or restoring the radiance of the skin, in particular by mesotherapy.

[0031] FIGURES

[0032] Figure 1 shows the photo after thawing of the gel from prototype A of example 2.

[0033] Figure 2 shows the photo after thawing of the gel from prototype B of example 2.

[0034] DEFINITIONS

[0035] "Atmospheric pressure" is the pressure exerted by the air constituting the atmosphere on any surface in contact with it. It varies according to altitude. At an altitude of 0 m, the average atmospheric pressure is 101,325 Pa.

[0036] The term "gel" refers to a polymer network that is expanded throughout its volume by a fluid. This means that a gel is formed of two media, one "solid" and one "liquid," dispersed in each other. The so-called "solid" medium consists of long polymer molecules connected to each other by weak bonds (e.g., hydrogen bonds) or by covalent bonds (crosslinking). The liquid medium consists of a solvent. A gel generally corresponds to a product that has a phase angle 5 less than or equal to 45° at 1 Hz for a strain of 0.1% or a pressure of 1 Pa, advantageously a phase angle 5 ranging from 2° to 45° or ranging from 20° to 45°.

[0037] The term "hydrogel" designates a gel as defined above in which the solvent constituting the liquid medium is predominantly water (for example at least 90%, in particular at least 95%, in particular at least 99% by weight of the liquid medium). Preferably, the liquid medium comprises, in particular consists of, a buffer solution, advantageously allowing a pH of the liquid medium of between 6.8 and 7.8, in particular a saline phosphate buffer.

[0038] The term “injectable gel” refers to a gel that can flow and be injected manually using a syringe equipped with a needle with a diameter ranging from 0.1 to 0.5 mm, for example a 30 G, 27 G, 26 G, 25 G hypodermic needle. Preferably, an “injectable gel” is a gel having an average extrusion force of less than or equal to 25 N, preferably ranging from 5 to 25 N, more preferably ranging from 8 to 15 N, when measured with a dynamometer, at a fixed speed of approximately 12.5 mm / min, in syringes with an external diameter greater than or equal to 6.3 mm, with a needle with an external diameter less than or equal to 0.4 mm (27 G) and a length of 1”, at room temperature.

[0039] The “stringiness” of a product refers to its ability to be stretched between two surfaces to which it has adhered. Stringiness can be determined using a texturometer, sensory analysis carried out by a panel, or rheological and mechanical measurements including the measurement of the phase angle (5), G', G”, or tensile tests. In particular, this characteristic can be measured as described by P. Micheels et al. (Micheels et al., Comparison of two Swiss-designed hyaluronic acid gels: six-month clinical follow-up, Journal of Drug in Dermatology, 2017, 16:154-161, “Resistance to stretching”) or by performing a Tack test and measuring the length of the gel threads in tension.

[0040] The "stickiness" of a product refers to its ability to adhere to a surface. It can be determined qualitatively using sensory analysis using a panel or by moving a bolus across a surface. It can also be determined quantitatively by measuring the strength of adhesion to a surface, using a tensile strength machine or mechanical analysis.

[0041] The term "polysaccharide" refers to a polymer composed of monosaccharides (preferably D-enantiomers) joined together by glycosidic bonds. The term "monosaccharide", also called "ose", refers to an unmodified or modified monosaccharide.

[0042] An “unmodified monosaccharide” means a compound of the formula H-(CHOH) X-CO- (CHOH)yH with x and y representing, independently of each other, an integer ranging from 0 to 5 provided that 2 < x+y < 5, the monosaccharide being able to be in a linear form represented by the above-mentioned formula or being able to be in a cyclized form by reaction of the CO function (aldehyde or ketone) with one of the OH groups to form a hemiacetal or hemiketal group. Preferably, the monosaccharide is in cyclized form. There are two types of ose: aldoses which carry an aldehyde function (when x or y is 0) and ketoses which carry a ketone function (when neither x nor y is 0). Monosaccharides are classified by number of carbons. For example, 6-carbon monosaccharides (x+y=5) are hexoses with the formula CeH^Oe and can be ai lose, altrose, glucose, mannose, gulose, idose, galactose or talose.The 5-carbon monosaccharides (x+y=4) are pentoses of formula C5H10O5 and may be ribose, arabinose, xylose, or lyxose. Preferably, the monosaccharide is a hexose, i.e. x+y = 5. A monosaccharide further comprises x+y asymmetric carbons and therefore 2. (x+y-1) pairs of enantiomers. Each pair of enantiomers is designated by a different name and the enantiomers of the same pair are called D and L enantiomers respectively.

[0043] A “modified monosaccharide” means an unmodified monosaccharide as defined above, including, for example:

[0044] - one or more of the OH functional groups have been replaced by another functional group, for example:

[0045] (i) a group OR with R representing a (Ci-C6)alkyl group such as methyl or ethyl; hydroxy-(Ci-C6)alkyl such as hydroxyethyl (-CH2CH2OH) or hydroxypropyl (-CH2- CH(OH)-CH3); carboxy-(Ci-C6)alkyl such as carboxymethyl (-CH2COOH); or CO-(Ci-C6)alkyl such as acetyl; and / or

[0046] (ii) a group NR'R” with R' and R” representing, independently of each other, H, (Ci-C6)alkyl or CO-(Ci-C6)alkyl such as acetyl; and / or

[0047] (iii) an OSO3H group; and / or

[0048] - the terminal CH2OH function(s) have been replaced by a COOH or CHO group;

[0049] - a -CH(OH)-CH(OH)- bond is oxidized to give two terminal -CHO (aldehyde) groups in place of this bond; and / or

[0050] - a terminal CH2OH function was condensed with an OH functional group to form an -O-CH2- chain.

[0051] The term "repeating unit" of a polysaccharide refers to a structural unit consisting of one or more (usually 1 or 2) monosaccharides whose repetition produces the complete polysaccharide chain.

[0052] Some or all monosaccharides may be in a modified form. Monosaccharides, when modified, may be in different modified forms.

[0053] The term "physiologically acceptable" means that which is generally safe, non-toxic, and neither biologically nor otherwise undesirable and is acceptable for human or veterinary cosmetic (i.e., non-therapeutic) or therapeutic use, including use by injection into the human or animal body or for topical application to the skin.

[0054] The “salts” useful in the context of the present invention are preferably physiologically acceptable salts. The terms “physiologically acceptable salts” designate in particular:

[0055] (1) pharmaceutically acceptable acid addition salts formed with pharmaceutically acceptable inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like;or formed with pharmaceutically acceptable organic acids such as formic acid, acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxynaphthoic acid, 2-hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, muconic acid, 2-naphthalenesulfonic acid, propionic acid, salicylic acid, succinic acid, dibenzoyl-L-tartaric acid, tartaric acid, p-toluenesulfonic acid, trimethylacetic acid, trifluoroacetic acid and the like, and;

[0056] (2) pharmaceutically acceptable base addition salts formed when an acid proton present in the parent compound is either replaced by a metal ion, for example an alkali metal ion (eg Na, K), an alkaline earth metal ion (eg, Ca, Mg), a zinc ion, a silver ion or an aluminum ion; or coordinated with a pharmaceutically acceptable organic base such as diethanolamine, ethanolamine, N-methylglucamine, triethanolamine, tromethamine and the like; or with a pharmaceutically acceptable inorganic base such as aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide and the like.

[0057] The “degree of modification” (MOD), expressed in %, of a polysaccharide, such as hyaluronic acid, corresponds to the molar quantity of modifying agent, such as the quantity of crosslinking agent and / or functionalizing agent linked to the polysaccharide, by one or more of its ends, expressed per 100 moles of repeating units of the polysaccharide. It can be determined by methods known to those skilled in the art such as Nuclear Magnetic Resonance (NMR) spectroscopy.

[0058] The “molar crosslinking rate” (TR), expressed in %, designates the molar ratio of the quantity of crosslinking agent to the quantity of repeating unit of the polysaccharide introduced into the crosslinking reaction medium expressed per 100 moles of repeating units of the polysaccharide in the crosslinking medium.

[0059] The "molar functionalization rate", expressed in %, means the molar ratio of the quantity of functionalizing agent to the quantity of repeating unit of the polysaccharide introduced into the crosslinking reaction medium expressed per 100 moles of repeating units of the polysaccharide in the functionalization medium. The expression "therapeutic active ingredient" means a substance for curing, alleviating the symptoms of and / or preventing a disease; a substance having curative or preventive properties with respect to human or animal diseases, as well as any substance which may be used in humans or animals or which may be administered to them, with a view to establishing a medical diagnosis or restoring, correcting or modifying their physiological functions by exerting a pharmacological, immunological or metabolic action.

[0060] The expression "cosmetic active ingredient" means any non-therapeutic substance intended in particular to be placed in contact with various superficial parts of the human body, such as the epidermis, hair and capillary systems, nails, lips, chest and teeth, with a view, exclusively or mainly, to cleaning them, protecting them, perfuming them, maintaining them in good condition, modifying their appearance or odor.

[0061] The term "approximately" means that the value concerned may be 10% lower or higher, in particular 5%, in particular 1%, than the indicated value.

[0062] An “aqueous reaction medium” means a reaction medium whose solvent is predominantly water (for example at least 90%, in particular at least 95%, notably at least 99% by weight of the total solvent) or even is water.

[0063] The term "spacer group" refers to a fragment comprising at least one atom intended to link together two chemical groups within the same molecule. Preferably, the spacer group contains at least one carbon atom.

[0064] The term "halogen" refers to an atom of fluorine, chlorine, bromine or iodine.

[0065] An "epoxide" group is an ethylene oxide residue linked to the rest of the molecule by one of its carbon atoms.

[0066] An “N-succinimidyloxycarbonyl” group is a group with the formula Chem. GR1 below:

[0067] Chem. GR1

[0068] An “N-sulfosuccinimidyloxycarbonyl” group is a group with the formula Chem.

[0069] GR2 below:

[0070] Chem. GR2

[0071] A "halocarbonyl" group is a group of formula -CO-Hal with Hal representing a halogen, such as Cl or Br.

[0072] A “carbodiimide” group is a group comprising a -N=C=N- motif, and more particularly a group of formula -N=C=NR a with R a representing an aliphatic hydrocarbon group comprising from 1 to 20 carbon atoms, preferably a (C1-C6)alkyl group, one or more carbon atoms of which are optionally replaced by a heteroatom chosen from O, S and N, in particular N.

[0073] An “acid anhydride residue” is a group comprising a -C(O)-OC(O)- unit, and more particularly a monovalent cyclic group comprising the -C(O)-O-C(O)- unit, such as a saturated monovalent hydrocarbon monocyclic group comprising 5 to 10, in particular 5 or 6, carbon atoms of which three successive carbon atoms are replaced by C(O)-OC(O) and optionally of which one or more, in particular one, additional carbon atoms, preferably not consecutive to the three carbon atoms substituted by CO-O-CO, are each replaced by a heteroatom such as N, O or S, in particular N. The acid anhydride residue may correspond in particular to the following Chem. GR3 formula:

[0074] Chem. GR3 The acid anhydride residue may also be selected from a maleic anhydride residue or a succinic anhydride residue.

[0075] The expression "aliphatic hydrocarbon chain" or "aliphatic hydrocarbon group" designates a linear, branched and / or cyclic hydrocarbon group, saturated or unsaturated but not aromatic, advantageously comprising from 1 to 50, in particular from 1 to 20, for example from 1 to 12 or from 1 to 6 carbon atoms. These will in particular be alkyl groups.

[0076] The term "branched aliphatic hydrocarbon chain" specifically refers to a main aliphatic hydrocarbon chain comprising at least one secondary aliphatic hydrocarbon chain.

[0077] The term "star-shaped aliphatic hydrocarbon chain" means a branched aliphatic hydrocarbon chain comprising several secondary aliphatic hydrocarbon chains all starting from a single branch point.

[0078] The expression "C1-Cx alkyl" or "(Cl-Cx)alkyl" or "alkyl containing 1 to x carbon atoms" means a saturated, linear or branched monovalent hydrocarbon group containing 1 to x carbon atoms, with x being an integer, such as, for example, a methyl, ethyl, isopropyl, tert-butyl, n-pentyl, cyclopropyl, cyclohexyl, etc. group.

[0079] The expression "(Cl-Cx)alkylene" denotes a saturated, linear or branched, divalent hydrocarbon group containing from 1 to x carbon atoms, with x being an integer, such as, for example, a methane-1,1-diyl, ethane-1,1-diyl, ethane-1,2-diyl, propane-1,3-diyl, butane-1,4-diyl, butane-1,3-diyl, butane-1,2-diyl, pentane-1,5-diyl, hexane-1,6-diyl, hexane-1,5-diyl, heptane-1,7-diyl, octane-1,8-diyl, nonane-1,9-diyl, decane-1,10-diyl, etc. These include a methane-1,1-diyl or propane-1,3-diyl group.

[0080] The expression “hydroxy-(C1-Cx)alkyl” denotes a (Cl-Cx)alkyl group as defined above substituted by a hydroxyl group (OH) such as for example a hydroxyethyl (-CH2CH2OH) or a hydroxypropyl (e.g. -CH2-CH(OH)-CH3).

[0081] The expression “carboxy-(C1-Cx)alkyl” designates a (Cl-Cx)alkyl group as defined above substituted by a carboxyl group (COOH) such as for example a carboxymethyl (-CH2COOH).

[0082] The term "aryl" denotes a monovalent aromatic hydrocarbon group, preferably comprising from 6 to 10 carbon atoms, comprising one or more rings, such as for example a phenyl or naphthyl group. The term "arylene" denotes a divalent aromatic hydrocarbon group, preferably comprising from 6 to 10 carbon atoms, comprising one or more rings, such as a phenylene group.

[0083] The expression “aryl-(C1-Cx)alkyl” designates an aryl group as defined above, linked to the rest of the molecule via a (Cl-Cx)alkyl chain as defined above with x an integer, such as for example the benzyl or phenylethyl group.

[0084] The term "polyvalent group" means a group that can form multiple covalent bonds with other groups in the same compound or in two different compounds. The bonds to the other groups can be formed from the same atom of the polyvalent group or from different atoms of the polyvalent group, and preferably from different atoms of the polyvalent group. In particular, the polyvalent group is a divalent group and can therefore form two covalent bonds with two other groups in the same compound or in two different compounds. The number of covalent bonds that can be formed is referred to as the "valence" of the polyvalent group.The expression "partly concomitant" as used in expressions of the type "steps b) and c) are partly concomitant" means that the two steps are carried out partly, at the same time, under the same reaction conditions, but that at least one of the two steps is initiated or completed under reaction conditions different from the common reaction conditions.

[0085] The expressions "between X and XX" or "from X to XX", X and XX representing a numerical value, are synonyms used equivalently. In all cases, the values ​​of the limits are included in the range considered.

[0086] DETAILED DESCRIPTION OF THE INVENTION

[0087] The inventors have developed a process for preparing a polysaccharide-based hydrogel that meets the expressed needs.

[0088] The proposed preparation method makes it possible to obtain a hydrogel based on crosslinked polysaccharides, advantageously weakly crosslinked, in particular crosslinked hyaluronic acid, advantageously weakly crosslinked, which have mechanical properties suitable for use in filling soft tissues.

[0089] The proposed method comprises the following steps: a) providing at least one polysaccharide; b) providing at least one crosslinking agent and / or at least one functionalizing agent, the functionalizing agent enabling crosslinking of the polysaccharide by sol-gel reaction; c) preparing a reaction medium comprising a solvent, the polysaccharide(s) and the crosslinking agent(s) and / or functionalizing agent(s); d) crosslinking the polysaccharide: d1) by reaction of the polysaccharide with the crosslinking agent(s); or d2) by sol-gel reaction of the functionalized polysaccharide, the functionalized polysaccharide being obtained by reaction of the polysaccharide with the functionalizing agent(s);wherein the crosslinking of the polysaccharide according to d1) or d2) is carried out under conditions which do not allow the sublimation of water, at a pressure P and at a temperature T higher than the temperature of the eutectic point of the reaction medium as measured at pressure P and lower than the temperature of the freezing point of the reaction medium as measured at pressure P.;

[0090] In other words, at temperature T, the reaction medium is in frozen form. Crosslinking the polysaccharide in the frozen state allows the polysaccharide chains to be preserved, even in acidic or basic environments, thus allowing for an extension of the reaction time. This extension of the reaction time makes it possible to reduce the quantity of crosslinking and / or modifying agent used and thus to obtain increasingly natural products.

[0091] Furthermore, carrying out the crosslinking step under conditions that do not allow the sublimation of water surprisingly makes it possible to avoid the formation of a gel comprising inhomogeneous, white and brittle crosslinked zones with a low swelling capacity, which may appear when the polysaccharide is kept in a frozen state for a prolonged period of time, of at least 1 hour, preferably at least 3 hours, preferably at least 72 hours. For example, said period may range from 2 to 27 weeks, preferably from 2 to 20 weeks, preferentially from 2 to 17 weeks, even more preferably from 3 to 8 weeks, even more preferably from 4 to 7 weeks. Gels having such inhomogeneous, white and brittle zones are unsuitable for filling soft tissues.

[0092] The hydrogels obtained by the process of the present invention are biocompatible and advantageously have a sticky character allowing the gel to adhere to the tissues so as not to migrate. The present invention also relates to a hydrogel capable of being obtained by the process according to the invention.

[0093] The present invention also relates to a composition comprising a hydrogel according to the invention, as well as the therapeutic, cosmetic or aesthetic applications of the hydrogels or compositions according to the invention.

[0094] PROCESS

[0095] The steps of the method of the present invention may be as described below.

[0096] Provision of at least one

[0097] Step a) of the method according to the invention consists of providing at least one polysaccharide. The polysaccharide may be in salt form.

[0098] Polysaccharide can be any polymer composed of monosaccharides joined together by glycosidic bonds.

[0099] Preferably, the polysaccharide is selected from pectin and pectic substances; chitosan; chitin; cellulose and its derivatives; agarose; glycosaminoglycans such as hyaluronic acid, heparosan, dermatan sulfate, keratan sulfate, chondroitin and chondroitin sulfate; and mixtures thereof. “Pectic substances”, including “pectin”, are polysaccharides composed of a skeleton of D-galacturonic acid in acid form possibly esterified by methanol, and L-rhamnose capable of forming branches with other oses.

[0100] Chitosan and chitin are each a polysaccharide composed of repeating D-glucosamine units linked together in a B-(1,4) manner, a portion of which is N-acetylated. Chitosan specifically has an acetylation degree of less than 50%, while chitin specifically has an acetylation degree of greater than 50%. Cellulose is a polysaccharide composed of a linear chain of D-glucose molecules.

[0101] "Cellulose derivatives" include methylcellulose, ethylcellulose, ethylmethylcellulose, hydroxypropylmethylcellulose (HPMC), hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), and carboxymethylcellulose (CMC). "Agarose" is a polysaccharide comprising as a repeating unit a disaccharide of D-galactose and 3,6-anhydro-L-galactopyranose.

[0102] Glycosaminoglycans are linear polysaccharides composed of repeating disaccharide units, said disaccharides containing a hexosamine (glucosamine (GIcN) or galactosamine (GaIN)) and another ose (glucuronic acid (GIcA), iduronic acid (IdoA) or galactose (Gai)). The hexosamine and the other ose may optionally be sulfated and / or acetylated. The glycosaminoglycan may be, in particular, hyaluronic acid, heparosan, dermatan sulfate, keratan sulfate, chondroitin or chondroitin sulfate.

[0103] "Hyaluronic acid" is a glycosaminoglycan whose repeating unit is a disaccharide composed of D-glucuronic acid and N-acetyl-D-glucosamine, linked together by alternating glycosidic bonds |3-(1,4) and |3-(1,3). When hyaluronic acid is in the form of a salt, it is also referred to as "hyaluronate" or "hyaluronan". In the context of the present invention, hyaluronic acid may have a weight-average molecular mass of between 0.5 and 10 MDa, preferably between 0.5 and 5 MDa, preferably between 0.5 and 4 MDa, preferably between 0.5 and 3 MDa, preferably between 1 and 5 MDa, preferably between 1 and 4 MDa, preferably between 1 and 3 MDa. Hyaluronic acid may be in salt form, particularly in physiologically acceptable salt form such as sodium salt, potassium salt, zinc salt, calcium salt, magnesium salt, silver salt, calcium salt and mixtures thereof.More specifically, hyaluronic acid is in acid form or in sodium salt form (NaHA).

[0104] "Heparosan" or "heparosan" is a glycosaminoglycan whose repeating unit is a disaccharide composed of glucuronic acid (GIcA) linked by an α-(1,4) bond to an N-acetyl glucosamine (GIcNAc). Each disaccharide repeating unit is linked to the next by a α-(1,4) bond.

[0105] Chondroitin sulfate or chondroitin sulfate is a glycosaminoglycan whose repeating unit is a disaccharide composed of glucuronic acid linked in P(1,3) to sulfated N-acetyl galactosamine, i.e. it includes at least one sulfate substituent. Each disaccharide repeating unit is linked to the next by a P-(1,4) bond.

[0106] "Dermatan sulfate" or "dermatan sulfate" is a glycosaminoglycan whose repeating unit is a sulfated disaccharide, i.e. comprising at least one sulfate substituent, of L-iduronic acid and N-acetyl-galactosamine- linked by a(1-3) bonds. Advantageously, the disaccharide is sulfated at the C-4 position of N-acetyl-galactosamine, at the C-6 position of N-acetyl-galactosamine, at the C-2 position of L-iduronic acid, or at a combination of these positions. Each disaccharide repeating unit is linked to the next by a |3-(1,4) bond.

[0107] "Keratan sulfate" or "keratan sulfate" is a glycosaminoglycan whose repeating unit is a sulfated disaccharide, i.e. comprising at least one sulfate substituent, composed of D-galactose and N-acetylglucosamine linked by alternating P(1-4) and P(1-3) bonds.

[0108] The polysaccharide may be in the form of a salt, in particular in the form of a physiologically acceptable salt such as sodium salt, potassium salt, zinc salt, calcium salt, magnesium salt, silver salt and mixtures thereof, more particularly in the form of sodium or potassium salt.

[0109] Advantageously, the polysaccharide is a glycosaminoglycan or a salt thereof, preferably hyaluronic acid or a salt thereof, more preferably hyaluronic acid or one of its physiologically acceptable salts such as the sodium salt, the potassium salt, the zinc salt, the silver salt and mixtures thereof, even more preferably hyaluronic acid or its sodium salt.

[0110] The polysaccharide generally has a weight average molecular mass ranging from 0.03 to 10 MDa.

[0111] Preferably, if the polysaccharide is hyaluronic acid, it has a weight average molecular weight (Mw) ranging from 0.5 to 10 MDa, preferably from 0.5 to 5 MDa, preferably from 0.5 to 4 MDa, preferably from 0.5 to 3 MDa, preferably from 1 to 5 MDa, preferably from 1 to 4 MDa, preferably from 1 to 3 MDa.

[0112] The polysaccharide may be supplied in hydrated form, wholly or partially, or in dry form, such as powder or fiber.

[0113] In some embodiments, in step a), the polysaccharide is provided in dry form such as powder or fiber.

[0114] When the polysaccharide is provided in hydrated form, it is in the form of a non-crosslinked gel or a solution. In particular, when the polysaccharide is in hydrated form, it is an aqueous non-crosslinked gel or an aqueous solution. More particularly, the polysaccharide is mixed with water, optionally with the addition of a phosphate buffer or a supplemented phosphate buffer, i.e. possibly comprising additional components as described in the "optional steps" section. Provision of at least one crosslinking agent and / or at least one functionalizing agent (Step b))

[0115] Step b) of the method according to the invention consists of providing at least one crosslinking agent and / or at least one functionalizing agent, the functionalizing agent allowing crosslinking of the polysaccharide by sol-gel reaction.

[0116] The crosslinking agent and the functionalizing agent are as described above.

[0117] Crosslinking agent

[0118] The “crosslinking agent”, still commonly and interchangeably referred to as a “crosslinking agent”, is typically a compound comprising at least two functional groups capable of covalently bonding with functional groups present on the polysaccharide, such as OH, CHO, NH2 or COOH groups carried by the polysaccharide, and thus inducing bonds between the polysaccharide chains (crosslinking) and / or bonds on the same polysaccharide chain.

[0119] The crosslinking agent may be in salt form, particularly in the form of a physiologically acceptable salt.

[0120] The crosslinking agent useful in the context of the present invention comprises at least two, preferably from 2 to 8, in particular 2, functional groups (designated "Z group") preferably chosen independently from the isocyanate (-N=C=O), amino (-NH2), epoxide, carboxyl (-COOH), N-succinimidyloxycarbonyl, N-sulfosuccinimidyloxycarbonyl, halocarbonyl, isothiocyanate (-N=C=S), vinyl (-CH=CH2), formyl (-CH=O), hydroxyl (-OH), sulfhydryl (-SH), hydrazino (-NH-NH2), acylhydrazino (-CO-NH-NH2), aminoxy (-O-NH2), carbodiimide groups, and an acid anhydride residue. Preferably, the functional groups are identical.

[0121] The isocyanate group can react with an OH or NH2 group of the polysaccharide to form a carbamate or urea function. The amino group can react with a COOH group of the polysaccharide to form an amide function. The epoxide group can react with an OH or COOH group of the polysaccharide to form an ether or ester function. The carboxyl group can react with an OH or NH2 group of the polysaccharide to form an ester or amide function. The N-succinimidyloxycarbonyl and N-sulfosuccinimidyloxycarbonyl groups can react with an OH or NH2 group of the polysaccharide to form an ester or amide function. The halocarbonyl group can react with an OH or NH2 group of the polysaccharide to form an ester or amide function. The isothiocyanate group can react with an OH or NH2 group of the polysaccharide to form a thiocarbamate or thiourea function.The vinyl group can react with an OH group of the polysaccharide to form an ether function. The formyl group can react with an OH or NH2 group of the polysaccharide to form a hemiacetal or hemiaminal function. The hydroxyl group can react with a COOH group of the polysaccharide to form an ester function. The sulfhydryl group can react with a COOH group of the polysaccharide to form a thioester function. The hydrazino group (-NH-NH2) can react with a CHO group of the polysaccharide to form a hydrazone function. The acylhydrazino group can react with a CHO group of the polysaccharide to form a carbonylated hydrazone function =NNHC(O)-. The aminoxy group can react with a CHO group of the polysaccharide to form an oxime function =NO-. The carbodiimide group can react with a COOH group of the polysaccharide to give a CO-NR function. a- CO-NH, and an acid anhydride residue can react with an OH or NH2 group of the polysaccharide to form an ester or amide function.

[0122] Preferably, the functional groups Z are identical and represent an epoxide or vinyl group, more preferably epoxide.

[0123] According to another advantageous embodiment, the functional groups Z are identical and chosen from amino, vinyl, formyl, and carbodiimide groups, preferably are amino groups.

[0124] In particular, the crosslinking agent is selected from hexamethylene diisocyanate, 4,4'-diphenylmethylene diisocyanate, PEG20K-4-arm isocyanate, spermine (or 1,12-diamino-5,9-diazadodecane), spermidine (or 1,8-diamino-5-azaoctane), cadaverine (or 1,5-diaminopentane), putrescine (or 1,4-diaminobutane), poly(ethylene glycol) diamine, ethylenediamine, 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxy-octane, poly(ethylene glycol) diglycidyl ether (PEGDGE), 1,2-bis(2,3-epoxypropoxy)ethane (EGDGE), 1 ,3-bis(3-glycidyloxypropyl)tetramethyldisiloxane, poly(dimethylsiloxane) terminated at each end with a diglycidyl ether (CAS number: 130167-23-6), poly(ethylene glycol) diacid, disuccinimidyl suberate, bis(sulfosuccinimidyl)suberate, sebacoyl chloride, 1,4-butane diisothiocyanate, divinylsulfone (DVS), glutaraldehyde, polyethylene glycol, 1,5-pentanedithiol, adipic acid dihydrazide,bis-aminooxy-poly(ethylene glycol), diethylenetriaminepentaacetic acid dianhydride, and mixtures thereof.,

[0125] When the functional groups Z are epoxide groups, the crosslinking agent is preferably chosen from 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxy-octane, poly(ethylene glycol) diglycidyl ether (PEGDGE), 1,2-bis(2,3-epoxypropoxy)ethane (EGDGE), 1,3-bis(3-glycidyloxypropyl)tetramethyldisiloxane, poly(dimethylsiloxane) terminated at each end by a diglycidyl ether (CAS number: 130167-23-6), hydroxyapatite beads modified to carry epoxy groups and mixtures thereof.

[0126] More preferably, the crosslinking agent is chosen from 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxy-octane, poly(ethylene glycol) diglycidyl ether (PEGDGE), 1,2-bis(2,3-epoxypropoxy)ethane (EGDGE), and mixtures thereof. More preferably, the crosslinking agent is 1,4-butanediol diglycidyl ether (BDDE). When the functional groups Z are amino groups, the crosslinking agent is preferably a polyamine chosen from spermine (or 1,12-diamino-5,9-diazadodecane), spermidine (or 1,8-diamino-5-azaoctane), cadaverine (or 1,5-diaminopentane), putrescine (or 1,4-diaminobutane), their salts or a mixture thereof, more preferably the crosslinking agent is a polyamine chosen from spermine, spermidine, their salts and their mixtures.

[0127] The crosslinking agent may be selected from hydroxyapatite beads modified to carry epoxy groups, a compound of formula Chem. I as described below, and mixtures thereof.

[0128] Preferably, the crosslinking agent is a compound of formula Chem. I:

[0129] Y-(Z) n in which the functional groups Z, identical or different, are as defined above, n is an integer greater than or equal to 2, in particular ranging from 2 to 8, preferably equal to 2,

[0130] Y is a polyvalent hydrocarbon group, notably aliphatic, having a valence of n and containing from 1 to 150 carbon atoms:

[0131] - in which one or more (for example 1 to 150, or 1 to 50 or 1 to 15 or 1 or 2) CH2 units are optionally replaced by one or more divalent units chosen from arylenes; -O-; -S-; -S(O)-; -C(=O)-; -SO2-; -N(R 1 )- ; and -[SiR2 R 3 O] m -SiR 2 R 3 - with: R 1 representing a hydrogen atom, an aliphatic hydrocarbon group containing from 1 to 6 carbon atoms, or an aryl-(C1-C6)alkyl; m an integer between 1 and 20 and the R 2 and the Rs 3 , identical or different, representing a hydrogen atom; a halogen atom; a -OR group 11 with R 11 representing a hydrogen atom, an aryl group or an aliphatic hydrocarbon group containing from 1 to 6 carbon atoms; an aryl; or an aliphatic hydrocarbon group containing from 1 to 6 carbon atoms optionally substituted by one or more groups chosen from a halogen atom, an aryl or a hydroxyl,

[0132] - said polyvalent group being unsubstituted or substituted by one or more monovalent groups chosen from a halogen atom, a hydroxyl, and an aryl-(C1-C6)alkyl, preferably unsubstituted.

[0133] In particular n is an integer ranging from 2 to 8, preferably n represents 2, 3 or 4, even more preferably n is equal to 2.

[0134] Advantageously, R 1 represents a hydrogen atom or a (C1-C6)alkyl group.

[0135] In particular, the R 2 and the Rs 3 , identical or different, represent an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms, more particularly a (C1-C6)alkyl group.

[0136] Preferably, in the definition of Y, the polyvalent hydrocarbon group may be a polyvalent aliphatic or aromatic hydrocarbon group, preferably aliphatic and in particular saturated, having a valence of n and comprising from 1 to 150 carbon atoms, preferably from 1 to 50 carbon atoms, more preferably from 1 to 20 carbon atoms, even more preferably from 2 to 20 carbon atoms.

[0137] In particular, in the definition of Y, the polyvalent hydrocarbon group is a polyvalent aliphatic, saturated, in particular linear, hydrocarbon group.

[0138] Preferably, Y is a polyvalent hydrocarbon group as described above in which one or more CH2 units are optionally replaced by one or more divalent units selected from -O-, -SO2-, -[SiR 2 R 3 O] m -SiR 2 R 3 - and -NH-, with R 2 , R 3 and m as described above.

[0139] In particular, Y is a polyvalent hydrocarbon group as described above, preferably aliphatic and saturated, and in particular linear, branched, or star-shaped, and optionally in which:

[0140] - at least two CH2 units are replaced by -O-, particularly between 1 and 50 CH2 units, more particularly between 1 and 15 CH2 units, or - at least one, preferably one or two, CH2 units is replaced by an -NH- unit, or

[0141] - at least one, preferably one, CH2 unit is replaced by an -SO2- unit, or

[0142] - at least two, preferably two, CH2 units are replaced by -O- and at least one, preferably one, CH2 unit is replaced by a -[SiR 2 R 3 O] m -SiR 2 R 3 - with R 2 , R 3 and m as described above.

[0143] More particularly, when one or more CH2 units are replaced by -O-, the replaced unit(s) are such that Y comprises one or more -CH2-CH2-O- units. In particular, Y comprises from 1 to 50 -CH2-CH2-O- units, advantageously from 2 to 25 -CH2-CH2-O- units, more advantageously from 2 to 15 -CH2-CH2-O- units. Y may comprise only -CH2-CH2-O- units.

[0144] More preferably, Y is an alkyl group comprising 1 to 150, in particular 1 to 50, in particular 1 to 20, for example 1 to 12, in particular 1 to 6 carbon atoms, preferably linear, in which optionally one or more CH2 units are replaced by one or more divalent units chosen from -O- and -NH-, more particularly between 1 and 50, in particular between 1 and 15, for example 1 or 2, divalent units chosen from -O- and -NH-.

[0145] According to a first embodiment, the R 2 and the Rs 3, identical or different, represent a -OR group 11 with R 11 as described above. In particular, R 11 represents an aliphatic hydrocarbon group containing from 1 to 6 carbon atoms, more particularly a (C1-C6)alkyl group.

[0146] According to a second embodiment, the R 2 and the Rs 3 , identical or different, represent an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms optionally substituted (preferably unsubstituted) by one or more groups chosen from a halogen atom, an aryl or a hydroxyl, more preferably an unsubstituted (C1-C6)alkyl group such as a methyl or an ethyl. Advantageously, the crosslinking agent is a compound of Chem formula, the following: Z 1 -Y 1 -Z 2 in which the Z groups 1 and Z 2, identical or different, are chosen from isocyanate, amino, epoxide, carboxyl, N-succinimidyloxycarbonyl, N-sulfosuccinimidyloxycarbonyl, halocarbonyl, isothiocyanate, vinyl, formyl, hydroxyl, sulfhydryl, hydrazino, acylhydrazino, aminoxy, carbodiimide groups, and an acid anhydride residue, and Y 1 represents a divalent hydrocarbon chain, in particular aliphatic, comprising from 1 to 50 carbon atoms: - in which one or more (e.g. 1 to 15 or even 1 or 2) CH2 units are optionally replaced by one or more divalent units chosen from arylenes, -O-, -S-, -S(O)-, -C(=O)-, -SO2-, -N(R 1 )- , and -[SiR 2 R 3 O]m-SiR 2 R 3 - with

[0147] R 1 representing a hydrogen atom, an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms, or an aryl-(C1-C6)alkyl, m an integer between 2 and 20, and the R 2and the Rs 3 , identical or different, representing a hydrogen atom; a halogen atom; a -OR group 11 with R 11 representing a hydrogen atom, an aryl group or an aliphatic hydrocarbon group containing from 1 to 6 carbon atoms; an aryl; or an aliphatic hydrocarbon group containing from 1 to 6 carbon atoms optionally substituted by one or more groups chosen from a halogen atom, an aryl or a hydroxyl,

[0148] - said chain being unsubstituted or substituted by one or more monovalent groups chosen from a halogen atom, a hydroxyl, an aryl-(C1-C6)alkyl.

[0149] Z groups 1 and Z 2 have the same definition as group Z defined above.

[0150] Y 1 has the same definition as Y defined above with valence n being equal to 2. In particular Y 1may comprise only -CH2-CH2-O- units, as defined above.

[0151] Preferably, the crosslinking agent of formula Chem. I or Chem. Ia does not comprise -[SiR 2 R 3 O] m -SiR 2 R 3 -.

[0152] Functionalizing agent

[0153] The functionalizing agent enables crosslinking of the polysaccharide by sol-gel reaction. Thus, the functionalizing agent typically comprises a single function capable of reacting with a functional group of the polysaccharide and comprises a silylated group capable of reacting with another silylated group via a sol-gel reaction so as to enable crosslinking of the polysaccharide and form a hydrogel.

[0154] The functionalizing agent is typically a molecule with the formula Chem. It as presented below:

[0155] Chem. It or a salt thereof, in which:

[0156] T represents an isocyanate, amino, epoxide, carboxyl, N-succinimidyloxycarbonyl, N-sulfosuccinimidyloxycarbonyl, halocarbonyl, isothiocyanate, vinyl, formyl, hydroxyl, sulfhydryl, hydrazino, acylhydrazino, aminoxy, carbodiimide group, or an acid anhydride residue;

[0157] A represents a chemical bond or a spacer group;

[0158] R 5 and R 6 , identical or different, represent a hydrogen atom; a halogen atom; a -OR group 4 with R 4 representing a hydrogen atom, an aryl group or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms; an aryl; or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms optionally substituted by one or more group(s) chosen from a halogen atom, an aryl and a hydroxyl;

[0159] R 10represents a hydrogen atom, an aryl group or an aliphatic hydrocarbon group containing from 1 to 6 carbon atoms.

[0160] Preferably, in the formula Chem. II, T represents an isocyanate, sulfhydryl, amino, epoxide, vinyl, formyl, or carbodiimide group, more preferably, T represents an epoxide or amino group, even more preferably T represents an epoxide group.

[0161] Preferably, in the formula Chem. II, A represents a spacer group, more preferably a divalent aliphatic hydrocarbon chain, in particular linear or branched and saturated, comprising from 1 to 12 carbon atoms:

[0162] - in which are optionally intercalated, between two carbon atoms of said chain, one or more (in particular 1, 2, 3 or 4) divalent units chosen from arylenes, -O-, -S-, -S(O)-, -C(=O)-, -SO2- and -N(R 9 )- with R 9representing a hydrogen atom, an aliphatic hydrocarbon group containing from 1 to 6 carbon atoms, or an aryl-(C1-C6)alkyl,

[0163] - said chain being unsubstituted or substituted by one or more monovalent groups chosen from a halogen atom, a hydroxyl, an aryl-(C1-C6)alkyl. Advantageously, A is a divalent aliphatic hydrocarbon chain, in particular linear or branched and saturated, in which are optionally intercalated, between two carbon atoms of said chain, one or more divalent units -O-, more advantageously from 1 to 4 divalent units -O-, even more advantageously a divalent unit O. Preferably, A is a (C1-C12)alkylene chain in which are optionally intercalated, between two carbon atoms of said chain, one or more divalent units -O-, more preferably from 1 to 4 divalent units -O-, even more preferably a divalent unit -O-.

[0164] In particular, A represents a divalent chain -(C1-C6)alkylene-O-(C1-C6)alkylene-, in particular -(C1-C4)alkylene-O-(C1-C4)alkylene-, more particularly a divalent chain -CH2-O-(CH2)3-, the CH2 group being linked to T and the (CH2)3 group being linked to Si in the molecule of formula Chem. II.

[0165] Advantageously, the spacer group will also avoid steric hindrance between the silylated group and the T group of the molecule of formula Chem. Il, while ensuring a stable bond between these two groups.

[0166] Preferably, in the formula Chem. II, R 5 and R 6 , identical or different, represent a -OR group 4 with R 4representing a hydrogen atom, an aryl group or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms; or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms optionally substituted by one or more groups chosen from a halogen atom, an aryl and a hydroxyl.

[0167] In particular, R 5 and R 6 , identical or different, represent a -OR group 4 with R 4 representing a (C1-C6)alkyl group; or a (C1-C6)alkyl group.

[0168] Advantageously, R 5 and R 6 , identical or different, represent a -OR group 4 with R 4 representing a hydrogen atom, an aryl group or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms, preferably with R 4 representing an aliphatic hydrocarbon group having from 1 to 6 carbon atoms, such as a (C1-C6)alkyl group.

[0169] Preferably, in the formula Chem. II, R 10 represents a hydrogen atom or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms such as a (C1-C6)alkyl group, more advantageously R 10 represents an aliphatic hydrocarbon group having from 1 to 6 carbon atoms such as a (C1-C6)alkyl group.

[0170] Preferably, the molecule of formula Chem. It is such that:

[0171] - T is as defined above and advantageously represents an amino or epoxide group, preferably an epoxide group;

[0172] - A is a divalent chain -(C1-C6)alkylene-O-((C1-C6)alkylene-, in particular -(C1-C4)alkylene-O-(C1-C4)alkylene-, such as -CH2-O-(CH2)3-, the CH2 group preferably being linked to T and the (CH2)3 group being linked to Si in the molecule of formula Chem. II; - R 5 and R 6 , identical or different, are each a -OR grouping 4with R 4 representing a (C1-C6)alkyl group, preferably a methyl or an ethyl; or a (C1-C6)alkyl group, preferably a methyl or an ethyl; and

[0173] - R 10 is a (C1-C6)alkyl group, preferably methyl or ethyl; the R groups 5 , R 6 and GOLD 10 which may be identical.

[0174] In particular, the molecule of formula Chem. It is chosen from (3-aminopropyl)triethoxysilane (APTES), (3-glycidyloxypropyl)trimethoxysilane (GPTMS), 3-Glycidoxypropyldimethoxymethylsilane, 3- Glycidoxypropyldimethylethoxysilane, (3-glycidyloxypropyl)ethoxydimethoxysilane, (3-glycidyloxypropyl)triethoxysilane, diethoxy(3-glycidyloxypropyl)methylsilane, and mixtures thereof; preferably from (3-glycidyloxypropyl)trimethoxysilane (GPTMS), (3-glycidyloxypropyl)ethoxydimethoxysilane, (3-glycidyloxypropyl)triethoxysilane, diethoxy(3-glycidyloxypropyl)methylsilane, and mixtures thereof; even more preferably (3-aminopropyl)triethoxysilane (APTES), (3-glycidyloxypropyl)trimethoxysilane (GPTMS) and mixtures thereof.

[0175] Preparation of the reaction medium (Step c))

[0176] Step c) of the process according to the invention comprises the preparation of a reaction medium comprising a solvent, the polysaccharide(s) and the crosslinking agent(s) and / or functionalizing agent(s).

[0177] The solvent is typically water or a mixture comprising water and an organic solvent (typically a mixture comprising at least 90% by weight of water, or at least 95% or at least 99% by weight of water relative to the total weight of the solvent). For example, an organic solvent such as an alcohol, in particular ethanol, or DMSO, may be used to solubilize the crosslinking agent, for example when it is poly(dimethylsiloxane) terminated at each end by a diglycidyl ether (CAS number: 130167-23-6), before its addition to form the reaction medium.

[0178] The reaction medium may further comprise salts, pH adjusters, for example a Bronsted base, more preferably a hydroxide salt, such as sodium or potassium hydroxide, additional components as described above and mixtures thereof.

[0179] The addition of a Bronsted base may be particularly necessary when the Z functional groups of the crosslinking agent, such as Z 1 or Z 2 , represent an epoxide group or a vinyl group. In these cases, crosslinking generally takes place at a pH greater than or equal to 10, more advantageously greater than or equal to 12, which requires the addition of a Bronsted base to the reaction medium, typically at a concentration between 0.10M and 0.30M.

[0180] The total amount of crosslinking agent in the reaction medium typically varies from 0.001 to 0.15 mole per 1 mole of repeating unit of the polysaccharide, preferably from 0.001 to 0.08 mole per 1 mole of repeating unit of the polysaccharide, more preferably from 0.001 to 0.05 mole per 1 mole of repeating unit of the polysaccharide, even more preferably from 0.001 to 0.03 mole per 1 mole of repeating unit of the polysaccharide.

[0181] In particular, the total amount of crosslinking agent in the reaction medium varies from 0.001 to 0.02 mole per 1 mole of repeating unit of the polysaccharide, preferably from 0.001 to 0.015 mole, more preferably from 0.001 to 0.01 mole per 1 mole of repeating unit of the polysaccharide, even more preferably from 0.001 to 0.008 mole or from 0.001 to 0.005 mole or from 0.001 to 0.004 per 1 mole of repeating unit of the polysaccharide. When the polysaccharide is a glycosaminoglycan such as hyaluronic acid, the repeating unit is a disaccharide unit.

[0182] The total amount of functionalizing agent in the reaction medium typically varies from 0.01 to 0.50, preferably from 0.05 to 0.45, in particular from 0.10 to 0.25 mole per 1 mole of repeating unit of the polysaccharide. For example, the total amount of formula Chem. II or a salt thereof in the reaction medium typically varies from 0.01 to 0.50, preferably from 0.05 to 0.45, in particular from 0.10 to 0.25 mole per 1 mole of repeating unit of the polysaccharide.

[0183] Typically, the higher the weight-average molecular mass Mw of the polysaccharide, the lower the functionalization rate will be in order to obtain a hydrogel having equivalent mechanical properties, in particular similar viscoelastic properties (in particular elastic modulus G', stress at the intersection of G' and G” and / or phase angle 5). In other words, the higher the weight-average molecular mass Mw of the polysaccharide, the lower the molar quantity of functionalizing agent, for example in molecule of formula Chem. II, in the reaction medium will be.

[0184] The functionalization of the polysaccharide is typically carried out in an aqueous reaction medium.

[0185] The mass concentration of polysaccharide or polysaccharide salt in the reaction medium advantageously varies from 50 to 300 mg / g of solvent, preferably from 100 to 250 mg / g, more preferably from 100 to 200 mg / g. Tl

[0186] Step c) of the process according to the invention typically comprises a step of homogenization of the reaction medium. Homogenization is generally carried out by three-dimensional stirring, stirring with a mixer, stirring with blades or stirring with a spatula.

[0187] Step c) is typically carried out at a temperature ranging from 4 to 35°C, preferably ranging from 10°C to 30°C, more preferably ranging from 15°C to 25°C.

[0188] Step c) is typically carried out at atmospheric pressure.

[0189] In particular, step c) is carried out at atmospheric pressure and at a temperature ranging from 4 to 35°C, preferably ranging from 10°C to 30°C, more preferably ranging from 15°C to 25°C.

[0190] The duration of the reaction medium preparation step typically does not exceed 5 hours. It generally varies from 15 minutes to 4 hours, preferably from 30 minutes to 2 hours.

[0191] The reaction medium is typically prepared from the polysaccharide or polysaccharides in a dry form. When the reaction medium is prepared from the polysaccharide or polysaccharides in a hydrated form, the aqueous uncrosslinked gel or aqueous polysaccharide solution used for the preparation of the reaction medium typically does not comprise sodium hydroxide. Thus, the maximum contact time of the polysaccharide with sodium hydroxide before initiating step d), whether the polysaccharide is provided in a dry or hydrated form, is generally 5 hours, for example 15 minutes to 4 hours or 30 minutes to 2 hours.

[0192] Crosslinking of the polysaccharide (step d))

[0193] Step d) of the process according to the invention consists of the crosslinking of the polysaccharide(s).

[0194] Crosslinking can be carried out by reaction of the polysaccharide(s) with the crosslinking agent (step d1)) or by sol-gel reaction of the functionalized polysaccharide by reaction of the polysaccharide with the functionalizing agent (step d2)).

[0195] Crosslinking of the polysaccharide according to step d) (d1) or d2)) is carried out at pressure P.

[0196] The crosslinking of the polysaccharide according to d1) or d2) is carried out under conditions which do not allow the sublimation of water and at a temperature T higher than the temperature of the eutectic point of the reaction medium as measured at pressure P and lower than the temperature of the freezing point of the reaction medium as measured at pressure P.

[0197] The pressure P is advantageously equal to atmospheric pressure or less than atmospheric pressure.

[0198] The crosslinking of the polysaccharide according to step d) (d1) or d2)) can be carried out under vacuum, in particular at a pressure P lower than atmospheric pressure, preferably at a pressure P of between 0.7.10 5 and 0.9.10 5 Pa (between 0.7 and 0.9 bar), preferably between 0.7.10 5 and 0.8.10 5 Pa (between 0.7 and 0.8 bar).

[0199] The freezing point temperature of the reaction medium refers to the temperature at which, at the pressure P considered, the mixture of the components of the reaction medium, on a macroscopic scale, solidifies, that is to say it becomes non-fluid. Below the freezing point, the mixture is in a frozen state which is characterized by the coexistence of components in solid and liquid form. The frozen state is maintained up to the eutectic point temperature of the reaction medium.

[0200] The eutectic point temperature of the reaction medium refers to the temperature below which, at the pressure P considered, the mixture of the components of the reaction medium passes from a frozen state (coexistence of liquid and solid phases) to a completely solid state, i.e. a state in which all the components of the mixture are in solid form.

[0201] The freezing point and eutectic point of a mixture depend on the pressure to which the mixture is subjected, therefore the freezing point and eutectic point are measured at pressure P.

[0202] The freezing point and eutectic point can be determined by differential scanning calorimetry. This method allows phase transitions to be determined. To do this, the product to be studied is gradually cooled until its phase transitions are observed.

[0203] Using a device in which gas exchange is not possible allows the crosslinking of the polysaccharide according to d1) or d2) to be carried out under conditions which do not allow the sublimation of water. The method may comprise a step prior to step d) of degassing to remove air bubbles from the mixture once it is placed in the device.

[0204] In some embodiments, the crosslinking is thus carried out in a sealed flexible airtight container (for example a flexible airtight pouch) in which an air vacuum has been created. The crosslinking is then said to be carried out under vacuum or in a flexible airtight container under vacuum. Typically, when the crosslinking of the polysaccharide according to d1) or d2) is carried out under vacuum, the reaction medium is placed or prepared in the flexible airtight container. At least 90% by volume, preferably at least 95% by volume, even more preferably at least 99% by volume, of the air contained in the container comprising the reaction medium is then removed, for example by means of a vacuum sealer. Before the vacuuming and sealing step, the method may comprise a degassing step to remove air bubbles from the mixture. A flexible airtight pouch suitable for the method according to the invention is for example described in patent EP 2 429 486 B1.

[0205] In some embodiments, the crosslinking is thus carried out in a closed, hermetically sealed rigid container. In the case of a rigid, hermetically sealed container, sublimation of water is prevented by saturating any free volume left by the reaction medium in the container with water vapor or by ensuring that the reaction medium occupies at least 95% by volume, or at least 98% by volume, or at least 99% by volume of the volume of the container.

[0206] The airtight container is advantageously placed at a temperature ranging from -35°C to -10°C at atmospheric pressure, preferably at a temperature of approximately -20°C at atmospheric pressure.

[0207] Step d) is typically carried out at least partially (i.e. in part or in full), under conditions which do not allow the sublimation of water and at temperature T, at pressure P. Advantageously according to the invention, during step d), the mass variation of the hydrogel is zero or represents at most 1% by mass, preferably at most 0.5% by mass, more preferably at most 0.1% by mass, even more preferably at most 0.05% by mass relative to the total mass of the hydrogel resulting from step d). The mass variation of the hydrogel can be verified by weighing or by gravimetric analysis. A zero or almost zero mass variation of the hydrogel makes it possible to confirm that there has been no sublimation of water.

[0208] Step d) is typically carried out for a period of at least 1 hour, preferably at least 3 hours, preferably at least 72 hours, preferably at most 27 weeks. Preferably, step d) is carried out for a period of from 2 to 25 weeks, preferably from 2 to 20 weeks or 2 to 17 weeks, even more preferably from 3 to 8 weeks or 4 to 7 weeks under conditions not allowing the sublimation of water and at temperature T, at pressure P. The crosslinking of the polysaccharide by reaction of the polysaccharide with the crosslinking agent mainly takes place during step d) but it can nevertheless begin as early as step c).

[0209] Step d1) makes it possible to crosslink the polysaccharide chains together. The functional groups of the crosslinking agent react with functional groups present on the polysaccharides, in particular the functional groups naturally present on the polysaccharides, so as to link the polysaccharide chains together and crosslink them by forming intermolecular bonds. The crosslinking agent can also react with functional groups present on the same polysaccharide molecule so as to form intramolecular bonds. In particular, the functional groups of the crosslinking agent react with the -OH or -COOH groups, or even -CHO, naturally present on polysaccharides such as hyaluronic acid. Crosslinked polysaccharides comprising at least one crosslinking link between two polysaccharide chains, said crosslinking link being the residue of the crosslinking agent, are thus obtained.In particular, following step d), the crosslinked polysaccharides comprise at least one crosslinking link between two polysaccharide chains, said crosslinking link more particularly comprising the polyvalent group Y as described above, preferably the divalent group Y. 1 as described above. Certain functional groups Z (such as Z 1 and Z 2 ) of the crosslinking agent may however not react with a polysaccharide chain. In particular, when the crosslinking agent comprises two functional groups Z 1 and Z 2 , one of the Z functional groups 1 can react with a polysaccharide while the other functional group Z 2 does not react with any polysaccharide. A dangling bond is then formed.

[0210] Crosslinking may be carried out in the presence of several crosslinking agents. When crosslinking is carried out in the presence of several crosslinking agents, the crosslinking agents may be added simultaneously or separately over time to the reaction medium. Step d1) may thus comprise repeated crosslinking steps.

[0211] The crosslinking is then typically carried out in the presence of a total amount of crosslinking agents (or their salts) ranging from 0.001 to 0.15 moles per 1 mole of repeating unit of the polysaccharide, preferably from 0.001 to 0.08 moles per 1 mole of repeating unit of the polysaccharide, more preferably from 0.001 to 0.05 moles per 1 mole of repeating unit of the polysaccharide, even more preferably from 0.001 to 0.03 moles of crosslinking agents per 1 mole of repeating unit of the polysaccharide. In particular, the crosslinking is carried out in the presence of a total amount of crosslinking agents (or their salts) ranging from 0.001 to 0.02 mole per 1 mole of repeating unit of the polysaccharide, preferably from 0.001 to 0.015 mole, more preferably from 0.001 to 0.01 mole per 1 mole of repeating unit of the polysaccharide, even more preferably from 0.001 to 0.008 mole or from 0.001 to 0.005 mole or from 0.001 to 0.004 per 1 mole of repeating unit of the polysaccharide.The crosslinking conditions, in particular the crosslinking agent contents, duration and temperatures as well as the weight-average molecular masses (Mw) of the polysaccharide, used are interdependent.

[0212] In particular, the higher the temperature of the crosslinking reaction, the shorter the reaction time can be to obtain the same degree of modification of the polysaccharide by the crosslinking agent.

[0213] The lower the crosslinking agent content, the longer the reaction time must be to obtain similar mechanical properties of the resulting gel. In other words, the lower the molar percentage of crosslinking agent, the fewer reactive functions there are in the reaction medium and the lower the probability that 2 groups meet and react together, thus the longer the reaction time must be to allow the functions to react with each other and form crosslinking bonds, and thus obtain a gel with desirable properties.

[0214] Conversely, too much crosslinking agent and too long a crosslinking time will result in a brittle and too hard gel, therefore not suitable for filling and / or cosmetic applications.

[0215] The higher the weight-average molecular mass (Mw) of the polysaccharide, the lower the degree of modification required to obtain gels with given mechanical properties.

[0216] For the same molar percentage of crosslinking agent, the lower the weight-average molecular mass (Mw) of the polysaccharide, the longer the reaction time is required to obtain gels with given mechanical properties.

[0217] When the functional groups Z of the crosslinking agent are amino groups, the crosslinking reaction with the polysaccharide is advantageously carried out in the presence of at least one activator, and where appropriate associated with at least one coupling auxiliary.

[0218] In this regard, the activator may be selected from water-soluble carbodiimides such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 1-ethyl-3-[3-(trimethylamino)propyl]carbodiimide hydrochloride (ETC), 1-cyclohexyl-3-(2-morphilinoethyl)carbodiimide (CMC), their salts and mixtures thereof, preferably is represented by EDC.

[0219] With respect to the coupling aid, when present, it may be selected from N-hydroxy succinimide (NHS), N-hydroxybenzotriazole (HOBt), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazole (HOOBt), 1-hydroxy-7-azabenzotriazole (Hat) and N-hydroxysylfosuccinimide (sulfo NHS), and mixtures thereof, preferably is represented by HOBt.

[0220] In certain embodiments, during step d1) the reaction medium obtained at the end of step c) is placed, for a period of at least 1 hour, preferably at least 3 hours, preferably at least 72 hours, preferably at most 27 weeks in conditions which do not allow the sublimation of water (e.g., a flexible, vacuum-sealed container or a rigid, airtight container whose free volume is saturated with water vapor or in which the reaction medium occupies at least 95% of the volume) and at a temperature T higher than the temperature of the eutectic point of the reaction medium (i.e., of the mixture comprising the polysaccharide(s), the crosslinking agent(s), the solvent and any salts, pH adjusters and additional components) as measured at pressure P and lower than the freezing point temperature of the reaction medium as measured at pressure P.

[0221] In certain embodiments, during step d1) the reaction medium obtained at the end of step c) is placed, for a period ranging from 2 to 27 weeks, preferably ranging from 2 to 20 or 2 to 17 weeks, even more preferably from 3 to 8 weeks or from 4 to 7 weeks, in conditions which do not allow the sublimation of water (e.g.flexible vacuum-sealed container or rigid airtight container whose free volume is saturated with water vapor or in which the reaction medium occupies at least 95% of the volume) and at a temperature T higher than the temperature of the eutectic point of the reaction medium (i.e. of the mixture comprising the polysaccharide(s), the crosslinking agent(s), the solvent and any salts, pH adjusters and additional components) as measured at pressure P and lower than the freezing point temperature of the reaction medium as measured at pressure P The pressure P is advantageously lower than or equal to atmospheric pressure.

[0222] The crosslinking of the polysaccharide according to step d1) can be carried out under vacuum, in particular at a pressure P lower than atmospheric pressure, preferably at a pressure P of between 0.7.10 5 and 0.9.10 5Pa (between 0.7 and 0.9 bar), preferably between 0.7.10 5 and 0.8.10 5 Pa (between 0.7 and 0.8 bar).

[0223] In step d1), the reaction medium obtained at the end of step c) is advantageously placed in a hermetic flexible container. After a possible degassing step, the hermetic flexible container is placed under vacuum and advantageously sealed. This hermetic flexible container under vacuum is left at atmospheric pressure at a temperature greater than or equal to -55°C and less than or equal to -5°C, preferably at a temperature ranging from -35°C to -10°C, even more preferably, at a temperature of approximately -20°C.

[0224] Step d2) consists of crosslinking by sol-gel reaction of the functionalized polysaccharide by reaction of the polysaccharide with the functionalizing agent. In other words, step d2) therefore comprises a functionalization of the polysaccharide by reaction of the polysaccharide with the functionalizing agent and a sol-gel reaction of the functionalized polysaccharide. The functionalization of the polysaccharide and the sol-gel reaction may be sequential or at least partly concomitant.

[0225] Functionalization of the polysaccharide

[0226] The polysaccharide is typically functionalized with at least one molecule of formula Chem. It as presented above so as to become the bearer of Si-OR groups which will be able to react together and lead to a crosslinked polysaccharide. The molecule of formula Chem. It comprising a single reactive function with respect to the polysaccharide and allowing crosslinking only via a sol-gel reaction, it does not present the toxicity of conventional crosslinking agents: the molecule of formula Chem. It cannot directly crosslink biological molecules (proteins, DNA, etc.). More specifically, the functional group T of the molecule of formula Chem. It reacts with a functional group present on the polysaccharides so as to functionalize the polysaccharide chains. In particular, the functional group T of the molecule Chem.It thus reacts with a -OH or -COOH group, or a CHO function, present on polysaccharides such as hyaluronic acid. This produces functionalized polysaccharides comprising pendant links on a polysaccharide chain, said pendant links comprising a -A-Si(R) group. 5 )(R 6 )GOLD 10 , the group -A-Si(R 5 )(R 6 )GOLD 10 from the molecule of formula Chem. It can provide biological properties to the hydrogel. The solvent is typically water or a mixture comprising water and an organic solvent (e.g. an alcohol, in particular ethanol, or DMSO; typically a mixture comprising at least 90% by weight of water, or at least 95% or at least 99% by weight of water relative to the total weight of the solvent).

[0227] The reaction medium typically comprises from 0.01 to 0.50, preferably from 0.05 to 0.45, in particular from 0.10 to 0.25 mole of molecule of formula Chem. Il or a salt thereof per 1 mole of repeating unit of the polysaccharide.

[0228] The mass concentration of polysaccharide in the reaction medium is advantageously between 50 and 300 mg / g of solvent, preferably between 100 and 200 mg / g.

[0229] In certain embodiments, in particular when T is an epoxide, the functionalization is carried out at a pH greater than or equal to 9, or greater than or equal to 10, more advantageously greater than or equal to 12, and in particular at a pH less than 14, for example less than or equal to 13.5. For this, the reaction medium preferably comprises a Bronsted base, more preferably a hydroxide, even more preferably sodium or potassium hydroxide. Advantageously, the reaction medium comprises sodium or potassium hydroxide at a concentration of between 0.10M and 0.30M.

[0230] In certain embodiments, in particular when T is an amino group, the functionalization is carried out at a pH of less than 7, more advantageously greater than or equal to 4.5 and less than 7 or less than or equal to 6.5. For this, the reaction medium preferably comprises a Bronsted acid, more preferably hydrochloric acid, sulfuric acid, or acetic acid.

[0231] In certain embodiments, the functionalization of the polysaccharide is carried out at atmospheric pressure and at a temperature between 4°C and 60°C, more preferably between 10°C and 50°C. In these embodiments, the duration of the functionalization reaction can vary from 1 hour to 2 weeks, more particularly from 3 hours to 1 week, even more particularly from 3 hours to 96 hours, for example from 3 hours to 80 hours, in particular from 3 hours to 75 hours.

[0232] In certain embodiments, in particular when the functionalization and the crosslinking of the polysaccharide are concomitant or partly concomitant, the functionalization of the polysaccharide can be, at least partly, carried out under conditions which do not allow the sublimation of water and at a temperature T higher than the temperature of the eutectic point of the reaction medium as measured at the pressure P and lower than the temperature of the freezing point of the reaction medium as measured at the pressure P. Here, the pressure P is advantageously atmospheric pressure.

[0233] The higher the functionalization temperature, the shorter the functionalization time can be to obtain the same degree of modification by functionalization.

[0234] Sol-gel reaction

[0235] The "sol-gel reaction" consists of forming Si-O-Si bonds from Si-OR groups with R representing a hydrogen atom, an aryl group or an aliphatic hydrocarbon group containing 1 to 6 carbon atoms. This reaction takes place as follows:

[0236] (i) if R is not a hydrogen atom, a step of hydrolysis of at least part of the Si-OR groups to give Si-OH groups; then

[0237] (ii) a step of condensation of the Si-OH groups two by two or of a Si-OH group with a Si-OR group to form Si-O-Si bonds.

[0238] The functionalized polysaccharide is crosslinked by sol-gel reaction to give a hydrogel.

[0239] This step allows the polysaccharide chains to be crosslinked together when they are functionalized with molecules of formula Chem. II. Indeed, during this step, at least part of the Si-OR groups 10and optionally at least part of the SiOR groups 4 will react two by two, possibly after hydrolysis of these groups, to form Si-O-Si bonds. This implies that two molecules of formula Chem. Il grafted onto polysaccharide chains will react together via their Si-OR terminal groups 10 (or even SiOR 4 where applicable) and bind covalently via the formation of Si-O-Si bonds, thus allowing the polysaccharide chains to be bound together and crosslinked.

[0240] Crosslinked polysaccharides are thus obtained comprising crosslinking links between two polysaccharide chains, said crosslinking links comprising a divalent group -Si-O-Si-.

[0241] Step d2) is carried out at least partially (i.e. in part or in full), under conditions which do not allow the sublimation of water and at a pressure P and at a temperature T higher than the temperature of the eutectic point of the reaction medium as measured at pressure P and lower than the temperature of the freezing point of the reaction medium as measured at pressure P.

[0242] The pressure P is advantageously less than or equal to atmospheric pressure. The crosslinking of the polysaccharide according to step d2) can be carried out under vacuum at a pressure P lower than atmospheric pressure, preferably at a pressure P of between 0.7.10 5 and 0.9.10 5 Pa (between 0.7 and 0.9 bar), preferably between 0.7.10 5 and 0.8.10 5 Pa (between 0.7 and 0.8 bar).

[0243] The pressure P is advantageously atmospheric pressure.

[0244] In some embodiments, the sol-gel reaction is carried out in a sealed, airtight container that may be rigid or flexible, as described previously.

[0245] Thus, during step d2), the reaction medium obtained at the end of step c) can advantageously be placed in a hermetic flexible container. After a possible degassing step, the hermetic flexible container is placed under vacuum and advantageously sealed. This hermetic flexible container under vacuum is left at atmospheric pressure at a temperature greater than or equal to -55°C and less than or equal to -5°C, preferably at a temperature ranging from -35°C to -10°C, even more preferably, at a temperature of approximately -20°C. The pressure P is then lower than atmospheric pressure, preferably at a pressure P of between 0.7.10 5 and 0.9.10 5 Pa (between 0.7 and 0.9 bar), preferably between 0.7.10 5 and 0.8.10 5Pa (between 0.7 and 0.8 bar).

[0246] The hermetic container is advantageously a rigid hermetic container. In the case of a rigid hermetic container, the sublimation of water is prevented by saturating any free volume left by the reaction medium in the container with water vapor or by ensuring that the reaction medium occupies at least 95% by volume, or at least 98% by volume, or at least 99% by volume of the volume of the container. The pressure P is advantageously atmospheric pressure.

[0247] Step d2) is typically carried out for a duration of at least 1 hour, preferably at least 3 hours, preferably at least 72 hours, preferably at most 27 weeks.

[0248] The duration of maintaining these conditions depends on the pH of the reaction medium. Thus, when the pH of the reaction medium is greater than or equal to 9, or greater than or equal to 10, and less than 14, the temperature T is maintained for a duration t ranging from at least 1 hour, preferably at least 3 hours, preferably at least 72 hours, preferably at most 27 weeks. Advantageously, the temperature T is maintained for a duration t ranging from 2 to 27 weeks, in particular from 2 to 20 weeks or from 2 to 17 weeks, for example from 3 to 8 weeks or from 4 to 7 weeks. When the pH of the reaction medium is greater than or equal to 6.8 and less than or equal to 7.8, the temperature T is maintained for a duration t ranging from 1 to 48 hours, preferably greater than or equal to 6 hours and less than or equal to 36 hours, in particular greater than or equal to 7 hours and less than or equal to 36 hours.It should be understood that the reaction conditions (pH, T, P, conditions not allowing the sublimation of water) set out above may correspond to the conditions applied throughout the duration of the crosslinking step (step d2)) or may correspond to the conditions applied only during part of the duration of step d2). In other words, the duration of the crosslinking step d2) may be greater than the durations t indicated above, the reaction conditions (pH, P or T) applied in the additional time then being different from those set out above.

[0249] The sol-gel reaction typically takes place in an aqueous reaction medium.

[0250] The mass concentration of polysaccharide in the reaction medium is advantageously between 50 and 300 mg / g of sol-gel reaction medium, preferably between 100 and 250 mg / g, more preferably between 100 and 200 mg / g.

[0251] Preferably, the pressure P is atmospheric pressure and the temperature T is greater than or equal to -55°C and less than or equal to -5°C, preferably it ranges from -35°C to -10°C, in particular from -30°C to -10°C or from -25°C to -15°C. Even more preferably, the temperature T is approximately -20°C at atmospheric pressure.

[0252] Advantageously, the reaction medium is placed and maintained at temperature T at atmospheric pressure by contact of the container comprising the reaction medium with air or a liquid L at temperature T. The liquid L may in particular be ethylene glycol, glycerol or an azeotropic mixture of these with water. The liquid L will be chosen according to the desired temperature T so as to be liquid at this temperature T at atmospheric pressure. More advantageously, the reaction medium is left at temperature T at atmospheric pressure by contact of the container comprising the reaction medium with air at temperature T.

[0253] Typically, the lower the temperature T, the longer the time t is required to obtain hydrogels with similar mechanical properties. Indeed, the lower the temperature T, the lower the kinetics of the sol-gel reaction.

[0254] Similarly, the lower the functionalization rate, the longer the time t is to obtain hydrogels with similar mechanical properties.

[0255] In other words, the lower the molar quantity of molecule of formula Chem. Il or a salt thereof per 1 mole of repeating unit of the polysaccharide, the fewer SiOH functions there are in the reaction medium and the lower the probability that 2 groups meet and react together, thus the longer the duration t must be to allow the Si-OH functions to react with each other and form crosslinks, and thus obtain a gel with desirable properties.

[0256] For the same molar quantity of molecule of formula Chem. Il or a salt thereof per 1 mole of repeating unit of the polysaccharide, the lower the weight-average molecular mass Mw of the polysaccharide, the longer the time t is to obtain hydrogels with similar mechanical properties.

[0257] When the crosslinking by sol-gel reaction at temperature T is carried out in a reaction medium at a pH greater than or equal to 9 or greater than or equal to 10 and less than 14, the reaction medium preferably comprises a Bronsted base, more preferably a hydroxide, even more preferably sodium or potassium hydroxide. Advantageously, the reaction medium comprises sodium or potassium hydroxide at a concentration of between 0.10M and 0.30M.

[0258] Preferably, at the end of the duration t (crosslinking carried out under conditions not allowing the sublimation of water and temperature T, at pressure P), the pH of the reaction medium is adjusted to a physiological pH, preferably to a pH of approximately 6.8 to 7.8. It should be understood that at the end of the duration t, the reaction medium is brought back to room temperature at atmospheric pressure.

[0259] When the crosslinking by sol-gel reaction at temperature T, at pressure P, under conditions which do not allow the sublimation of water (e.g. flexible hermetic container under vacuum or rigid hermetic container whose free volume is saturated with water vapor or in which the reaction medium occupies at least 95% of the volume) is carried out in a reaction medium at physiological pH (pH greater than or equal to 6.8 and less than or equal to 7.8) and the functionalization is carried out in a basic medium (pH greater than or equal to 9, or greater than or equal to 10, and less than 14), the pH of the reaction medium will be brought to a physiological pH before the temperature is brought to temperature T under conditions which do not allow the sublimation of water.In this case, the process will advantageously comprise a step of neutralizing the gel to reach this physiological pH, before the reaction medium is brought to temperature T under conditions which do not allow the sublimation of water. For this, a Bronsted acid is preferably added to the reaction medium, preferably an aqueous solution of hydrochloric acid, an aqueous solution of sulfuric acid or an aqueous solution of acetic acid. When crosslinking by sol-gel reaction at temperature T, at pressure P, under conditions which do not allow the sublimation of water (e.g.flexible vacuum-sealed container or rigid airtight container whose free volume is saturated with water vapor or in which the reaction medium occupies at least 95% of the volume) is carried out in a reaction medium at physiological pH (pH greater than or equal to 6.8 and less than or equal to 7.8) and the functionalization is carried out at a pH lower than 7, for example greater than or equal to 4.5 and less than 7 or less than or equal to 6.5, the pH of the reaction medium will be brought to a physiological pH before the temperature is brought to temperature T under conditions which do not allow the sublimation of water.

[0260] In step d2), the pressure P is advantageously atmospheric pressure.

[0261] Very generally, the process of the present invention comprises the concomitant or partly concomitant carrying out of steps c) and d2). Concomitant carrying out of steps c) and d2) makes it possible to shorten the duration of the process for preparing the hydrogel and to simplify it.

[0262] The method of the present invention then comprises steps a) to d2) as described above and is characterized in that steps c) and d2) are concomitant or partly concomitant. The method then comprises:

[0263] - functionalization and crosslinking by sol-gel reaction carried out in a reaction medium at a pH greater than or equal to 9, or greater than or equal to 10, and less than 14, under conditions which do not allow the sublimation of water (e.g.: rigid hermetic container whose free volume is saturated with water vapor or in which the reaction medium occupies at least 95% of the volume), at pressure P and at a temperature T greater than the temperature of the eutectic point of the reaction medium as measured at pressure P and less than the temperature of the freezing point of the reaction medium as measured at pressure P, for a duration t ranging from 2 weeks to 27 weeks, in particular from 2 to 20 weeks or from 2 to 17 weeks, for example from 3 to 8 weeks or 4 to 7 weeks, or

[0264] - functionalization and crosslinking by sol-gel reaction carried out in a reaction medium at a pH greater than or equal to 6.8 and less than or equal to 7.8 under conditions which do not allow the sublimation of water (e.g. rigid hermetic container whose free volume is saturated with water vapor or in which the reaction medium occupies at least 95% of the volume), at pressure P and at a temperature T greater than the temperature of the eutectic point of the reaction medium as measured at pressure P and less than the temperature of the freezing point of the reaction medium as measured at pressure P, for a duration t of between 1 h and 48 hours, preferably greater than or equal to 6 hours and less than or equal to 36 hours, in particular greater than or equal to 7 hours and less than or equal to 36 hours.

[0265] The pressure P is advantageously atmospheric pressure.

[0266] It should be understood from the above that steps c) and d) are at least partially (in part or in full) carried out under the conditions set out above (T, P, pH, conditions not allowing the sublimation of water, t).

[0267] In some embodiments, the functionalization of the polysaccharide and the crosslinking of the functionalized polysaccharide are then carried out in the following manner:

[0268] 1) preparation of a reaction medium comprising the polysaccharide(s), the molecule(s) of formula Chem. II and a solvent, the pH of the reaction medium being greater than or equal to 9, or greater than or equal to 10, and less than 14;

[0269] 2) optionally placing the reaction medium at a temperature ranging from 4°C to 60°C, preferably from 10°C to 50°C, at atmospheric pressure, typically for a period ranging from 1 hour to 2 weeks, more particularly from 3 hours to 1 week, for example from 3 hours to 80 hours, in particular from 3 hours to 75 hours;

[0270] 3) optionally adjusting the pH of the reaction medium to a pH greater than or equal to 6.8 and less than or equal to 7.8;

[0271] 4) placement of the reaction medium:

[0272] - under conditions that do not allow the sublimation of water (e.g.: rigid airtight container whose free volume is saturated with water vapor or in which the reaction medium occupies at least 95% of the volume), at atmospheric pressure, and at a temperature T higher than the temperature of the eutectic point of the reaction medium as measured at atmospheric pressure and lower than the temperature of the freezing point of the reaction medium as measured at atmospheric pressure, for a duration t ranging from 2 weeks to 27 weeks, when the pH of the reaction medium is greater than or equal to 9, or greater than or equal to 10, and less than 14, in particular from 2 to 20 weeks or from 2 to 17 weeks, for example from 3 to 8 weeks or 4 to 7 weeks, or

[0273] - under conditions which do not allow the sublimation of water (e.g.: rigid airtight container whose free volume is saturated with water vapour or in which the reaction medium occupies at least 95% of the volume), at atmospheric pressure, and at a temperature T higher than the temperature of the eutectic point of the reaction medium as measured at atmospheric pressure and lower than the temperature of the freezing point of the reaction medium as measured at atmospheric pressure, for a duration t of between 1 h and 48 hours, preferably greater than or equal to 6 hours and less than or equal to 36 hours, in particular greater than or equal to 7 hours and less than or equal to 36 hours, when the pH of the reaction medium is greater than or equal to 6.8 and less than or equal to 7.8.

[0274] In other words, the method of the present invention can then be defined as follows: a) providing at least one polysaccharide; b) providing at least one molecule of formula Chem. II as described above; c) functionalizing the polysaccharide with at least one molecule of formula Chem. II as described above; d) crosslinking by sol-gel reaction of the functionalized polysaccharide to give a hydrogel; wherein the functionalization and crosslinking of the functionalized polysaccharide are carried out as follows:

[0275] 1) preparation of a reaction medium comprising the polysaccharide(s), the molecule(s) of formula Chem. II and a solvent, the pH of the reaction medium being greater than or equal to 9, or greater than or equal to 10, and less than 14;

[0276] 2) optionally placing the reaction medium at atmospheric pressure and at a temperature ranging from 4°C to 60°C, preferably from 10°C to 50°C, typically for a period ranging from 1 hour to 2 weeks, more particularly from 3 hours to 1 week, for example from 3 hours to 80 hours, in particular from 3 hours to 75 hours;

[0277] 3) optionally adjusting the pH of the reaction medium to a pH greater than or equal to 6.8 and less than or equal to 7.8;

[0278] 4) placement of the reaction medium:

[0279] - under conditions which do not allow the sublimation of water (e.g.: rigid airtight container whose free volume is saturated with water vapour or in which the reaction medium occupies at least 95% of the volume), at atmospheric pressure and at a temperature T higher than the temperature of the eutectic point of the reaction medium as measured at atmospheric pressure and lower than the temperature of the freezing point of the reaction medium as measured at atmospheric pressure, for a duration t ranging from 2 weeks to 27 weeks, in particular from 2 to 20 weeks or from 2 to 17 weeks, for example from 3 to 8 weeks or 4 to 7 weeks, when the pH of the reaction medium is greater than or equal to 9, or greater than or equal to 10, and less than 14, or ...: rigid airtight container whose free volume is saturated with water vapor or in which the reaction medium occupies at least 95% of the volume), at atmospheric pressure and at a temperature T higher than the temperature of the eutectic point of the reaction medium as measured at atmospheric pressure and lower than the temperature of the freezing point of the reaction medium as measured at atmospheric pressure, for a duration t of between 1 h and 48 hours, preferably greater than or equal to 6 hours and less than or equal to 36 hours, in particular greater than or equal to 7 hours and less than or equal to 36 hours, when the pH of the reaction medium is greater than or equal to 6.8 and less than 7.8.

[0280] In certain embodiments, the functionalization and crosslinking are carried out in a reaction medium whose pH is greater than or equal to 9, or greater than or equal to 10, and less than 14. For this, the reaction medium preferably comprises a Bronsted base, more preferably a hydroxide, even more preferably sodium or potassium hydroxide. Advantageously, the reaction medium comprises sodium or potassium hydroxide at a concentration of between 0.10M and 0.30M.

[0281] In these embodiments, according to a first variant, the reaction medium prepared in step 1) can be placed at atmospheric pressure and at a temperature ranging from 4°C to 60°C, preferably from 10°C to 50°C, typically for a period ranging from 1 hour to 2 weeks, more particularly from 3 hours to 1 week, for example from 3 hours to 80 hours, in particular from 3 hours to 75 hours (step 2) before being placed at temperature T, at atmospheric pressure, under conditions not allowing the sublimation of water for a period t (step 4). During step 2), the polysaccharide will be functionalized and a portion of the Si-OR groups 10 and optionally Si-OR groups 4 will condense together (pre-condensation), more Si-OR groups 10 and optionally Si-OR groups 4 condensing during step 4) (advanced condensation).

[0282] In these embodiments, according to a second variant, the reaction medium prepared in step 1) can be placed directly at the end of step 1) at temperature T, at atmospheric pressure under conditions which do not allow the sublimation of water for a duration t (step 4).

[0283] At the end of time t (first and second variant), the temperature of the reaction medium is typically raised to room temperature. The pH of the reaction medium is then preferably brought to a physiological pH (approximately 6.8 to 7.8). For this, a Bronsted acid is preferably added to the reaction medium, preferably an aqueous hydrochloric acid solution, an aqueous sulfuric acid solution or an aqueous acetic acid solution.

[0284] In certain embodiments, the crosslinking is carried out partially in a reaction medium whose pH is greater than or equal to 6.8 and less than 7.8. In these embodiments, a reaction medium having a pH greater than or equal to 9, or greater than or equal to 10, and less than 14 and comprising the polysaccharide(s), the molecule(s) of formula Chem. II and a solvent is prepared (step 1)). The reaction medium is placed at a temperature ranging from 4°C to 60°C, preferably from 10°C to 50°C, typically for a period ranging from 1 hour to 2 weeks, more particularly from 3 hours to 1 week, for example from 3 hours to 80 hours, in particular from 3 hours to 75 hours (step 2). Then, the pH of the reaction medium is brought to a physiological pH (step 3)) before the temperature is brought to T at atmospheric pressure under conditions that do not allow the sublimation of water.In this case, the process will advantageously comprise a step of neutralizing the gel to reach this physiological pH, before the reaction medium is brought to atmospheric pressure at temperature T under conditions that do not allow the sublimation of water. For this, a Bronsted acid is preferably added to the reaction medium, preferably an aqueous solution of hydrochloric acid, an aqueous solution of sulfuric acid or an aqueous solution of acetic acid. The reaction medium is then placed at temperature T at atmospheric pressure under conditions that do not allow the sublimation of water for a duration t of between 1 h and 48 hours (step 4).

[0285] Preferably, the process of the present invention comprises only a single step of placing the reaction medium at temperature T at atmospheric pressure under conditions which do not allow the sublimation of water.

[0286] The method according to the invention may comprise one or more additional steps, such as for example steps of adding one or more additional components, purification, sterilization, swelling screening and / or conditioning.

[0287] They can be carried out:

[0288] - before the reaction medium is at temperature T during step d), - or after step d), i.e. after the reaction medium has been maintained at temperature T.

[0289] The method according to the invention may comprise a step of adding at least one additional component. The additional component may be chosen from lubricating agents; cosmetic active ingredients such as antioxidants, coenzymes, amino acids, vitamins, minerals, and nucleic acids; therapeutic active ingredients such as anesthetics, antibiotics, antifungals and adrenaline and its derivatives, and mixtures thereof. The additional components may be as described above.

[0290] The method according to the invention may comprise at least one purification step. The purification may be carried out by dialysis.

[0291] The method according to the invention may comprise a step of sterilizing the hydrogel. Sterilization is preferably carried out by heat. Sterilization is generally carried out by increasing the temperature of the sterilization medium to a temperature called "plateau temperature", which is maintained for a determined period of time called "plateau time". Sterilization is preferably carried out at a plateau temperature ranging from 121°C to 135°C, preferably with a plateau time ranging from 1 minute to 20 minutes with FO > 15. The sterilizing value FO corresponds to the time required, in minutes, at 121°C, to inactivate 90% of the population of microorganisms present in the product to be sterilized. Alternatively, sterilization may be carried out in particular by gamma ray, UV radiation or by means of ethylene oxide.

[0292] The method may comprise a step of sieving the hydrogel, more particularly with a sieve with a porosity of between 50 and 2000 μm. This sieving step makes it possible to obtain a more homogeneous hydrogel with the most constant extrusion force possible, i.e. the most regular possible. A person skilled in the art knows how to select a sieve with a suitable pore size depending on the mechanical properties of the hydrogel being prepared.

[0293] The method may comprise a step of swelling the hydrogel. During the step of swelling the hydrogel, the polysaccharide concentration of the hydrogel is adapted. In particular, a solvent is added, for example, water, a phosphate buffer, water for injection. More particularly, the added solvent has a pH around physiological pH. The polysaccharide concentration obtained following the swelling step advantageously varies from 1 mg / g of gel to 50 mg / g of the hydrogel, more advantageously from 5 mg / g to 35 mg / g of the hydrogel, even more advantageously from 10 mg / g to 30 mg / g of the hydrogel gel.

[0294] The method may include a step of conditioning the hydrogel.

[0295] Where appropriate, the step of adding one or more additional components preferably takes place after the purification step.

[0296] Where appropriate, the step of adding one or more additional components preferably takes place before the sterilization step.

[0297] In particular, the step of adding one or more additional components may also comprise the addition of at least one therapeutic active ingredient, or at least one cosmetic active ingredient, or their mixture. When at least one therapeutic active ingredient and / or at least one cosmetic active ingredient is added, the step of adding one or more additional components preferably takes place after step d).

[0298] Where appropriate, the purification step preferably takes place after the step of adding one or more additional components.

[0299] Where applicable, the purification step preferably takes place before the sterilization step.

[0300] Where appropriate, the purification step preferably takes place before the sieving step.

[0301] The sterilization step is preferably carried out after steps a) to d) and any additional steps. In particular, the hydrogel is sterilized after being packaged in its injection device and the packaging of the gel takes place following all the steps of the process and before sterilization.

[0302] When the polysaccharide is crosslinked according to step d2), the method according to the invention may also comprise an additional step of crosslinking the polysaccharide with a conventional crosslinking agent, and more particularly the crosslinking of the polysaccharide provided in step a) or of the crosslinked polysaccharide obtained following step d2), in the presence of at least one crosslinking agent or a salt thereof, said crosslinking agent comprising at least two functional groups Z as described above.

[0303] The method according to the invention may also comprise a step of adding a molecule of the following formula Chem. Ill:

[0304] R 7 GOLD 12 R 13 SiO] p -R 8 or a salt thereof in which:

[0305] - p is an integer from 1 to 20;

[0306] - R 12 and the Rs 13 , identical or different, represent a hydrogen atom; a halogen atom; a -OR group 14 with R 14 representing a hydrogen atom, an aryl group or an aliphatic hydrocarbon group containing from 1 to 6 carbon atoms; an aryl; or an aliphatic hydrocarbon group containing from 1 to 6 carbon atoms optionally substituted by one or more groups chosen from a halogen atom, an aryl or a hydroxyl; and

[0307] - R 7 and R 8, identical or different, represent a hydrogen atom, an aryl group or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms. According to one embodiment, the step of adding a molecule of formula Chem. Ill is carried out before the temperature of the reaction medium reaches temperature T under conditions which do not allow the sublimation of water during step d), in particular before, during or after step c).

[0308] According to a variant, this step can be carried out after step d), that is to say after the reaction medium has been maintained at temperature T under conditions which do not allow the sublimation of water.

[0309] Where step c) and step d) are concurrent, this step may be carried out before steps c) and d), in particular between step a) and step c).

[0310] Preferably, R 12 and the Rs 13, identical or different, represent a -OR group 14 with R 14 representing a hydrogen atom, an aryl group or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms; an aryl; or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms optionally substituted by one or more groups chosen from a halogen atom, an aryl or a hydroxyl.

[0311] In particular, R 12 and the Rs 13 , identical or different, represent a -OR group 14 with R 14 representing a hydrogen atom, an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms, preferably a (C1-C6)alkyl group; or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms, preferably a (C1-C6)alkyl group.

[0312] Advantageously, R 7 and R 8, identical or different, represent a hydrogen atom, or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms, preferably a (C1-C6)alkyl group.

[0313] This molecule with the formula Chem. Ill contains Si-OR groups (Si-OR 7 , Si-OR 8 and possibly Si-OR 14 ) capable of reacting with Si-OR groups (Si-OR 10 and possibly Si-OR 4 ) of the molecule of formula Chem. II. Thus, during the sol-gel reaction allowing the formation of Si-O-Si bonds, a molecule of formula Chem. Ill can bind to two molecules of formula Chem. Il grafted onto polysaccharide chains so as to form crosslinking bonds resulting from the coupling of a molecule of formula Chem. Ill with two molecules of formula Chem. II.

[0314] For example, the molecule with the formula Chem. Ill is orthosilicic acid, tetraethyl orthosilicate (TEOS), polydimethylsiloxane (PDMS), oligomerized TEOS / orthosilicic acid, or methyl silanetriol (preferably used in the form of its sodium salt called sodium methyl siliconate - NAMS).

[0315] Advantageously, the step of adding a molecule of formula Chem. Ill takes place at a pH greater than or equal to 9, in particular greater than or equal to 10, more advantageously greater than or equal to 12, and in particular less than 14, for example less than or equal to 13.5, in particular when the molecule of formula Chem. Ill is sodium methyl siliconate (NAMS).

[0316] For this, the reaction medium preferably comprises a Bronsted base, more preferably a hydroxide, even more preferably a sodium or potassium hydroxide. In particular, the reaction medium comprises a Bronsted base, more preferably a hydroxide, even more preferably a sodium or potassium hydroxide at a concentration of between 0.10M and 0.30M.

[0317] HYDROGEL

[0318] The present invention also relates to a hydrogel capable of being obtained by the method of the present invention. Such a hydrogel may also be designated by the term “cryogel” in the present description.

[0319] Preferably, the liquid medium of the hydrogel is an aqueous medium chosen from an aqueous solution or a mixture of aqueous solutions, preferentially chosen from water for injection, phosphate buffered saline or a mixture of the two, more preferentially phosphate buffered saline in the context of therapeutic, cosmetic and aesthetic applications according to the invention.

[0320] The hydrogel comprises one or more polysaccharides as defined above. The polysaccharide is crosslinked. The molar crosslinking rate of the polysaccharide is greater than 0 and less than or equal to 15%, preferably less than or equal to 8%, preferably less than or equal to 5%, preferably less than or equal to 3%, preferably less than or equal to 2%, preferably less than or equal to 1.5%, preferably less than or equal to 1%, in particular between 0.1% and 0.8%, more preferably less than or equal to 0.5%, in particular between 0.1% and 0.4% (number of moles of crosslinking agent(s) per 100 moles of repeating unit of the polysaccharide(s).

[0321] The crosslinking agent(s) are as defined above.

[0322] The crosslinking agent is preferably a crosslinking agent whose functional groups are epoxide groups. Thus, preferably the crosslinking agent is 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxy-octane, poly(ethylene glycol) diglycidyl ether (PEGDGE), 1,2-bis(2,3-epoxypropoxy)ethane (EGDGE), and mixtures thereof.

[0323] The hydrogel of the present invention advantageously has a sticky character. The hydrogel of the present invention advantageously has a stringy character. The hydrogel is preferably an injectable hydrogel.

[0324] It is preferably sterile, in particular heat sterilized at a plateau temperature of 121°C to 135°C, preferably with a plateau time of 1 minute to 20 minutes with FO > 15.

[0325] This hydrogel is preferably homogeneous.

[0326] This hydrogel may also comprise an additional component selected from lubricating agents; cosmetic active ingredients such as antioxidants, co-enzymes, amino acids, vitamins, minerals, and nucleic acids; and mixtures thereof, as described below.

[0327] This hydrogel may also comprise at least one therapeutic active ingredient advantageously chosen from anesthetics, antibiotics, antifungals, adrenaline and its derivatives, and mixtures thereof, as described below.

[0328] The polysaccharide of this hydrogel is preferably as defined above, within the framework of the description of step a) of the method according to the invention.

[0329] Preferably, a hydrogel according to the present invention, acceptable for the therapeutic and / or cosmetic applications targeted by the present invention, has a cross-over stress (or stress at the crossing of the moduli G' and G”) greater than or equal to 50 Pa, preferably between 50 and 5000 Pa and more preferably between 100 and 1000 Pa and an elastic modulus G' greater than or equal to 20 Pa, preferably from 100 Pa to 2000 Pa, more preferably from 100 Pa to 1000 Pa.

[0330] Preferably, a hydrogel according to the present invention, acceptable for the therapeutic and / or cosmetic applications targeted by the present invention, has a cohesiveness of 1 N to 30 N. This cohesiveness is measured by mechanical compression using a rheometer. For this, the gel is deposited on a Peltier plane with an initial air gap of 2.60 mm; it is then compressed at a constant speed of 100 pm / s up to 70% of the initial air gap, at 25°C; finally, the cohesiveness of the gel is measured at the end of the compression stroke. The more cohesive a gel is, i.e. the higher the cohesiveness value, the more it is able to withstand stresses, such as those it may encounter after its administration to a subject.

[0331] COMPOSITIONS

[0332] The present invention also relates to a composition comprising the hydrogel according to the present invention. It is preferably a cosmetic or pharmaceutical composition. It may further comprise physiologically acceptable excipients.

[0333] The hydrogel according to the invention comprises a polysaccharide, preferably hyaluronic acid, crosslinked. The composition may further comprise a polysaccharide, preferably hyaluronic acid, not crosslinked.

[0334] Non-crosslinked hyaluronic acid may be present in the composition as a lubricant.

[0335] The composition according to the present invention may thus comprise from 0.1 to 10% by weight, preferably from 0.1 to 5% by weight, preferably from 1 to 3% by weight of polysaccharide, preferably hyaluronic acid, relative to the total weight of said composition, the polysaccharide such as hyaluronic acid, being present in crosslinked and optionally non-crosslinked form. In particular, the content of non-crosslinked polysaccharide, in particular hyaluronic acid, varies from 0 to 40% by weight, preferably from 1 to 40% by weight, more preferably from 5 to 30% by weight, relative to the total weight of polysaccharide, in particular hyaluronic acid, present in the composition. The composition according to the present invention is preferably a sterile composition, in particular sterilized by heat at a plateau temperature of between 121°C and 135°C, preferably with a plateau duration of between 1 minute and 20 minutes with FO > 15.It is preferably an injectable composition. The composition according to the invention then preferably comprises a physiologically acceptable medium, preferably a physiologically acceptable aqueous medium.

[0336] The physiologically acceptable aqueous medium may comprise a physiologically acceptable solvent or mixture of solvents and preferably comprises water, preferably the solvent is water.

[0337] The physiologically acceptable medium may also include isotonic agents such as oses, sodium chloride and their mixture.

[0338] The physiologically acceptable medium may further comprise at least one isotonic and physiologically acceptable saline solution.

[0339] Preferably, said balanced saline solution is a phosphate-buffered saline solution, and particularly a KH2PO4 / K2HPO4 saline buffer. The composition according to the invention may further comprise at least one additional compound chosen from lubricating agents; cosmetic active ingredients such as antioxidants, coenzymes, amino acids, vitamins, minerals, and nucleic acids; and mixtures thereof. Preferably, the additional compound is water-soluble or modified to be soluble in an aqueous medium. The composition according to the invention may also comprise at least one therapeutic active ingredient advantageously chosen from anesthetics, antibiotics, antifungals, adrenaline and its derivatives, and mixtures thereof, as described below. Preferably, the therapeutic active ingredient is water-soluble.

[0340] Examples of anesthetics include Ambucaine, Amoxecaine, Amylein, Aprindine, Aptocaine, Articaine, Benzocaine, Betoxycaine, Bupivacaine, Butacaine, Butamben, Butanilicaine, Chlorobutanol, Chloroprocaine, Cinchocaine, Clodacaine, Cocaine, Cryofluorane, Cyclomethycaine, Dexivacaine, Diamocaine, Diperodon, Dyclonine, Etidocaine, Euprocine, Febuvérine, Fomocaine, Guafécainol, Heptacaine, Hexylcaine, Hydroxyprocaine, Hydroxytetracaine, Isobutamben, Leucinocaine, Levobupivacaine, Levoxadrol, Lidamidine, Lidocaine, Lotucaine, Menglytate, Mepivacaine, Meprylcaine, Myrtecaine, Octacaine, Octodrine, Oxetacaine, Oxybuprocaine, Parethoxycaine, Paridocaine, Phenacaine, Piperocaine, Piridocaine, Polidocanol, Pramocaine, Prilocaine, Procaine, Propanocaine, Propipocaine, Propoxycaine, Proxymetacaine, Pyrrocaine, Quatacaine,Quinisocaine, Risocaine, Rodocaine, Ropivacaine, Tetracaine, Tolycaine, Trimecaine or a salt thereof, in particular a hydrochloride thereof, and a mixture thereof. Advantageously, the anesthetic is selected from lidocaine or a salt thereof, in particular a hydrochloride, and mepivacaine or a salt thereof, in particular a hydrochloride, or mixtures thereof.

[0341] Examples of antioxidants include, but are not limited to, glutathione, reduced glutathione, ellagic acid, spermine, resveratrol, retinol, L-carnitine, polyols, polyphenols, flavonols, theaflavins, catechins, caffeine, ubiquinol, ubiquinone, alpha-lipoic acid and their derivatives, and a mixture thereof.

[0342] Examples of amino acids include, but are not limited to, arginine (eg, L-arginine), isoleucine (eg, L-isoleucine), leucine (eg, L-leucine), lysine (eg, L-lysine or L-lysine monohydrate), glycine, valine (eg, L-valine), threonine (eg, L-threonine), proline (eg, L-proline), methionine, histidine, phenylalanine, tryptophan, cysteine, their derivatives (eg, N-acetylated derivatives such as N-acetyl-L-cysteine) and a mixture thereof. Examples of vitamins and their salts include, but are not limited to, vitamins E, A, C, B, especially vitamins B6, B8, B4, B5, B9, B7, B12, and more preferably pyridoxine and its derivatives and / or salts, preferably pyridoxine hydrochloride.

[0343] Examples of minerals include, but are not limited to, zinc salts (e.g., zinc acetate, in particular dehydrated), magnesium salts, calcium salts (e.g., hydroxyapatite, in particular in bead form), potassium salts, manganese salts, sodium salts, copper salts (e.g., copper sulfate, in particular pentahydrate), optionally in a hydrated form, and mixtures thereof.

[0344] As nucleic acids, mention may be made in particular of adenosine, cytidine, guanosine, thymidine, cytodine, their derivatives and a mixture thereof.

[0345] Examples of coenzymes include coenzyme Q10, CoA, NAD, NADP, and mixtures thereof. Examples of adrenaline derivatives include noradrenaline.

[0346] The amounts of additional compounds depend of course on the nature of the compound in question, the desired effect, and the intended purpose of the composition as described herein. APPLICATIONS

[0347] The hydrogel or composition according to the invention may have therapeutic, cosmetic or aesthetic applications.

[0348] The present invention therefore also relates to a hydrogel or a composition according to the invention for its use in filling and / or replacing tissues, in particular soft tissues, in particular by injecting the hydrogel or the composition into the tissue.

[0349] The hydrogel or composition may be intended for superficial application. Superficial application refers to the administration of a composition into the upper layers of the skin, i.e., into or onto the skin, for example, by mesotherapy, for example, to reduce superficial wrinkles and / or to improve skin quality (such as skin radiance, density, or structure) and / or to rejuvenate the skin.

[0350] The hydrogel or composition may be intended for deep application. Deep application refers to the administration of a composition into the deeper layers of the skin and / or beneath the skin (above the periosteum) to increase soft tissue volume, such as to fill deep wrinkles and / or partially atrophied regions of the face and / or body.

[0351] The hydrogel or composition may be versatile, i.e., be used for both deep and superficial application.

[0352] Preferably, when the hydrogel or the composition according to the invention comprises at least one therapeutic active ingredient, the present invention relates to the hydrogel or a composition according to the invention for its use in the modified, delayed or prolonged release of therapeutic active ingredients.

[0353] In particular, the hydrogel or composition according to the invention is used in oral care and more particularly in the treatment of gingival recession, or to fill periodontal pockets. More particularly, the hydrogel or composition according to the invention is used to treat defects in gingival architecture that may occur with tooth loss, with aging, with periodontal diseases and disorders, or after the placement of tooth implants, crowns or bridges.The hydrogel or the composition according to the invention can also be used in ophthalmology, more particularly to protect ocular structures during eye surgery such as for example ophthalmic surgery of the anterior or posterior segment, cataract extraction possibly with implantation of an intraocular lens, corneal transplant surgery, glaucoma filtering surgery, or even implantation of a secondary lens. In this case, the hydrogel or the composition according to the invention will more particularly be injected into the eye.

[0354] The hydrogel or composition according to the invention can also be used in orthopedics or rheumatology, for example by injection into the synovial cavity. The hydrogel or composition according to the invention is then used as viscosupplementation.

[0355] The hydrogel or composition according to the invention can also be used in the treatment of lipodystrophy.

[0356] The hydrogel or composition according to the invention can be used in cosmetic surgery, in particular for gynecoplasties and / or penoplasties.

[0357] The hydrogel or composition according to the invention is administered more particularly by injection.

[0358] The hydrogel or composition according to the invention can also be used for the modified, delayed or prolonged release of therapeutic active ingredients, in particular the therapeutic active ingredients as described above. The longer the hydrogel is left at temperature T° and pressure P in the presence of the active ingredient(s), the greater the sol-gel reaction will be and the slower the release of the active ingredient will be. Thus, it is possible to adapt the duration and / or intensity of release of the active ingredient to the request. This also applies to the modified, delayed or prolonged release of cosmetic active ingredients.

[0359] The present invention also relates to the aesthetic, and therefore non-therapeutic, use of a hydrogel or a composition according to the invention to prevent and / or treat the alteration of the viscoelastic or biomechanical properties of the skin, and in particular to regenerate, moisturize, firm or restore the radiance of the skin, in particular by mesotherapy; to fill volume defects of the skin, and in particular to fill wrinkles, fine lines or scars (in particular hollow scars); to reduce the appearance of wrinkles and fine lines; or when said hydrogel or said composition comprises at least one cosmetic active ingredient, for the modified, delayed or prolonged release of cosmetic active ingredients, in particular as defined above.

[0360] For example, the present invention relates to the aesthetic use of a hydrogel or a composition according to the invention to reduce nasolabial folds and bitterness lines; to increase the volume of the cheekbones, chin or lips; to restore the volumes of the face, in particular the cheeks, temples, the oval of the face, and the area around the eyes; or to regenerate, hydrate, firm or restore the radiance of the skin, in particular by mesotherapy. In particular, the hydrogel or the composition according to the invention is an anti-aging hydrogel or composition. The hydrogel or the composition according to the invention is administered more particularly by injection.

[0361] The present invention also relates to a method for the cosmetic treatment, preferably anti-aging, of keratin materials, in particular of the skin, comprising at least one step of administering a hydrogel or a composition according to the invention on or through said keratin materials, more particularly by injection.

[0362] The administration may be an injection, in particular an intra-epidermal and / or intradermal and / or subcutaneous injection. The administration by intra-epidermal and / or intradermal and / or subcutaneous injection according to the invention aims to inject a hydrogel or a composition of the invention into an epidermal, dermo-epidermal and / or dermal region. The hydrogel or the composition according to the invention may also be administered by a supra-periosteal injection.

[0363] The hydrogel or composition according to the invention may be injected using any of the methods known to those skilled in the art. In particular, a hydrogel or composition according to the invention may be administered by means of an injection device suitable for intra-epidermal and / or intradermal and / or subcutaneous and / or supra-periosteal injection. The injection device may in particular be chosen from a syringe, a set of microsyringes, a laser or hydraulic device, an injection gun, a needle-free injection device, or a micro-needle roller.

[0364] The injection device may comprise any commonly used injection means suitable for intraepidermal and / or intradermal and / or subcutaneous and / or supraperiosteal injection. Preferably, such means may be a hypodermic needle or a cannula.

[0365] A needle or cannula according to the invention may have a diameter varying from 18 to 34 G, preferably between 25 and 32 G, and a length varying from 4 to 70 mm, and preferably from 4 to 25 mm. The needle or cannula is advantageously for single use.

[0366] Advantageously, the needle or cannula is associated with a syringe or any other device making it possible to deliver said hydrogel or said injectable composition through the needle or cannula.

[0367] According to an alternative embodiment, a catheter can be inserted between the needle / cannula and the syringe. In a known manner, the syringe can be operated manually by the practitioner or by a syringe holder such as guns.

[0368] Preferably, the injection device may be chosen from a syringe or a set of microsyringes. In an alternative embodiment, the injection device may be adapted to the mesotherapy technique.

[0369] Mesotherapy is a treatment technique involving intra-epidermal and / or intradermal and / or subcutaneous injection of a composition or hydrogel. The composition or hydrogel is administered using this technique by injection in the form of multiple small droplets at the level of the epidermis, the dermo-epidermal junction and / or the dermis in order, in particular, to create a subcutaneous coating. The mesotherapy technique is described in particular in the book "Traité de mésothérapie" by Jacques LE COZ, published by Masson, 2004. Mesotherapy performed on the face is also called mesolift, or also under the Anglo-Saxon term "mesoglow". Administration can also be topical.

[0370] Preferably, it is a topical application to the surface of the skin, more particularly to the epidermis, even more particularly to the facial epidermis.

[0371] The present invention thus also relates to an injection device as described previously comprising a hydrogel or a composition according to the invention.

[0372] The additional accessory biological effects of the hydrogels according to the invention can be studied in vitro and / or in vivo and / or ex vivo; said in vivo tests can for example include administration tests in small animals of a composition according to the invention vs. a comparative composition in order to monitor the appearance of biological effects with in particular the evaluation of the improvement in the quality of the skin in the animal, in particular living humans (e.g. its hydration and / or its elasticity) and, after sacrifice of the animal, histological sections to study the possible modification of protein expression at the administration site (coloration).

[0373] These in vivo tests may also include the evaluation of skin quality in humans following the administration of a composition according to the invention vs. a comparative composition.

[0374] Said in vitro tests may include tests on dermal cells (such as fibroblasts) for cytotoxicity, viability, protein expression (ELISA) in particular for the expression of hyaluronic acid, elastin, fibrillin, aquaporin and / or collagens of different types and gene expression (eg, genes coding for hyaluronic acid, elastin, fibrillin, aquaporin and / or collagens of different types). Ex vivo tests, including the aforementioned tests, are carried out for example on explants of animal or human skin. The present invention is illustrated by the non-limiting examples below.

[0375] EXAMPLES

[0376] 1.1. MATERIALS

[0377] - Uncrosslinked sodium hyaluronate of 1.5MDa, 3MDa and 4MDa

[0378] - BDDE

[0379] - NaOH 0.25M

[0380] - 1 M HCl

[0381] - Phosphate Buffer

[0382] - Three-dimensional agitator

[0383] - DHR-2 Rheometer

[0384] - Dynamometer and test bench

[0385] - Homogenizer Paddle mill

[0386] - Sterile polyethylene bag

[0387] 1.2. METHODS

[0388] 1.2.1. MEASUREMENT OF VISCOELASTIC PROPERTIES

[0389] The viscoelastic properties of the obtained hydrogels were measured using a rheometer (DHR-2) having a stainless steel cone (1° - 40 mm) with cone-plate geometry and an anodized aluminum peltier plane (42 mm) (air gap 24 μm). 0.5 g of sterilized hydrogel was deposited between the peltier plane and the said cone. Then a stress scan was performed at 1 Hz and 25°C. The elastic modulus G', the viscous modulus G” and the phase angle 5 were reported for a stress of 5 Pa.

[0390] The stress at the intersection of G' and G”, T, is determined at the intersection of the curves of the modules G' and G” and is expressed in Pascal.

[0391] 1.2.2. MEASUREMENT OF EXTRUSION FORCE

[0392] The extrusion forces (in Newton) of the gels packaged in syringes were conducted using a test bench equipped with a dynamometer at a constant speed of 12.5 mm / min, through a 27G% needle and at room temperature. The extrusion force results correspond to the average of the average extrusion forces on at least 2 samples. 1.3 EXAMPLES

[0393] Unless otherwise stated, the steps described below are carried out at room temperature (21°C).

[0394] 1.3.1 EXAMPLE 1

[0395] Prototypes No. 1 to 2 are prepared as follows:

[0396] - BDDE (crosslinking rate given in Table 1) and sodium hyaluronate (1.5 MDa, 120 mg / g) are dissolved in a 0.25 M sodium hydroxide solution in a sterile, sealed bag,

[0397] - The mixture is homogenized in a paddle mill for 3 cycles of 15 min at 210 rpm, the pH of the mixture is approximately 13,

[0398] - The bag containing the mixture is degassed to remove air bubbles from the mixture and vacuum-sealed at 800 mbar and sealed using a vacuum sealer (Solis Vac Premium Typ 574),

[0399] - The bag containing the vacuum mixture is placed and maintained at -20°C, for a duration given in Table 1,

[0400] - The bag containing the vacuum-packed mixture is left to defrost at room temperature for 3 hours before continuing with the preparation,

[0401] - A 1 N HCI solution is added to the sterile bag until a pH of 7.3 ± 0.5 is obtained,

[0402] -In a container that may be different from the bag, the mixture is diluted to a concentration of 23 mg of hyaluronic acid per gram of mixture with PBS phosphate buffer,

[0403] - Sodium hyaluronate (4 MDa) is added as a lubricant and then the mixture is homogenized,

[0404] - The product thus obtained is sieved to the order of microns and sterilized in an autoclave.

[0405] Table 1: Crosslinking conditions After sterilization, the prototypes are analyzed, the elastic modulus G', the phase angle 5 and the stress at the intersection of G' and G”, T, are determined. The results are reported in Table 2 below.

[0406] Table 2: Results

[0407] The prototype gels 1 to 2 prepared according to the invention have good mechanical properties and have a phase angle 5 less than or equal to 45°. The gels obtained in accordance with the method according to the invention 1 and 2 are transparent, with a smooth appearance and an absence of filament.

[0408] 1.3.2 EXAMPLE 2

[0409] Prototypes A and B are prepared as follows:

[0410] A common gel base was prepared for both trials (prototypes A and B) as follows: 10 g of hyaluronic acid (3 MDa) was dissolved in 73 g of 0.25 M NaOH. The mixture was then homogenized in a paddle mill for 4 cycles of 15 min at 210 rpm. Subsequently, 0.160 g of a BDDE solution diluted 1 / 8 in 0.25 M NaOH was added to the hyaluronic acid solution.

[0411] The HA-BDDE-NaOH mixture of prototype A is placed in a sealed, sterile bag and does not undergo any further processing, in particular no vacuum.

[0412] The HA-BDDE-NaOH mixture of prototype B is placed in a sterile, airtight bag. The mixture is degassed to remove air bubbles from the mixture. Then, the bag containing the degassed mixture is evacuated to 800 mbar and sealed using a vacuum heat sealer (Solis Vac Premium Typ 574). The bag is as described in patent EP2429486B1.

[0413] The bags containing the mixtures of prototypes A and B are placed for 3 months (100 days) at -20°C to allow the crosslinking of the mixtures. Photos are taken after thawing (after 3 hours). Results:

[0414] The results are presented in Figures 1 and 2.

[0415] The gel obtained in accordance with the process according to the invention (figure 2 - prototype B) is transparent, it is characterized by a smooth appearance and an absence of filaments. The gel obtained in figure 1 (prototype A) has inhomogeneous, white and brittle crosslinked zones.

Claims

DEMANDS 1. A process for preparing a polysaccharide-based hydrogel comprising the following steps: a) supplying at least one polysaccharide; b) supplying at least one crosslinking agent and / or at least one functionalizing agent, the functionalizing agent enabling crosslinking of the polysaccharide by sol-gel reaction; c) preparing a reaction medium comprising a solvent, the polysaccharide(s) and the crosslinking agent(s) and / or functionalizing agent(s); d) crosslinking the polysaccharide: d1) by reaction of the polysaccharide with the crosslinking agent(s); or d2) by sol-gel reaction of the functionalized polysaccharide, the functionalized polysaccharide being obtained by reaction of the polysaccharide with the functionalizing agent(s);in which the crosslinking of the polysaccharide according to d1) or d2) is carried out under conditions which do not permit the sublimation of water, at a pressure P and at a temperature T which is higher than the eutectic point temperature of the reaction medium as measured at pressure P and lower than the freezing point temperature of the reaction medium as measured at pressure P.; 2. A process according to claim 1 wherein the crosslinking agent comprises at least two identical or different Z functional groups selected from the following groups: isocyanate, amino, epoxide, carboxyl, N-succinimidyloxycarbonyl, N-sulfosuccinimidyloxycarbonyl, halocarbonyl, isothiocyanate, vinyl, formyl, hydroxyl, sulfhydryl, hydrazino, acylhydrazino, aminoxy, carbodiimide, and an acid anhydride residue.

3. A process according to claim 1 or 2, wherein the functionalizing agent is a chemical molecule having the following formula in which: T represents an isocyanate, amino, epoxide, carboxyl, N-succinimidyloxycarbonyl, N-sulfosuccinimidyloxycarbonyl, halocarbonyl, isothiocyanate, vinyl, formyl, hydroxyl, sulfhydryl, hydrazino, acylhydrazino, aminoxy, carbodiimide, or acid anhydride residue group; A represents a chemical bond or a spacer group; R5 and R6, identical or different, represent a hydrogen atom; a halogen atom; an -OR4 group with R4 representing a hydrogen atom, an aryl group or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms; an aryl; or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms possibly substituted by one or more group(s) chosen from a halogen atom, an aryl and a hydroxyl; R10 represents a hydrogen atom, an aryl group, or an aliphatic hydrocarbon group containing 1 to 6 carbon atoms.

4. A method according to claim 2 in which the functional groups Z are identical and represent an epoxy or vinyl group, more preferably epoxy.

5. A process according to any one of the preceding claims wherein the crosslinking agent is selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxy-octane, poly(ethylene glycol) diglycidyl ether (PEGDGE), 1,2-bis(2,3-epoxypropoxy)ethane (EGDGE), 1,3-bis(3-glycidyloxypropyl)tetramethyldisiloxane, poly(dimethylsiloxane) terminated at each end by a diglycidyl ether (CAS number: 130167-23-6), hydroxyapatite beads modified to bear epoxy groups and mixtures thereof.

6. A method according to any one of the preceding claims wherein the amount of crosslinking agent varies from 0.001 to 0.15 moles per 1 mole of repeating unit of the polysaccharide, preferably from 0.001 to 0.08 moles per 1 mole of repeating unit of the polysaccharide, even more preferably from 0.001 to 0.05 moles per 1 mole of repeating unit of the polysaccharide.

7. A process according to any one of the preceding claims wherein the crosslinking of the polysaccharide according to d1) or d2) is carried out for a period of at least 1 hour, preferably at least 3 hours, preferably at least 72 hours, preferably at most 27 weeks, under conditions not permitting the sublimation of water at temperature T.

8. A process according to any one of the preceding claims wherein the crosslinking of the polysaccharide according to d1) or d2) is carried out for a period of 2 to 25 weeks, preferably from 2 to 20 weeks or 2 to 17 weeks, even more preferably from 3 to 8 weeks or from 4 to 7 weeks, under conditions not permitting the sublimation of water at temperature T.

9. A method according to any one of the preceding claims, wherein the pressure P is less than or equal to atmospheric pressure, advantageously between 0.7 x 10 5 Pa and 0.9.10 5 Pa or equal to atmospheric pressure.

10. A method according to any one of the preceding claims wherein step d) is carried out in a closed hermetically sealed container which may be flexible or rigid, advantageously flexible.

11. Method according to the preceding claim wherein in step d) the hermetically sealed container is placed at a temperature ranging from -35°C to -10°C at atmospheric pressure, preferably at a temperature of about -20°C at atmospheric pressure.

12. Hydrogel obtained by the process of one of the preceding claims.

13. Cosmetic or pharmaceutical composition comprising a hydrogel according to claim 12.

14. Cosmetic use of a hydrogel according to claim 12 or of a composition according to claim 13 to prevent and / or treat the alteration of the viscoelastic or biomechanical properties of the skin; to fill wrinkles, fine lines and scars; to reduce nasolabial folds and marionette lines; to reduce the appearance of wrinkles and fine lines; or to stimulate, regenerate, hydrate, firm or restore the radiance of the skin, particularly through mesotherapy.

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