Method for preparing a sterile hydrogel comprising a cross-linked or non-crosslinked polysaccharide or a mixture thereof

Abstract

The present invention relates to a sterile hydrogel comprising a cross-linked polysaccharide, a non-cross-linked polysaccharide or a mixture thereof, in particular comprising a cross-linked hyaluronic acid, a non-cross-linked hyaluronic acid or a mixture thereof, and further comprising zinc and citrate ions, as well as to a method for preparing same.

Description

[0001] Process for preparing a sterile hydrogel comprising a crosslinked, non-crosslinked polysaccharide or their mixture

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a sterile hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide or a mixture thereof, in particular comprising a crosslinked hyaluronic acid, a non-crosslinked hyaluronic acid or a mixture thereof, and further comprising zinc and citrate ions as well as a method for its preparation.

[0004] TECHNOLOGICAL BACKGROUND

[0005] 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. To improve skin quality, hydrogels prepared from unmodified hyaluronic acid are of interest because they have the advantage of being perfectly biocompatible.

[0006] It is also possible to use hydrogels based on modified hyaluronic acid, where hyaluronic acid is usually modified by crosslinking. This crosslinking has the advantage of increasing the in vivo durability and resistance to in vivo degradation of the hydrogels. Crosslinked hyaluronic acid hydrogels can be obtained by different preparation methods.

[0007] Today, there is a growing need for hydrogels capable of improving their biocompatibility profile and delivering beneficial biological effects for skin quality. In this context, zinc is proving to be an element of choice. It is a micronutrient that exhibits numerous beneficial biological effects as a cofactor for numerous enzymes, particularly those involved in the healing and extracellular matrix reconstruction processes. In addition to healing activities, anti-inflammatory and anti-infectious activities have also been associated with zinc. Zinc may therefore be of interest in reducing potential side effects due to the inflammatory response associated with hydrogel administration.

[0008] However, the preparation of hydrogels comprising zinc is not easy, as zinc can precipitate in the presence of certain salts, in particular phosphates, carbonates and / or sulfates. Its incorporation into hydrogels therefore remains difficult. Thus, a need remains for the provision of a process for preparing hydrogels comprising a crosslinked and / or non-crosslinked polysaccharide and further comprising zinc, in particular at concentrations where zinc is biologically active, without precipitation of the latter. Advantageously, the proposed process will be as respectful as possible of the properties of the hydrogels, that is to say it will cause the least possible degradation of the rheological properties of the hydrogels during heat sterilization.

[0009] BRIEF DESCRIPTION OF THE INVENTION

[0010] The present invention relates to a method for preparing a sterile hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide or a mixture thereof and further comprising zinc ions, the method comprising the following steps:

[0011] (1) preparing a hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide or a mixture thereof, the preparation of the hydrogel comprising the following steps:

[0012] - bringing the crosslinked polysaccharide, the non-crosslinked polysaccharide, or their mixture into contact with a physiological saline solution, preferably buffered, the physiological saline solution, preferably buffered, comprising phosphate or carbonate or sulfate salts or their mixtures,

[0013] - addition of citrate ions to the crosslinked polysaccharide, to the non-crosslinked polysaccharide, or to their mixture, in an amount sufficient to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel,

[0014] - addition of zinc ions to the crosslinked polysaccharide, to the non-crosslinked polysaccharide, or to their mixture, in an amount sufficient to achieve a zinc ion concentration of at most 20 mM in the hydrogel, the addition of the citrate ions and the zinc ions being carried out in a molar ratio [citrate ions] / [zinc ions] ranging from 1 to 20, and on the condition that the addition of the zinc ions is not carried out before the addition of the citrate ions when the contact with the physiological saline solution, preferably buffered, is carried out before the addition of the citrate ions;

[0015] (2) sterilization, preferably by heat, of the hydrogel obtained at the end of step (1) to obtain a sterile hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide or their mixture and further comprising zinc ions. The invention also relates to a method for preparing a sterile hydrogel comprising a crosslinked polysaccharide and optionally a non-crosslinked polysaccharide and further comprising zinc ions, the method comprising the following steps:

[0016] (0) preparation of a crosslinked polysaccharide from a crosslinking reaction medium comprising one or more polysaccharide(s), one or more crosslinking agent(s), a solvent and zinc ions in an amount allowing the preparation of a hydrogel comprising at most 20 mM of zinc ions;

[0017] (1) preparation of a hydrogel from the crosslinked polysaccharide obtained at the end of step (0) and optionally from a non-crosslinked polysaccharide, the preparation of the hydrogel comprising a step of bringing the crosslinked polysaccharide into contact with a physiological saline solution, preferably buffered, comprising phosphate or carbonate or sulfate salts or mixtures thereof;

[0018] (2) sterilization, preferably by heat, of the hydrogel obtained at the end of step (1) to obtain a sterile hydrogel; in which:

[0019] - the crosslinking reaction medium further comprises citrate ions in an amount sufficient to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel, the molar ratio [citrate ions present in the reaction medium] / [zinc ions present in the reaction medium] ranging from 1 to 20; or

[0020] - step (1) further comprises, before the step of bringing the crosslinked polysaccharide into contact with the physiological saline solution, preferably buffered, a step of adding citrate ions in a quantity sufficient to reach a citrate ion concentration of at least 0.1 mM in the hydrogel, the molar ratio [citrate ions added] / [zinc ions present in the reaction medium] ranging from 1 to 20; or

[0021] - the physiological saline solution, preferably buffered, further comprises citrate ions in a quantity sufficient to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel, the molar ratio [citrate ions present in the physiological saline solution, preferably buffered] / [zinc ions present in the reaction medium] ranging from 1 to 20.

[0022] The invention also relates to a method for preparing a sterile hydrogel comprising a crosslinked polysaccharide and optionally a non-crosslinked polysaccharide and further comprising zinc ions, the method comprising the following steps:

[0023] (0') preparation of a crosslinking reaction medium comprising:

[0024] - one or more polysaccharide(s), - one or more crosslinking agent(s),

[0025] - citrate ions in an amount sufficient to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel,

[0026] - zinc ions in an amount allowing the preparation of a hydrogel comprising at most 20 mM of zinc ions, and

[0027] - a physiological saline solution, preferably buffered, comprising phosphate or carbonate or sulfate salts or mixtures thereof; the molar ratio [citrate ions present in the reaction medium] / [zinc ions present in the reaction medium] ranging from 1 to 20, the preparation of the reaction medium being carried out by adding the citrate ions before any contact of the zinc ions with the physiological saline solution;

[0028] (0) preparation of a crosslinked polysaccharide from the reaction medium obtained at the end of step (0');

[0029] (1) preparation of a hydrogel from the crosslinked polysaccharide obtained at the end of step (0) and optionally from a non-crosslinked polysaccharide;

[0030] (2) sterilization, preferably by heat, of the hydrogel obtained at the end of step (1) to obtain a sterile hydrogel.

[0031] The invention also relates to a sterile hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide or a mixture thereof, in particular comprising a crosslinked hyaluronic acid, a non-crosslinked hyaluronic acid or a mixture thereof, and further comprising zinc and citrate ions obtained by the methods according to the invention.

[0032] The invention relates to the use of citrate ions for protecting a hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide or their mixture, optionally an anesthetic agent, and further zinc ions, from the degradation of its rheological properties during its sterilization, preferably by heat or for preserving the stability over time of the rheological properties of a hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide or their mixture, optionally an anesthetic agent, and further zinc ions.

[0033] Finally, the invention relates to the use of a solution comprising zinc ions and citrate ions to protect a hydrogel comprising a crosslinked and / or non-crosslinked polysaccharide, optionally an anesthetic agent, from the degradation of its rheological properties during its sterilization, preferably by heat, or to preserve the stability over time of a hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide or their mixture, optionally an anesthetic agent.

[0034] Other aspects of the invention are as described below and in the claims.

[0035] FIGURES

[0036] Fig. 1: hydrogel obtained according to the method of the invention (microscope: Olympus SZX16, software: OLYMPUS Stream Start)

[0037] Fig.2: hydrogel excluding invention (microscope: Olympus SZX16, software: OLYMPUS Stream Start)

[0038] DETAILED DESCRIPTION OF THE INVENTION

[0039] Definitions

[0040] 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 viscoelastic product that has a phase angle 5 of less than 90°, preferably less than or equal to 70°, preferably less than or equal to 45°, at 1 Hz for a strain of 0.1% or a pressure of 1 Pa, preferably a phase angle 5 ranging from 2° to 45° or ranging from 20° to 45°.

[0041] 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 97%, in particular at least 98% by weight of the liquid medium) and having a pH ranging from 6.8 to 7.8.

[0042] The term “injectable hydrogel” refers to a hydrogel 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 32G, 30G, 27G, 26G, 25G hypodermic needle. Preferably, an “injectable hydrogel” is a hydrogel having an average extrusion force of less than or equal to 25N, preferably ranging from 5 to 25N, more preferably ranging from 8 to 15N, 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 (27G) and a length of 1”, at room temperature.

[0043] A “superficial application” means the administration, for example by mesotherapy, of a composition superficially into the skin, or onto the skin, for the treatment of the superficial layers of the skin, the epidermis and the most superficial parts of the dermis, to reduce superficial wrinkles and / or improve the quality of the skin (such as its radiance, density or structure) and / or rejuvenate the skin.

[0044] A “midline application” means the administration of a composition into the midline of the skin to treat the midline layers of the skin, as well as to reduce midline wrinkles.

[0045] A “deep application” refers to the administration of a composition into the deepest layers of the skin, the hypodermis and the deepest part of the dermis, and / or beneath the skin (above the periosteum) to “add volume,” such as for filling the deepest wrinkles and / or partially atrophied regions of the facial and / or body contour. So-called “volumizing” hydrogels can typically be administered for deep application.

[0046] A "crosslinked polysaccharide" refers to a polysaccharide that has been modified during a crosslinking reaction.

[0047] Conversely, a “non-crosslinked polysaccharide” refers to a polysaccharide that has not been modified with a crosslinking agent and which therefore has not undergone a crosslinking reaction.

[0048] The term "crosslinking agent" refers to any compound capable of introducing crosslinking between different polysaccharide chains.

[0049] The "molar crosslinking ratio" (MR), expressed in %, refers to the molar ratio of the amount of crosslinking agent to the amount of polysaccharide repeating unit introduced into the crosslinking reaction medium expressed per 100 moles of polysaccharide repeating units in the crosslinking medium. For example, a molar crosslinking ratio of 1% means that there is one molecule of crosslinking agent introduced into the reaction medium per 100 moles of polysaccharide repeating units.

[0050] 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.

[0051] The “degree of modification” (MOD) of a polysaccharide, such as hyaluronic acid, corresponds to the molar quantity of crosslinking 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. For example, a degree of modification of 1% means that there is one molecule of crosslinking agent per 100 moles of repeating units of polysaccharide.

[0052] The term "polysaccharide" refers to a polymer composed of monosaccharides (preferably D-enantiomers) joined together by glycosidic bonds. "Room temperature" means a temperature ranging from 20 to 25°C, more particularly 21°C.

[0053] The Linear Viscoelastic Region (LVER) corresponds to the range of hydrogel deformations from an initial elastic modulus value G' to the elastic modulus value G' reduced by 10% of its initial value. The LVER measurement consists of an oscillatory stress sweep measurement in compression mode at a given oscillation frequency to determine the linear viscoelastic region.

[0054] Processes

[0055] The inventors have developed three alternative processes that meet the expressed needs.

[0056] According to a first variant (method 1), the preparation of a hydrogel comprising a crosslinked and / or non-crosslinked polysaccharide and further comprising zinc is made possible by the addition of citrate ions during the hydrogel preparation phase.

[0057] According to a second variant (method 2), when the hydrogel comprises a crosslinked polysaccharide, the preparation of a hydrogel comprising zinc is made possible by carrying out the crosslinking of the polysaccharide in a reaction medium comprising zinc ions and by adding citrate ions either during the crosslinking step or subsequently but before any contact of the zinc ions with a physiological saline solution, preferably buffered, comprising phosphate or carbonate or sulfate salts, alone or their mixtures.

[0058] According to a third variant (method 3), when the hydrogel comprises a crosslinked polysaccharide, the preparation of a hydrogel comprising zinc is made possible by carrying out the crosslinking of the polysaccharide in a reaction medium comprising zinc ions, citrate ions and a physiological saline solution, preferably buffered, comprising phosphate or carbonate or sulfate salts, alone or their mixtures, provided that the citrate ions are added before any contact of the zinc ions with the physiological saline solution.

[0059] It has been observed that the addition of citrate ions, in particular citric acid or sodium citrate, or calcium citrate, or potassium citrate or magnesium citrate, to zinc makes it possible to prevent the precipitation of zinc, in particular in the presence of a physiological saline solution, preferably buffered, comprising phosphate or carbonate or sulfate salts or mixtures thereof.

[0060] Unexpectedly, it was observed that the proposed method (variants 1, 2 and 3) also makes it possible to effectively protect the hydrogel from degradation of its rheological properties during sterilization, in particular during sterilization by heat. The hydrogels obtained by the method of the present invention thus exhibit lesser modifications of their rheological properties after sterilization (better preservation of the elastic modulus G', better preservation of the phase angle) compared to hydrogels prepared by an equivalent method without the addition of zinc and citrate ions, in particular without the addition of zinc and citrate ions in the form of a physiological saline solution, preferably buffered, comprising phosphate or carbonate or sulfate salts or their mixtures.

[0061] Unexpectedly, this protective effect is prolonged over time. The hydrogels obtained by the process of the present invention have better stability over time, that is to say they maintain their rheological properties more effectively over time, in particular after sterilization, than hydrogels prepared by an equivalent process without the addition of zinc and citrate ions, in particular without the addition of zinc and citrate ions in the form of a physiological saline solution, preferably buffered, comprising phosphate or carbonate or sulfate salts or their mixtures, and zinc ions and citrate ions. Indeed, the hydrogels according to the invention have better conservation over time of their rheological properties. ± especially after sterilization, and do not show zinc precipitation.

[0062] Furthermore, it is known that the additional presence of anesthetic in a hydrogel during sterilization, preferably by heat, leads to degradation of the rheological properties of hydrogels based on crosslinked and / or non-crosslinked polysaccharide, in particular hydrogels based on crosslinked and / or non-crosslinked hyaluronic acid. The addition of citrate and zinc ions makes it possible to limit these effects. The hydrogels obtained by the process of the present invention comprising an anesthetic agent exhibit less degradation of their rheological properties after sterilization compared to hydrogels, comprising an anesthetic agent, prepared by an equivalent process without the addition of zinc and citrate ions, in particular without the addition of zinc and citrate ions in the form of a physiological saline solution, preferably buffered, comprising phosphate or carbonate or sulfate salts or mixtures thereof.

[0063] METHOD 1

[0064] The present invention thus relates to a process for preparing a sterile hydrogel comprising a crosslinked and / or non-crosslinked polysaccharide and further comprising zinc ions, the process comprising the following steps:

[0065] (1) preparation of a hydrogel comprising a crosslinked and / or non-crosslinked polysaccharide, the preparation of the hydrogel comprising the following steps:

[0066] - bringing a crosslinked and / or non-crosslinked polysaccharide into contact with a physiological saline solution, preferably buffered, the saline solution, preferably buffered, comprising phosphate or carbonate or sulfate salts or mixtures thereof;

[0067] - additions of zinc ions and citrate ions to the crosslinked and / or non-crosslinked polysaccharide in a citrate ion / zinc ion molar ratio ranging from 1 to 20 and in an amount sufficient to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel and a zinc ion concentration of at most 20 mM in the hydrogel, provided that the addition of the zinc ions is not carried out before the addition of the citrate ions when contact with the physiological saline solution is carried out before the addition of the citrate ions;

[0068] (2) sterilization, preferably by heat, of the hydrogel obtained at the end of step (1) to obtain a sterile hydrogel.

[0069] It is understood that the preparation of the hydrogel from the crosslinked and / or non-crosslinked polysaccharide (step (1)) comprises at least the steps indicated above, the order of these steps being indifferent. Other steps can be implemented.

[0070] Crosslinked and / or non-crosslinked polysaccharide

[0071] The polysaccharide may be any polymer composed of monosaccharides joined together by glycosidic bonds or mixtures thereof. Preferably, the polysaccharide is chosen 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. Even more preferably, the polysaccharide is chosen from hyaluronic acid, heparosan, chondroitin and mixtures thereof, even more preferably the polysaccharide is hyaluronic acid or one of its salts, in particular a physiologically acceptable salt such as the sodium salt, the potassium salt, the zinc salt, the calcium salt, the magnesium salt, the silver salt, the calcium salt and mixtures thereof.More specifically, hyaluronic acid is in its acid form or in the form of sodium salt (NaHA). The hydrogel can thus be a hydrogel based on hyaluronic acid and / or one of its salts.

[0072] Preferably, if the polysaccharide is hyaluronic acid or one of its salts, it has a weight average molecular mass (Mw) ranging from 0.05 to 10 MDa, preferably ranging from 0.5 to 5 MDa, for example ranging from 2 to 4 MDa or ranging from 1 to 5 MDa.

[0073] The polysaccharide may be provided in hydrated form (fully or partially hydrated), or in dry form, such as powder or fiber. When the polysaccharide is provided in hydrated form, it is typically in the form of a gel.

[0074] A crosslinked polysaccharide may be prepared by any method known to those skilled in the art.

[0075] The crosslinked polysaccharide may result from the reaction of the polysaccharide with a crosslinking agent or result from the reaction of a polysaccharide modified to allow the formation of covalent intermolecular bonds.

[0076] For example, the crosslinked polysaccharide can be prepared as described in WO2010131175A1 or WO201277054A1.

[0077] The method of the present invention may thus comprise, before the step of preparing the hydrogel, a step of preparing a crosslinked polysaccharide.

[0078] The crosslinked polysaccharide is preferably a crosslinked polysaccharide whose molar crosslinking rate is less than or equal to 10%. Preferably, the crosslinked polysaccharide is a crosslinked polysaccharide whose molar crosslinking rate is greater than 0 and less than or equal to 6%. Even more preferably, the crosslinked polysaccharide is a crosslinked polysaccharide whose molar crosslinking rate is greater than 0 and less than or equal to 4%. Even more preferably, the crosslinked polysaccharide is a crosslinked polysaccharide whose molar crosslinking rate is greater than 0 and less than or equal to 2%, preferably less than or equal to 1%, still preferably less than or equal to 0.8%, in particular ranging from 0.1% to 0.5% (number of moles of crosslinking agent(s) per 100 moles of repeating unit of the polysaccharide(s).

[0079] The polysaccharide may be crosslinked by reacting a previously modified polysaccharide. The polysaccharide may have been modified by introducing functional groups capable of reacting with each other and forming covalent intermolecular bonds. The polysaccharide may have been modified by grafting using a molecule that allows subsequent crosslinking of the modified polysaccharide. For example, the polysaccharide may have been modified by grafting a silylated molecule, an amino acid, an amino acid derivative, or a protein.

[0080] The polysaccharide may be crosslinked by means of a crosslinking agent. The polysaccharide is preferably crosslinked by means of a crosslinking agent selected from epoxy or non-epoxy bi- or multifunctional crosslinking agents, i.e. prepared by reaction of the polysaccharide with a crosslinking agent. Among the epoxy agents, mention may be made of 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxy-octane, 1,2-bis(2,3-epoxypropyl)-2,3-ethane (EGDGE), poly(ethylene glycol)-diglycidyl ether (PEGDE), and mixtures thereof. Among the non-epoxy agents that may be mentioned are endogenous polyamines such as spermine, spermidine and putrescine, aldehydes such as glutaraldehyde, carbodiimides and divinylsulfone, hydrazide derivatives such as adipic acid dihydrazide, bisalkoxyamines, dithiols such as polyethylene glycol dithiol and mixtures thereof.Among the non-epoxy agents that may be mentioned are amino acids such as cysteine, lysine; peptides or proteins containing amino acids such as cysteine, lysine; poly(dimethylsiloxane); trimetaphosphates, such as sodium trimetaphosphate, calcium trimetaphosphate, or barium trimetaphosphate.

[0081] In some embodiments, the crosslinking agent is an epoxy agent, preferably 1,4-butanediol diglycidyl ether (BDDE) or polyethylene glycol diglycidyl ether. Preferably, the crosslinking agent is 1,4-butanediol diglycidyl ether (BDDE). In some embodiments, the crosslinking agent is a non-epoxy agent, preferably selected from endogenous polyamines, aldehydes, carbodiimides, divinyl sulfone, amino acids, peptides, and mixtures thereof.

[0082] The crosslinked polysaccharide is preferably a crosslinked polysaccharide having a degree of modification (MOD) of less than or equal to 10%, preferably less than or equal to 6%, preferably less than or equal to 4%, preferably less than or equal to 2%, more preferably less than or equal to 1%. Advantageously, the crosslinked polysaccharide is a crosslinked polysaccharide having a degree of modification (MOD) of less than or equal to 1.8%, more preferably less than or equal to 1.5%, preferably less than or equal to 1.2%, even more preferably less than 1%.

[0083] The crosslinked polysaccharide may in particular be prepared by a process comprising the following steps:

[0084] (a1) preparing a crosslinking reaction medium comprising one or more polysaccharide(s), one or more crosslinking agent(s) and a solvent; and

[0085] (a2) reacting the reaction medium to obtain a crosslinked polysaccharide.

[0086] The polysaccharide is as described above. Preferably, the polysaccharide is hyaluronic acid or a salt of hyaluronic acid, preferably a sodium salt.

[0087] In step (a1), the polysaccharide may be provided in dry form, such as powder or fiber, or in hydrated form. When the polysaccharide is provided in hydrated form, it is in the form of an uncrosslinked gel or a solution. In particular, when the polysaccharide is in hydrated form, it is an aqueous uncrosslinked gel or an aqueous solution.

[0088] The crosslinking agent is as described above.

[0089] 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 the aqueous reaction medium.

[0090] 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 below and mixtures thereof. The addition of a Bronsted base may be particularly necessary when the functional groups of the crosslinking agent have an epoxide group or a vinyl group. In these cases, the crosslinking 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 of between 0.10M and 0.30M.The total amount of crosslinking agent in the reaction medium typically varies from 0.001 to 0.10 mole per 1 mole of polysaccharide repeating unit, preferably from 0.001 to 0.08 mole or from 0.001 to 0.06 mole per 1 mole of polysaccharide repeating unit, preferably from 0.001 to 0.04 mole per 1 mole of polysaccharide repeating unit, preferably from 0.001 to 0.03 mole per 1 mole of polysaccharide repeating unit, preferably 0.001 to 0.02 mole per 1 mole of polysaccharide repeating unit, more preferably from 0.001 to 0.01 mole per 1 mole of polysaccharide repeating unit, even more preferably from 0.001 to 0.005 mole per 1 mole of polysaccharide repeating unit. When the polysaccharide is a glycosaminoglycan such as hyaluronic acid, the repeating unit is a disaccharide unit.

[0091] The mass concentration of polysaccharide or polysaccharide salt in the reaction medium advantageously varies from 50 to 300 mg / g of solvent, preferably from 80 to 200 mg / g.

[0092] Step (a1) of the process typically comprises a step of homogenizing the reaction medium. Homogenization is generally carried out by three-dimensional stirring, stirring with a mixer, stirring with paddles or stirring with a spatula.

[0093] Step (a1) is typically carried out at a temperature ranging from 4 to 35°C, preferably ranging from 15°C to 25°C. Preferably, the duration of step (1) does not exceed 5 hours. It generally varies from 15 minutes to 4 hours, preferably from 30 minutes to 2 hours.

[0094] Step (a2) consists of reacting the reaction medium to obtain a crosslinked polysaccharide. Advantageously, step (a2) is carried out directly after step (a1).

[0095] This step allows the polysaccharide chains to be crosslinked together. The functional groups of the crosslinking agent react with functional groups 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, 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. Crosslinking can be carried out in the presence of several crosslinking agents.When the crosslinking is carried out in the presence of several crosslinking agents, the crosslinking agents can be added simultaneously or separately in time to the reaction medium. Step (a2) can thus comprise repeated crosslinking steps, advantageously step (a2) comprises a single crosslinking step. The crosslinking is then carried out in the presence of a total quantity of crosslinking agents typically ranging from 0.1 to 10 moles, or from 0.1 to 8 moles, or from 0.1 to 6 moles, or from 0.1 to 4 moles, or from 0.1 to 3 moles, or from 0.1 to 2 moles or from 0.1 to 1 mole or from 0.1 to 0.8 moles, or from 0.1 to 0.5 moles of crosslinking agents (or their salts) per 100 moles 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.

[0096] The lower the content of crosslinking agent, the longer the reaction time must be to obtain similar mechanical properties of the resulting crosslinked polysaccharide, and ultimately of the prepared hydrogel. 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 crosslinked polysaccharide, and ultimately a hydrogel with desirable properties.

[0097] In certain embodiments, step (a2) can be carried out by placing the reaction medium directly obtained at the end of step (a1), at a temperature less than or equal to 30°C, preferably less than or equal to 25°C. The temperature is typically greater than 0°C or greater than 5°C or even greater than 10°C. Even more preferably, step (a2) can be carried out by placing the reaction medium directly obtained at the end of step (a1) at a temperature equal to room temperature. When step (a2) is carried out at a temperature less than or equal to 30°C and greater than 0°C, the crosslinking time is at least 1 minute, preferably at least 10 minutes, even more preferably at least 1 hour. Preferably, the crosslinking time is at most 5 days.

[0098] In certain embodiments, step (a2) can be carried out by placing the reaction medium directly obtained at the end of step (a1), at a temperature greater than 30°C, or greater than or equal to 35°C, or greater than or equal to 40°C, or greater than or equal to 45°C, or greater than or equal to 50°C. The temperature is typically less than 60°C. When the temperature is greater than 30°C, the duration of the crosslinking step is at least greater than or equal to 1 minute, preferably at least greater than or equal to 10 minutes, even more preferably at least 1 hour, preferably between 1 hour and 5 hours.

[0099] In certain embodiments, step (a2) can be carried out by placing the reaction medium directly obtained at the end of step (a1) at a temperature ranging from 0 to 15°C or from 1 to 10°C or from 1 to 9°C.

[0100] In certain embodiments, step (a2) can be carried out by placing the reaction medium directly obtained at the end of step (a1), at a pressure P less than or equal to atmospheric pressure and at a temperature T 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, preferably for a period of at least 1 hour. The crosslinked polysaccharide-based hydrogels prepared by such a method are highly biocompatible. Indeed, the crosslinked polysaccharides can be prepared with smaller amounts of crosslinking agent, for example amounts ranging from 0.001 to 0.02 mole per 1 mole of repeating unit of the polysaccharide.

[0101] The freezing point temperature of the reaction medium refers to the temperature at which the mixture of the components of the reaction medium, on a macroscopic scale, solidifies, i.e. it becomes non-fluid. Below the freezing point, the mixture is in a frozen state 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.

[0102] The eutectic point temperature of the reaction medium refers to the temperature below which 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. The freezing point and the eutectic point of a mixture depend on the pressure to which the mixture is subjected, therefore the freezing point and the eutectic point are measured at pressure P.

[0103] 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. The temperature T is preferably greater than or equal to -55°C and less than or equal to -5°C, preferably it ranges from -35°C to -10°C. Even more preferably, the temperature T is approximately -20°C.

[0104] The pressure P is preferably atmospheric pressure. "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. 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 T varies from -35°C to -10°C or is approximately -20°C.

[0105] Preferably, during the crosslinking step (a2), when the temperature T is greater than or equal to -55°C and less than or equal to -5°C, the reaction medium obtained at the end of step (1) 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 under these conditions. Preferably, the crosslinking step (a2) is carried out for a period of time 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 4 to 7 weeks and at the temperature T, at the pressure P.

[0106] At the end of step (a2), the crosslinked polysaccharide is typically in the form of a gel. This gel is generally directly used in the rest of the process of the invention (step (1)).

[0107] The crosslinked and / or non-crosslinked polysaccharides described above are useful for implementing the methods of the invention and thus preparing hydrogels comprising a crosslinked and / or non-crosslinked polysaccharide. The crosslinked or non-crosslinked polysaccharide, or their mixture, will constitute the polymer network of the hydrogel. The hydrogel comprising a crosslinked or non-crosslinked polysaccharide, or their mixture can thus be said to be based on a crosslinked polysaccharide, or a non-crosslinked polysaccharide, or their mixture. A hydrogel comprising, as the only polysaccharide, a non-crosslinked polysaccharide, is prepared from a non-crosslinked polysaccharide. A hydrogel comprising, as the only polysaccharide, a crosslinked polysaccharide, is prepared from a crosslinked polysaccharide. When the hydrogel comprises the mixture of a crosslinked and non-crosslinked polysaccharide, the hydrogel is prepared from a crosslinked polysaccharide and a non-crosslinked polysaccharide.The uncrosslinked polysaccharide is typically added to the crosslinked polysaccharide during hydrogel preparation. Hydrogel preparation (step (1)).

[0108] The method of the present invention according to method 1 comprises preparing a hydrogel comprising a crosslinked and / or non-crosslinked polysaccharide.

[0109] The preparation of the hydrogel includes the following steps:

[0110] - bringing the crosslinked and / or non-crosslinked polysaccharide into contact with a physiological saline solution, preferably buffered, the physiological saline solution, preferably buffered, comprising phosphate or carbonate or sulfate salts or mixtures thereof;

[0111] - addition of zinc and citrate ions to the crosslinked and / or non-crosslinked polysaccharide in a molar ratio of citrate ions to zinc ions ranging from 1 to 20 and in an amount sufficient to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel and a zinc ion concentration of at most 20 mM in the hydrogel, provided that the addition of the zinc ions is not carried out before the addition of the citrate ions when the contacting of the crosslinked and / or non-crosslinked polysaccharide with the physiological saline solution, preferably buffered, is carried out before the addition of the citrate ions.

[0112] When the physiological saline solution is buffered, it is buffered by phosphate and / or carbonate and / or sulfate salts and generally has a physiological pH (6.8-7.8). Generally, the buffered physiological saline solution is a buffered physiological saline solution comprising phosphate salts, preferably the buffered physiological saline solution may be a phosphate buffer. The phosphate buffer may be a PBS buffer with a pH around the physiological pH (6.8-7.8) (CAS No: 7647-14-5, 7447-40-7). Preferably, the buffer is a phosphate buffer, particularly a saline buffer of NaH2PO4 / Na2HPC>4 or KH2PO4 / K2HPO4.

[0113] When preparing a hydrogel, contacting the crosslinked and / or non-crosslinked polysaccharide with a physiological saline solution, preferably buffered, comprising phosphate or carbonate or sulfate salts or mixtures thereof may occur at different stages. For example, contacting the crosslinked and / or non-crosslinked polysaccharide with the physiological saline solution, preferably buffered, may occur at the time of adjusting the concentration of crosslinked and / or non-crosslinked polysaccharide in the prepared hydrogel. This step is commonly referred to as "dilution". Thus, when preparing the hydrogel, a physiological saline solution, preferably buffered, comprising phosphate or carbonate or sulfate salts or mixtures thereof is typically added during dilution.Alternatively or additionally, the contacting of the crosslinked and / or non-crosslinked polysaccharide with the physiological saline solution, preferably buffered, can occur at the time of pH adjustment or at the time of addition of one or more additional components (see below).

[0114] Preferably, in the method of the present invention, contacting with the physiological saline solution, preferably buffered, comprising phosphate or carbonate or sulfate salts or their mixtures is carried out at least during the step of adjusting the concentration of crosslinked and / or non-crosslinked polysaccharide (dilution).

[0115] The amount of citrate ions added allows a citrate ion concentration in the hydrogel of at least 0.1 mM and generally at most 150 mM, 100 mM, 50 mM, 20 mM or 15 mM to be achieved. Preferably, the amount of citrate ions added makes it possible to achieve a concentration of citrate ions in the hydrogel varying from 0.1 to 150 mM or from 0.1 to 100 mM or from 0.1 to 50 mM or from 0.1 to 20 mM or from 0.1 to 15 mM or from 0.3 to 15 mM or from 0.5 to 15 mM or from 1 to 10 mM or from 1.5 to 10 mM or from 2 to 10 mM or from 3 to 10 mM or from 3 to 8 mM or from 5 to 8 mM.

[0116] The molar ratio of citrate ions to repeating units of the polysaccharide can vary from 0.001 to 10 or from 0.001 to 5 or from 0.001 to 4.3, preferably from 0.001 to 4, even more preferably from 0.001 to 3. The polysaccharide may have been modified by the introduction of functional groups capable of reacting with each other and forming covalent intermolecular bonds.

[0117] For example, the molar ratio of citrate ions / disaccharide units of hyaluronic acid varies from 0.001 to 10 or from 0.001 to 4, preferably still from 0.01 to 3, more preferably from 0.01 to 2, for example from 0.01 to 1.00 or from 0.015 to 0.500.

[0118] The amount of zinc ions added makes it possible to achieve a zinc ion concentration in the hydrogel not exceeding 20 mM, or not exceeding 7 mM or not exceeding 5 mM or not exceeding 3.5 mM or not exceeding 2 mM or not exceeding 1.6 mM, or not exceeding 1.15 mM, or not exceeding 0.8 mM. The amount of zinc ions added makes it possible to achieve a zinc ion concentration in the hydrogel typically ranging from 0.10 to 20 mM or from 0.10 to 7 mM or from 0.10 to 5 mM or from 0.10 to 3.5 mM or from 0.10 to 2 mM or from 0.10 to 1.6 mM, or from 0.10 to 1.15 mM or from 0.10 to 0.8 mM. In certain embodiments, the amount of zinc ions added makes it possible to achieve a zinc ion concentration in the hydrogel ranging from 0.10 to 1.6 mM, more preferably from 0.10 to 1.15 mM, even more preferably from 0.1 to 0.8 mM, preferably from 0.3 to 0.8 mM.

[0119] The molar ratio of citrate ions to added zinc varies from 1 to 20, preferably from 5 to 20 or from 5 to 15 or from 6 to 15 or from 6 to 10.

[0120] The citrate and zinc ions may be added before or after contacting the crosslinked and / or non-crosslinked polysaccharide with the preferably buffered physiological saline solution comprising phosphate or carbonate or sulfate salts or mixtures thereof. The citrate and zinc ions may be added sequentially, either before or after contacting the crosslinked and / or non-crosslinked polysaccharide with the preferably buffered physiological saline solution. For example, the citrate ions may be added first and then the zinc ions, or the zinc ions may be added first and then the citrate ions. However, the addition of the zinc ions before the addition of the citrate ions may only be done when the addition of the citrate and zinc ions is done before contacting the crosslinked and / or non-crosslinked polysaccharide with the preferably buffered physiological saline solution.Alternatively, the citrate and zinc ions can be added concomitantly, either before or after the step of contacting the crosslinked and / or non-crosslinked polysaccharide with the physiological saline solution, preferably buffered.

[0121] In other embodiments, the addition of the citrate and zinc ions is concomitant with the contacting of the crosslinked and / or non-crosslinked polysaccharide with the physiological saline solution, preferably buffered. In this case, the physiological saline solution, preferably buffered, may comprise phosphate and / or carbonate and / or sulfate salts or mixtures thereof, and further comprise citrate and zinc ions.

[0122] The citrate ions may be added in powder form or in the form of a solution. The solution may be prepared by adding citric acid to water or to physiological saline, preferably buffered, for example to a buffered physiological saline solution comprising phosphate or carbonate or sulfate salts or mixtures thereof. The solution may be prepared by adding sodium citrate, calcium citrate, potassium citrate or magnesium citrate to water or to physiological saline, preferably buffered, for example to a buffered physiological saline solution comprising phosphate or carbonate or sulfate salts or mixtures thereof.

[0123] Zinc ions can be added in powder form or as a solution. The solution can be prepared by adding zinc acetate and / or zinc chloride and / or zinc sulfate and / or zinc oxide and / or zinc gluconate to water.

[0124] Preferably, the zinc and citrate ions may be added as a solution comprising zinc and citrate ions. A solution comprising both citrate and zinc ions is then added during the preparation of the hydrogel. The solution may be prepared by adding citric acid to water and then adding zinc salts (e.g., zinc acetate and / or zinc chloride and / or zinc sulfate and / or zinc oxide and / or zinc gluconate), preferably by adding zinc chloride. The solution may be prepared by adding sodium citrate, calcium citrate, potassium citrate, or magnesium citrate to water and then adding zinc salts (e.g., zinc acetate and / or zinc chloride and / or zinc sulfate and / or zinc oxide and / or zinc gluconate), preferably by adding zinc chloride.Alternatively, the solution may be prepared by adding citric acid to a physiological saline solution, preferably buffered, followed by the addition of zinc salts (e.g., zinc acetate and / or zinc chloride and / or zinc sulfate and / or zinc oxide and / or zinc gluconate). Alternatively, the solution may be prepared by adding sodium citrate or calcium citrate, potassium citrate or magnesium citrate to a physiological saline solution, preferably buffered, followed by the addition of zinc salts (e.g., zinc acetate and / or zinc chloride and / or zinc sulfate and / or zinc oxide and / or zinc gluconate). Preferably, the zinc salt is zinc chloride. The buffered physiological solution may be, for example, a phosphate buffer. The phosphate buffer can be PBS buffer with a pH around physiological pH (6.8-7.8) (CAS No: 7647-14-5, 7447-40-7).Preferably, the buffer is a phosphate buffer, particularly a saline buffer of NaH2PO4 / Na2HPC>4 or KH2PO4 / K2HPO4. If necessary, the pH of the solution can be adjusted to reach a physiological pH (6.8-7.8), for example by adding sodium hydroxide. Thus, in certain embodiments, a phosphate buffer solution comprising zinc and citric acid, the pH of which varies from 6.8 to 7.8, is added during the preparation of the hydrogel. When sodium citrate, calcium citrate, potassium citrate or magnesium citrate is used, it is typically not necessary to adjust the pH.

[0125] The preparation of a hydrogel from the crosslinked and / or non-crosslinked polysaccharide may be carried out in a conventional manner, except that zinc ions and citrate ions are added during the preparation of the hydrogel. Thus, the preparation of a hydrogel from the crosslinked and / or non-crosslinked polysaccharide may comprise one or more of the following conventional steps:

[0126] - pH adjustment (1);

[0127] Dilution (2);

[0128] Purification (3);

[0129] - Addition of at least one additional component (4); Extrusion (5).

[0130] These steps, well known to those skilled in the art, may be as described below. They may be at least partly concomitant.

[0131] The conventional steps can be carried out in the following sequential manner: possible pH adjustment (1) then possible dilution (2) then possible purification (3) then possible addition of an additional component (4) then possible extrusion (5). They can also be carried out in a different order. Advantageously, the extrusion step (5) is carried out last, when at least one of the other conventional steps is implemented. It can also be carried out several times and be inserted between the other conventional steps described.

[0132] For example, the conventional steps may be performed in the following sequential manner: (1), (2), (3), (4), (5); or (2), (1), (3), (4), (5); or (2) (1), (4), (5); or (2), (4), (5); or (1), (4), (5); or (2), (4), (3), (5); or (2), (4), (1), (5); or (2), (4), (5); or (4), (2), (1); or (4), (1), (2) or (2), (3), (4), (5); or (4), (2), (3), (5); or (2), (4), (1); or (1), (5), (3), (4); or (1), (5), (4); or (2), (4). Steps (2), (3), (4) and (5) may be concurrent. For example, the preparation of the hydrogel may comprise the following sequence: (2) and (4) are carried out concomitantly.

[0133] Citrate ions (in powder form or in solution) and zinc ions (in powder form or in solution) may be added at the time of, before, or after any of these conventional steps. Typically, when adding citrate ions in powder form, neutralization of the effect of citrate ions on the pH of the hydrogel may be achieved. Citrate and zinc ions are preferably added in the form of a solution comprising zinc and citrate ions. The solution is as described above.

[0134] Citrate ions (in powder form or in solution) and / or zinc ions (in powder form or in solution) are preferably added during step (2) or (4), preferably during step (4). Steps (2) and (4) can be carried out concomitantly.

[0135] In one variant, the citrate ions and / or the zinc ions are added before the extrusion step (5) so as to obtain a homogeneous gel.

[0136] When a purification step (3) is implemented, the citrate ions and / or the zinc ions are advantageously added after the purification step (3). The addition of the citrate ions and / or the zinc ions after the purification step ensures better control of the concentration of citrate ions and / or the zinc ions in the prepared hydrogel.

[0137] Preferably, citrate ions and / or zinc ions are added between the purification (3) and extrusion (5) steps.

[0138] The addition of citrate and zinc ions can be carried out after the dilution step (2) or during the dilution step (2), for example the citrate ions can be added into the aqueous dilution solvent.

[0139] Preferably, the citrate ions in powder form or in solution (advantageously the solution comprising citrate ions) and / or the zinc ions are (are) added during the dilution step (2) and / or during the step of adding at least one additional component (4), preferably during the step of adding at least one additional component (4). In particular, in certain embodiments, the addition of the citrate ions and / or the zinc ions (advantageously of the solution comprising citrate and zinc ions) is concomitant with the step of adding at least one additional component (4).

[0140] Preferably, the citrate ions in powder form or in solution (advantageously the solution comprising citrate ions) and / or the zinc ions are (are) added during the dilution step (2) and / or during the step of adding at least one additional component (4), preferably during the step of adding at least one additional component (4). In particular, in certain embodiments, the addition of the citrate ions and / or the zinc ions (advantageously of the solution comprising citrate and zinc ions) is concomitant with the step of adding at least one additional component (4).

[0141] In particular, in certain embodiments, the addition of zinc ions and citrate ions, preferably in the form of a solution comprising citrate and zinc ions, is concomitant with the addition of an anesthetic solution.

[0142] In particular, in certain embodiments, the addition of zinc ions and citrate ions, preferably in the form of a solution comprising citrate and zinc ions, is concomitant with the addition of a lubricating agent.

[0143] In some embodiments, the solution comprising zinc ions and added citrate ions may comprise other components, in particular a lubricating agent, for example, non-crosslinked hyaluronic acid, non-crosslinked heparosan or a mixture thereof.

[0144] The steps of dilution (2), addition of at least one additional component (4) and addition of citrate and / or zinc ions may be concomitant.

[0145] Citrate and / or zinc ions may be added after the pH adjustment step (1). Citrate ions may be added between the pH adjustment (1) and extrusion (5) steps when both steps are implemented.

[0146] pH adjustment (1)

[0147] The method of preparing the hydrogel may include a step of adjusting the pH of the hydrogel to achieve the desired pH (pH of 6.8-7.8).

[0148] Dilution (2)

[0149] The method for preparing the hydrogel may comprise a step of diluting the crosslinked and / or non-crosslinked polysaccharide. The dilution step makes it possible to adapt the concentration of crosslinked and / or non-crosslinked polysaccharide in the prepared hydrogel. In particular, an aqueous solvent is added to the crosslinked and / or non-crosslinked polysaccharide, for example, a physiological saline solution, possibly buffered by the presence of salts, such as phosphate or carbonate or sulfate salts or mixtures thereof. More particularly, the added aqueous solvent has a pH around the physiological pH (6.8-7.8). The polysaccharide concentration obtained following the dilution step advantageously varies from 1 mg / g to 50 mg / g of hydrogel, more advantageously from 5 mg / g to 35 mg / g of hydrogel, even more advantageously from 10 mg / g to 30 mg / g of hydrogel. Purification (3)

[0150] The process for preparing the hydrogel may comprise at least one purification step. The purification step aims to remove any undesirable impurities. These undesirable impurities may result from the crosslinking of the polysaccharide, for example from step (a2) described above. Such impurities may comprise, for example, the residual crosslinking agent, in particular of the epoxy type, which has not reacted. This step may also allow a liquid exchange to be carried out, for example a buffer exchange.

[0151] The purification step can therefore be particularly implemented when the hydrogel comprises a crosslinked polysaccharide.

[0152] Purification can be carried out by dialysis or by filtration, for example by dynamic tangential filtration (“DGF” for Dynamic Cross-flow Filtration).

[0153] Addition of additional components (4)

[0154] The method for preparing the hydrogel may comprise a step of adding at least one additional component. The additional component may be selected from anesthetic agents, antioxidants, lubricating agents, amino acids, peptides, proteins such as collagen and silk fibroin, vitamins, elements such as silicon (e.g. via the addition of orthosilicic acid), minerals, nucleic acids, nucleotides or polynucleotides such as PDRN, nucleosides, coenzymes, adrenergic derivatives, sodium dihydrogen phosphate monohydrate and / or dihydrate, sodium chloride and a mixture thereof.

[0155] Non-crosslinked polysaccharides, in particular non-crosslinked hyaluronic acid, non-crosslinked heparosan or their mixture, may be cited as an example of a lubricating agent.

[0156] Examples of anesthetics include, but are not limited to, 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, Guafecainol, 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, and one of their salts, in particular a hydrochloride salt, or a mixture thereof. Preferably, the hydrogel according to the invention comprises an anesthetic agent as defined above and in particular lidocaine, mepivacaine or one of their salts such as the hydrochloride.,

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] Examples of nucleic acids include, but are not limited to, adenosine, cytidine, guanosine, thymidine, cytodine, their derivatives, and a mixture thereof. As coenzymes, coenzyme Q10, CoA, NAD, NADP, and mixtures thereof may be cited.

[0162] As adrenaline derivatives, adrenaline, noradrenaline and a mixture thereof may be cited. Extrusion (5)

[0163] The process for preparing the hydrogel may comprise one or more extrusion steps. This extrusion step makes it possible to obtain a more homogeneous hydrogel, in particular with the most constant extrusion force possible, i.e., the most regular possible. For example, the extrusion step may be carried out using a sieve whose performance has a diameter of between 50 and 2000 μm. A person skilled in the art knows how to select the perforation diameter according to the desired mechanical properties of the hydrogel.

[0164] Sterilization of the hydrogel (step (2))

[0165] The method of the present invention comprises a step of sterilizing the prepared hydrogel. Sterilization is preferably carried out by heat, for example in an autoclave. 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 at a plateau time ranging from 1 minute to 20 minutes with F0 > 15. The sterilizing value F0 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 can be carried out in particular by gamma ray, UV radiation or by means of ethylene oxide.

[0166] The hydrogel obtained at the end of the process typically has a pH ranging from 6.8 to 7.8 (physiological pH).

[0167] METHOD 2

[0168] The present invention also relates to a method for preparing a sterile hydrogel comprising a crosslinked polysaccharide and optionally a non-crosslinked polysaccharide, and further comprising zinc ions, the method comprising the following steps:

[0169] (0) preparation of a crosslinked polysaccharide from a crosslinking reaction medium comprising one or more polysaccharide(s), one or more crosslinking agent(s), a solvent and zinc ions in an amount allowing the preparation of a hydrogel comprising at most 20 mM of zinc ions;

[0170] (1) preparation of a hydrogel from the crosslinked polysaccharide obtained at the end of step (0) and optionally from a non-crosslinked polysaccharide, the preparation of the hydrogel comprising bringing the crosslinked polysaccharide into contact with a physiological saline solution, preferably buffered, the physiological saline solution, preferably buffered, comprising phosphate or carbonate or sulfate salts or mixtures thereof;

[0171] (2) sterilization, preferably by heat, of the hydrogel obtained at the end of step (1) to obtain a sterile hydrogel; in which:

[0172] - the crosslinking reaction medium further comprises citrate ions in an amount sufficient to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel, the molar ratio [citrate ions present in the reaction medium] / [zinc ions present in the reaction medium] ranging from 1 to 20; or

[0173] - step (1) further comprises, before bringing the crosslinked polysaccharide into contact with the physiological saline solution, preferably buffered, a step of adding citrate ions in a quantity sufficient to reach a citrate ion concentration of at least 0.1 mM in the hydrogel, the molar ratio [citrate ions added] / [zinc ions present in the reaction medium] ranging from 1 to 20; or

[0174] - the physiological saline solution, preferably buffered, further comprises citrate ions in a quantity sufficient to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel, the molar ratio [citrate ions present in the physiological saline solution, preferably buffered] / [zinc ions present in the reaction medium] ranging from 1 to 20.

[0175] The crosslinked polysaccharide may in particular be prepared by a process comprising the following steps:

[0176] (a1) preparing a crosslinking reaction medium comprising one or more polysaccharide(s), one or more crosslinking agent(s), a solvent, zinc ions in an amount allowing the preparation of a hydrogel comprising at most 20 mM of zinc ions and optionally citrate ions in an amount sufficient to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel; and

[0177] (a2) reacting the reaction medium to obtain a crosslinked polysaccharide.

[0178] When the crosslinked polysaccharide is prepared from a polysaccharide previously modified by the introduction of functional groups capable of reacting with each other and forming covalent intermolecular bonds (i.e. in the absence of a crosslinking agent), step (0) comprises the preparation of a crosslinked polysaccharide from a crosslinking reaction medium comprising one or more modified polysaccharide(s), a solvent and zinc ions in an amount allowing the preparation of a hydrogel comprising at most 20 mM of zinc ions.

[0179] The crosslinked polysaccharide may in particular be prepared by a process comprising the following steps:

[0180] (a1) preparing a crosslinking reaction medium comprising one or more modified polysaccharide(s), a solvent, zinc ions in an amount allowing the preparation of a hydrogel comprising at most 20 mM zinc ions and optionally citrate ions in an amount sufficient to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel; and

[0181] (a2) reacting the reaction medium to obtain a crosslinked polysaccharide.

[0182] Steps (0), (a1) and (a2) of the process according to method 2 are as described previously in the section “The crosslinked and / or non-crosslinked polysaccharide”, except that the crosslinking reaction medium further comprises zinc ions and optionally citrate ions. When the citrate ions are not present in the crosslinking reaction medium, they are added before the step of bringing the crosslinked polysaccharide into contact with the physiological saline solution, preferably buffered, or in the physiological saline solution, preferably buffered. The polysaccharide is as described in the section “The crosslinked and / or non-crosslinked polysaccharide”.

[0183] The zinc ions are present in the reaction medium in an amount that does not exceed a zinc ion concentration in the hydrogel of 20 mM, or 7 mM or 5 mM or 3.5 mM or 2 mM or 1.6 mM, or 1.15 mM, or 0.8 mM. The zinc ions are typically present in the reaction medium in an amount that achieves a zinc ion concentration in the hydrogel ranging from 0.10 to 20 mM or 0.10 to 7 mM or 0.10 to 5 mM or 0.10 to 3.5 mM or 0.10 to 2 mM or 0.10 to 1.6 mM, or 0.10 to 1.15 mM or 0.10 to 0.8 mM. In certain embodiments, the zinc ions are present in the reaction medium in an amount making it possible to achieve a zinc ion concentration in the hydrogel varying from 0.10 to 1.6 mM, more preferably from 0.10 to 1.15 mM, even more preferably from 0.1 to 0.8 mM, preferentially from 0.3 to 0.8 mM.

[0184] When citrate ions are present in the reaction medium, they are present in an amount making it possible to achieve a citrate ion concentration in the hydrogel of at least 0.1 mM and generally at most 150 mM, 100 mM, 50 mM, 20 mM or 15 mM. Preferably, they are present in an amount making it possible to achieve a concentration of citrate ions in the hydrogel varying from 0.1 to 150 mM or from 0.1 to 100 mM or from 0.1 to 50 mM or from 0.1 to 20 mM or from 0.1 to 15 mM or from 0.3 to 15 mM or from 0.5 to 15 mM or from 1 to 10 mM or from 1.5 to 10 mM or from 2 to 10 mM or from 3 to 10 mM or from 3 to 8 mM or from 5 to 8 mM. No covalent bond is formed between the polysaccharide and the citrate ions.

[0185] When citrate ions are not present in the reaction medium, they are added before contact with the physiological saline solution, preferably buffered, or in the physiological saline solution, preferably buffered, in a quantity sufficient to reach the concentrations indicated above.

[0186] The molar ratio [citrate ions present in the reaction medium] / [zinc ions present in the reaction medium] or [added citrate ions] / [zinc ions present in the reaction medium] or [citrate ions present in the physiological saline solution, preferably buffered] / [zinc ions present in the reaction medium] varies from 1 to 20, preferably from 5 to 20 or from 5 to 15 or from 6 to 15 or from 6 to 10.

[0187] The zinc ions present in the reaction medium may result from the addition of zinc salts to the reaction medium such as zinc sulfate, zinc chloride, zinc gluconate, preferably zinc chloride.

[0188] The citrate ions present in the reaction medium can result from the addition of citric acid, in powder form or in the form of an aqueous solution to the reaction medium.

[0189] The citrate ions present in the reaction medium may result from the addition of sodium citrate, calcium citrate, potassium citrate or magnesium citrate, in powder form or in the form of an aqueous solution to the reaction medium.

[0190] When citrate ions are not present in the reaction medium, they are added before the step of contacting with the physiological saline solution, preferably buffered, or in the physiological saline solution, preferably buffered. They can be added in the form of a solution. The solution can be prepared by adding citric acid or sodium citrate, or calcium citrate, or potassium citrate or magnesium citrate in water or in a physiological saline solution, preferably buffered, for example a saline solution, preferably buffered comprising phosphate or carbonate or sulfate salts or mixtures thereof. In other embodiments, the physiological saline solution, preferably buffered, added during the preparation of the hydrogel comprises citrate ions.

[0191] At the end of step (0) or (a2), the crosslinked polysaccharide is typically in the form of a gel comprising zinc ions, and possibly citrate ions. This gel is generally directly used in the rest of the process of the invention (step (1)).

[0192] The preparation of a hydrogel (step (1)) from the crosslinked polysaccharide obtained at the end of step (0) or (a2) can be carried out in a conventional manner. In particular, the preparation of a hydrogel from the crosslinked polysaccharide obtained at the end of step (0) or (a2) typically comprises one or more of the following conventional steps:

[0193] - pH adjustment (1);

[0194] Dilution (2);

[0195] Purification (3);

[0196] - Addition of at least one additional component (4);

[0197] Extrusion (5).

[0198] These steps, well known to those skilled in the art, may be as described above in relation to method 1. They may be implemented in the sequential manners described above.

[0199] Sterilization (step (2)) is as described in relation to step (2) of method 1.

[0200] METHOD 3

[0201] The invention also relates to a method for preparing a sterile hydrogel comprising a crosslinked polysaccharide and optionally a non-crosslinked polysaccharide and further comprising zinc ions, the method comprising the following steps: (0') preparing a crosslinking reaction medium comprising:

[0202] - one or more polysaccharide(s),

[0203] - one or more crosslinking agent(s),

[0204] - citrate ions in an amount sufficient to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel, - zinc ions in an amount allowing the preparation of a hydrogel comprising at most 20 mM zinc ions, and

[0205] - a physiological saline solution, preferably buffered, comprising phosphate or carbonate or sulfate salts or mixtures thereof; the molar ratio [citrate ions present in the reaction medium] / [zinc ions present in the reaction medium] ranging from 1 to 20, the preparation of the reaction medium being carried out by adding the citrate ions before any contact of the zinc ions with the physiological saline solution;

[0206] (0) preparation of a crosslinked polysaccharide from the reaction medium obtained at the end of step (0');

[0207] (1) preparation of a hydrogel from the crosslinked polysaccharide obtained at the end of step (0) and optionally from a non-crosslinked polysaccharide;

[0208] (2) sterilization, preferably by heat, of the hydrogel obtained at the end of step (1) to obtain a sterile hydrogel.

[0209] When the crosslinked polysaccharide is prepared from a polysaccharide previously modified by the introduction of functional groups capable of reacting with each other and forming covalent intermolecular bonds (i.e. in the absence of a crosslinking agent), step (0') comprises the preparation of a crosslinking reaction medium comprising:

[0210] - one or more previously modified polysaccharide(s),

[0211] - citrate ions in an amount sufficient to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel,

[0212] - zinc ions in an amount allowing the preparation of a hydrogel comprising at most 20 mM of zinc ions, and

[0213] - a physiological saline solution, preferably buffered, comprising phosphate or carbonate or sulfate salts or mixtures thereof; the molar ratio [citrate ions present in the reaction medium] / [zinc ions present in the reaction medium] ranging from 1 to 20, the preparation of the reaction medium comprising an addition of the citrate ions before any contact of the zinc ions with the physiological saline solution.

[0214] The components of the reaction medium are as described previously. Step (0) of the process according to method 3 is as described previously in the section “The crosslinked and / or non-crosslinked polysaccharide”, except that the crosslinking reaction medium comprises zinc ions, citrate ions and a physiological saline solution, preferably buffered, comprising phosphate or carbonate or sulfate salts or mixtures thereof.

[0215] The preparation of a hydrogel (step (1)) from the crosslinked polysaccharide obtained at the end of step (0) can be carried out in a conventional manner. In particular, the preparation of a hydrogel from the crosslinked polysaccharide obtained at the end of step (0) typically comprises one or more of the following conventional steps:

[0216] - pH adjustment (1);

[0217] Dilution (2);

[0218] Purification (3);

[0219] - Addition of at least one additional component (4);

[0220] Extrusion (5).

[0221] These steps, well known to those skilled in the art, may be as described above in relation to method 1. They may be implemented in the sequential manners described above.

[0222] Sterilization (step (2)) is as described in relation to step (2) of method 1.

[0223] METHOD 1 OR 2 OR 3: Optional step

[0224] The method of the present invention (method 1 or 2 or 3) may further comprise a step of conditioning the hydrogel. The conditioning of the hydrogel is typically carried out in an injection device. The conditioning is preferably carried out just before the sterilization step (step (2)). Thus, the sterile hydrogel may be in the form of an injection device pre-filled with the hydrogel, for example a syringe pre-filled with the hydrogel.

[0225] Sterile hydrogel

[0226] The present invention also relates to a sterile hydrogel comprising a crosslinked and / or non-crosslinked polysaccharide obtained or obtainable by the method of the present invention (method 1 or 2 or 3). The sterile hydrogel comprises zinc ions and citrate ions in a citrate ion / zinc ion molar ratio ranging from 1 to 20, the citrate ion concentration in the hydrogel being at least 0.1 mM and the zinc concentration in the hydrogel not exceeding 20 mM.

[0227] Preferably, the zinc ion concentration in the hydrogel ranges from 0.10 to 20 mM or from 0.10 to 7 mM or from 0.10 to 5 mM or from 0.10 to 3.5 mM or from 0.10 to 2 mM or from 0.10 to 1.6 mM, or from 0.10 to 1.15 mM or from 0.10 to 0.8 mM. In some embodiments, the zinc ion concentration in the hydrogel ranges from 0.10 to 1.6 mM, more preferably from 0.10 to 1.15 mM, even more preferably from 0.1 to 0.8 mM, preferably from 0.3 to 0.8 mM.

[0228] Preferably, the concentration of citrate ions in the hydrogel varies from 0.1 to 150 mM or from 0.1 to 100 mM or from 0.1 to 50 mM or from 0.1 to 20 mM or from 0.1 to 15 mM or from 0.3 to 15 mM or from 0.5 to 15 mM or from 1 to 10 mM or from 1.5 to 10 mM or from 2 to 10 mM or from 3 to 10 mM or from 3 to 8 mM or from 5 to 8 mM.

[0229] The sterile hydrogel obtained or obtainable by the method of the present invention (method 1 or 2 or 3) has a physiological pH, Le., ranging from 6.8 to 7.8. The pH of the sterile hydrogel is preferably greater than or equal to 6.9 and less than or equal to 7.4; 7.3; 7.2; 7.1 or 7.

[0230] The sterile hydrogel obtained by the method of the present invention (method 1 or 2 or 3) and comprising a crosslinked polysaccharide, advantageously has a phase angle 5 less than or equal to 45°, at 1 Hz for a deformation of 0.1% or a pressure of 1 Pa, preferably a phase angle 5 ranging from 2° to 45° or ranging from 20° to 45°.

[0231] The hydrogel obtained or obtainable by the method of the present invention is preferably an injectable hydrogel, i.e. one which can flow and be injected manually by means of a syringe equipped with a needle with a diameter ranging from 0.1 to 0.5 mm, for example a 32G, 30G, 27G, 26G, 25G hypodermic needle. The hydrogel obtained or obtainable by the method of the present invention may comprise from 0.1 to 5% by weight, preferably from 1 to 3% by weight, of polysaccharide (total weight of polysaccharide, i.e. total weight of crosslinked and / or non-crosslinked polysaccharide, for example crosslinked and / or non-crosslinked hyaluronic acid), relative to the total weight of the hydrogel.Thus, when the hydrogel comprises, as the only polysaccharide, a non-crosslinked polysaccharide, the hydrogel obtained by the method of the present invention may therefore comprise from 0.1 to 5% by weight, preferably from 1 to 3% by weight, of non-crosslinked polysaccharide (for example non-crosslinked hyaluronic acid), relative to the total weight of the hydrogel. When the hydrogel comprises, as the only polysaccharide, a crosslinked polysaccharide, the hydrogel obtained by the method of the present invention may therefore comprise from 0.1 to 5% by weight, preferably from 1 to 3% by weight, of crosslinked polysaccharide (for example crosslinked hyaluronic acid), relative to the total weight of the hydrogel.When the hydrogel comprises the mixture of a crosslinked and non-crosslinked polysaccharide, the hydrogel obtained by the method of the present invention may therefore comprise from 0.1 to 5% by weight, preferably from 1 to 3% by weight, of a mixture of non-crosslinked and crosslinked polysaccharide (for example non-crosslinked and / or crosslinked hyaluronic acid), relative to the total weight of the hydrogel. In particular, the content of non-crosslinked polysaccharide (for example hyaluronic acid) may vary from 0.5 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 (for example hyaluronic acid) present in the hydrogel.

[0232] The total polysaccharide concentration in the hydrogel obtained by the method of the present invention advantageously varies from 1 mg / g to 50 mg / g of hydrogel, more preferably from 5 mg / g to 35 mg / g of hydrogel, even more preferably from 10 mg / g to 30 mg / g of hydrogel. Preferably the polysaccharide is hyaluronic acid, even more preferably sodium hyaluronate.

[0233] The total polysaccharide concentration in the hydrogel obtained by the method of the present invention advantageously varies from 1 mg / g to 50 mg / g of hydrogel, more preferably from 5 mg / g to 35 mg / g of hydrogel, even more preferably from 10 mg / g to 30 mg / g of hydrogel. Preferably the polysaccharide is hyaluronic acid, even more preferably sodium hyaluronate.

[0234] When the hydrogel comprises a crosslinked polysaccharide, the crosslinked polysaccharide preferably has a molar crosslinking rate of less than or equal to 10%. Preferably, the hydrogel comprises a crosslinked polysaccharide whose molar crosslinking rate is greater than 0 and less than or equal to 6%. Even more preferably, the hydrogel comprises a crosslinked polysaccharide whose molar crosslinking rate is greater than 0 and less than or equal to 4%. Even more preferably, the hydrogel comprises a crosslinked polysaccharide whose molar crosslinking rate is greater than 0 and less than or equal to 2%, preferably less than or equal to 1%, still preferably less than or equal to 0.8%, in particular ranging from 0.1% to 0.5% (number of moles of crosslinking agent(s) per 100 moles of repeating unit of the polysaccharide(s).

[0235] When the hydrogel comprises a crosslinked polysaccharide, the crosslinked polysaccharide preferably has a degree of modification (MOD) of less than or equal to 10%, preferably less than or equal to 6%, preferably less than or equal to 4%, preferably less than or equal to 2%, more preferably less than or equal to 1%. Advantageously, the crosslinked polysaccharide has a degree of modification (MOD) of less than or equal to 1.8%, more preferably less than or equal to 1.5%, preferably less than or equal to 1.2%, even more preferably less than 1%.

[0236] In some embodiments, the hydrogel comprises an anesthetic agent. The anesthetic agent may be as described above, in particular the anesthetic agent may be mepivacaine, lidocaine or a salt thereof; more particularly in the form of a hydrochloride salt; preferably in amounts ranging from 0.1 to 30 mg / ml, for example from 0.5 to 10 mg / ml or more preferably from 2 to 6 mg / ml.

[0237] The sterile hydrogels prepared according to the method of the invention are particularly useful for filling and / or replacing tissues, in particular soft tissues, in particular by injecting the hydrogel into the tissue. In addition to filling soft tissues, they can deliver biostimulant effects.

[0238] In some embodiments, the sterile hydrogel is injected into the subject subcutaneously. Ideally, the hydrogel allows for a slow release of zinc ions in the subject after injection. This release of zinc ions must remain significantly lower than the toxic dose of zinc ions. For example, for a hydrogel containing zinc ions, the subcutaneous release in the subject after injection must be less than 0.1 mmol / day. They can be injected using any of the methods known to those skilled in the art. In particular, they can be administered by means of an injection device suitable for intraepidermal and / or intradermal and / or subcutaneous and / or supraperiosteal injection. The injection device can in particular be chosen from a syringe, a set of micro-syringes, a wire, a laser or hydraulic device, an injection gun, a needle-free injection device, or a micro-needle roller.

[0239] The sterile hydrogels prepared according to the method of the invention are preferably injected subcutaneously.

[0240] They can involve deep applications, mid-line applications and / or superficial applications.

[0241] They may have therapeutic and / or cosmetic and / or cosmeceutical applications.

[0242] In the cosmetic field, hydrogels can be particularly useful for compensating for tissue volume losses due to aging. They can be used in the prevention and / or cosmetic treatment of an alteration in the surface appearance of the skin. For example, hydrogels can be used in the cosmetic field to prevent and / or treat the alteration of the viscoelastic or biomechanical properties of the skin; to fill volume defects of the skin, in particular to fill wrinkles, fine lines and scars; to reduce nasolabial folds and bitterness lines; to increase the volume of the cheekbones, chin or lips; to restore facial volume, in particular the cheeks, temples, the oval of the face, and the area around the eyes; to reduce the appearance of wrinkles and fine lines.

[0243] The present invention also relates to the cosmetic use of a hydrogel as described above for filling tissues, in particular soft tissues, in particular to compensate for losses of tissue volume due to aging.

[0244] The following examples are given for illustrative purposes, but should in no way be considered as limiting the present invention.

[0245] EXAMPLES

[0246] 1. Materials

[0247] - Non-crosslinked sodium hyaluronate

[0248] - BDDE (Sigma Aldrich)

[0249] - Citric acid (Sigma Aldrich) (CAS No: 5949-29-1)

[0250] - Sodium citrate (Sigma Aldrich) (CAS No: 6132-04-3)

[0251] - Zinc chloride (Sigma Aldrich) (CAS No: 7646-85-7)

[0252] - Commercial zinc citrate (Thermo Fisher) (CAS 5990-32-9)

[0253] - NaOH 0.25M

[0254] - 1 M HCl

[0255] - PBS Phosphate Buffer (BBraun),

[0256] - Lidocaine hydrochloride

[0257] - Three-dimensional agitator

[0258] - DHR-2 Rheometer

[0259] - Dynamometer and test bench

[0260] - Homogenizer Paddle mill

[0261] - Sterile polyethylene bag 2. Methods

[0262] Measurement of viscoelastic properties

[0263] The viscoelastic properties of the obtained hydrogels were measured using a rheometer (DHR-2) having a stainless steel cone (1° - 40 mm) with cone-plane geometry and an anodized aluminum peltier plane (42 mm) (air gap 24 μm).

[0264] 0.5 g of sterilized hydrogel is deposited between the Peltier plane and said cone. Then a stress scan is carried out at 1 Hz and 25°C. The elastic modulus G', the viscous modulus G” and the phase angle 5 are reported for a stress of 5 Pa. The measurements are carried out in the linear LVER domain.

[0265] 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.

[0266] 3. Examples

[0267] 3.1 Example 1a

[0268] A cross-linked hyaluronic acid hydrogel is prepared from a high molecular weight hyaluronic acid 4 MDa and BDDE in a 0.25M aqueous sodium hydroxide solution (cross-linking for 72 hours at 21°C). The cross-linked polysaccharide has a cross-linking rate of 2%. Phosphate buffer and 1 N HCl solution are then added to the cross-linked polysaccharide until a pH of 7.3 ± 0.5 is obtained. The resulting hydrogel is homogenized using a three-dimensional stirrer. The hydrogel is dialyzed. The resulting hydrogel has a concentration of 15 mg of hyaluronic acid per gram of hydrogel.

[0269] The following are then added to the hydrogels obtained:

[0270] - a solution of high molecular weight sodium hyaluronate as a lubricant (same quantity in the different mixtures);

[0271] - an aqueous solution of lidocaine hydrochloride to obtain 0.3% by weight of lidocaine hydrochloride relative to the weight of the final hydrogel;

[0272] - possibly a solution comprising zinc and citrate ions.

[0273] The solution comprising zinc and citrate ions is prepared as follows. Citric acid (in powder form) is first dissolved in phosphate buffer, then zinc chloride is dissolved (ZnCh powder), and finally 5M NaOH is added to adjust the pH to a physiological level. The objective is to make a solution concentrated 100 times (in zinc and citrate ions) compared to the actual concentration desired in the final hydrogel. This is to avoid too strong a dilution effect of the hydrogel due to the addition of the zinc and citric acid solution.

[0274] The solution containing zinc and citrate ions was added at the same time as the anesthetic solution, after the addition of the high molecular weight sodium hyaluronate solution. The resulting hydrogels were sieved and then packaged in a syringe.

[0275] Finally, the obtained hydrogels were sterilized in an autoclave (plateau temperature between 121°C and 135°C with FO > 15).

[0276] After sterilization, hydrogels A1-A6 were analyzed. No hydrogel showed precipitation. The elastic modulus G' and phase angle 5 were determined. The results are shown in Table 1 below.

[0277] The hydrogels have a molar crosslinking rate of 2%. Table 1

[0278] 1 AG' (%) = (G' after sterilization - G' before sterilization) / (G' before sterilization) * 100

[0279] 2At 5 (%) = (5 after sterilization - 5 before sterilization) / ( 5 before sterilization) *100 * it was considered that 1 mL of hydrogel weighs one gram.

[0280] It is observed that the hydrogels prepared from a process according to the invention comprising a step of adding a solution comprising zinc ions and citrate ions exhibit lesser modifications in their rheological properties after sterilization compared to hydrogels prepared by an equivalent process without addition of such a solution.

[0281] 3.2 Example 1b

[0282] A cross-linked hyaluronic acid hydrogel is prepared from a high molecular weight hyaluronic acid 4MDa and BDDE in a 0.25M aqueous sodium hydroxide solution (cross-linking for 72 hours at 21°C). The cross-linked polysaccharide has a modification rate of 2%. PBS phosphate buffer and a 1N HCl solution are then added to the cross-linked polysaccharide until a pH of 7.3 ± 0.5 is obtained. The resulting hydrogel is homogenized using a three-dimensional stirrer. The hydrogel is dialyzed. The resulting hydrogel has a concentration of 15 mg of hyaluronic acid per gram of product.

[0283] The following are then added to the hydrogels obtained:

[0284] - a solution of non-crosslinked high molecular weight sodium hyaluronate as a lubricant (same quantity in the different mixtures);

[0285] - possibly a solution comprising zinc and citrate ions.

[0286] The solution comprising zinc and citrate ions is prepared as follows. Citric acid (in powder form) is first dissolved in phosphate buffer, then zinc chloride is dissolved (ZnCh powder), and finally 5M NaOH is added to adjust the pH to a physiological level. The objective is to make a solution concentrated 100 times (in zinc and citrate ions) compared to the actual concentration desired in the final hydrogel. This is to avoid too strong a dilution effect of the hydrogel due to the addition of the zinc and citric acid solution. The solution comprising zinc and citrate ions is added after the addition of the high molecular weight sodium hyaluronate solution.

[0287] The hydrogels obtained were sieved and then packaged in a syringe.

[0288] Finally, the obtained hydrogels are sterilized in an autoclave (plateau temperature between 121°C and 135°C with FO > 15).

[0289] After sterilization, hydrogels B1-B4 were analyzed. No hydrogel showed precipitation. The elastic modulus G', phase angle 5 and stress at the intersection of G' and G”, T were determined. The results are shown in Table 2 below.

[0290] The hydrogels have a molar crosslinking rate of 2%.

[0291] Table 2

[0292] 1 AG' (%) = (G' after sterilization - G' before sterilization) / (G' before sterilization) * 100

[0293] 2 A 5 (%) = (5 after sterilization - 5 before sterilization) / (5 before sterilization) *100

[0294] * 1mL of hydrogel was considered to weigh one gram.

[0295] It is observed that hydrogels prepared from a process according to the invention comprising a step of adding a solution comprising zinc and citrate ions exhibit lesser modifications in their rheological properties after sterilization compared to hydrogels prepared by an equivalent process without addition of such a solution.

[0296] Furthermore, it was observed that the addition of a solution comprising zinc and citrate ions allows the structure of the hydrogels to be preserved from degradation over time (Table 3).

[0297] Table 3

[0298] 1 AG' (%)= (G' T3months - G' T0) / (G' TO) *100

[0299] 2 AG' (%)= (G' T6months - G' T0) / (G' T0) *100

[0300] After 3 months and 6 months at 40°C, the hydrogels prepared from a process according to the invention comprising a step of adding a solution comprising zinc and citrate ions exhibit lesser changes in their rheological properties after sterilization compared to hydrogels prepared by an equivalent process without the addition of such a solution. For hydrogel B4 prepared from a process according to the invention comprising a step of adding a solution comprising zinc and citrate ions, no precipitation is observed either after 3 months or after 6 months.

[0301] 3.3 Example 2

[0302] For comparison, a hydrogel according to example 1 b is made with the incorporation of 0.47 g of commercial zinc citrate (in powder form, molar ratio of citrate ions / zinc ions in the hydrogel = 0.667) in buffer.

[0303] The solution containing commercial zinc citrate is prepared as follows. Zinc citrate is first dissolved in phosphate buffer and then added to 5M NaOH to adjust the pH to a physiological level. The goal is to make a solution 100 times concentrated (in zinc and citrate ions) compared to the actual concentration desired in the final hydrogel. This avoids too strong a dilution effect of the hydrogel due to the addition of the solution containing commercial zinc citrate.

[0304] The solution comprising commercial zinc citrate is added after addition of the high molecular weight sodium hyaluronate solution.

[0305] The hydrogels obtained were sieved and then packaged in a syringe.

[0306] Finally, the obtained hydrogels were sterilized in an autoclave (plateau temperature between 121°C and 135°C with FO > 15).

[0307] The results of the hydrogels observed under the microscope are presented in Figures 1 and 2. The microscope used is: Olympus SZX16, the software: OLYMPUS Stream Start.

[0308] The hydrogel obtained in accordance with the method according to the invention (Fig. 1 - prototype C3) is transparent.

[0309] A precipitate is observed immediately after sterilization of the hydrogel incorporating commercial zinc citrate (Fig. 2), which is not desirable for a hydrogel.

[0310] 3.4 Example 3

[0311] A study conducted by the NAMSA medical analysis laboratory, according to ISO 10993-23 standard “Biological Evaluation of Medical Devices, Part 23 (2021): Tests for Irritations”, made it possible to evaluate the irritant potential of the hydrogels according to the invention after intradermal injection in rabbits.

[0312] The control hydrogels C1 and according to the invention 02 tested are prepared as follows: The crosslinked hyaluronic acid hydrogel is prepared from a high molecular weight hyaluronic acid 1.5MDa and BDDE in a 0.25M aqueous sodium hydroxide solution (crosslinking for 72 hours at 21°C). The crosslinked polysaccharide has a modification rate of 4%. PBS phosphate buffer and a 1N HCl solution are then added to the crosslinked polysaccharide until a pH of 7.3 ± 0.5 is obtained. The hydrogel obtained is homogenized using a three-dimensional stirrer. The hydrogel is subsequently dialyzed. The hydrogels obtained have a concentration of 23 mg of hyaluronic acid per gram of product.

[0313] The resulting cross-linked polysaccharide hydrogel is then divided into 2.

[0314] The following are then added to the hydrogels obtained:

[0315] - for hydrogel C1 and C2 an aqueous solution of lidocaine hydrochloride to obtain 0.3% by weight of lidocaine hydrochloride relative to the weight of the final hydrogel and a solution of non-crosslinked high molecular weight sodium hyaluronate as lubricant (same quantity in the different mixtures);

[0316] - for hydrogel C2, a solution comprising 0.46 mM zinc ions and 2.3 mM citrate ions.

[0317] The solution comprising zinc and citrate ions is prepared as follows. Citric acid (in powder form) is first dissolved in phosphate buffer, then zinc chloride is dissolved (ZnCh powder), and finally 5M NaOH is added to adjust the pH to a physiological level. The objective is to make a solution concentrated 100 times (in zinc and citrate ions) compared to the actual concentration desired in the final hydrogel. This avoids too strong a dilution effect of the hydrogel due to the addition of the zinc and citric acid solution.

[0318] The solution comprising zinc and citrate ions is added after addition of the high molecular weight sodium hyaluronate solution.

[0319] The hydrogels obtained were sieved and then packaged in a syringe.

[0320] Finally, the obtained hydrogels are sterilized in an autoclave (plateau temperature between 121°C and 135°C with F0 > 15). No hydrogel shows precipitation.

[0321] A 0.2 mL dose of each of the sterilized hydrogels C1 and C2 was injected intradermally into 5 separate sites on the sides of the back of three rabbits. The injection sites were observed at 24, 48, and 72 hours post-injection for signs of erythema and edema, and then daily for up to 28 days. Signs of erythema and edema were measured using a 0-4 score scale for each injection site and for each animal. The overall mean score was determined by dividing the sum of the scores by the total number of sites evaluated.

[0322] The hydrogel according to the invention C2 exhibits, over the entire duration of the test, an average irritation score lower than the control hydrogel C1. This thus indicates that the hydrogel C2 prepared according to the invention exhibits a less irritating character than the control hydrogel. 3.5 Example 4

[0323] Two cross-linked hyaluronic acid hydrogels were prepared from a high molecular weight hyaluronic acid 1.5 MDa and BDDE in a 0.25M aqueous sodium hydroxide solution (cross-linked for 1 month at -20°C). The cross-linked polysaccharide had a cross-linking rate of 0.5%. Phosphate buffer and a 1 N HCl solution were then added to the cross-linked polysaccharide until a pH of 7.3 ± 0.5 was obtained. The resulting hydrogel was homogenized using a three-dimensional stirrer. The hydrogel was dialyzed. The resulting hydrogels had a concentration of 23 mg of hyaluronic acid per gram of hydrogel.

[0324] The following are then added to the hydrogels obtained:

[0325] - a solution of high molecular weight sodium hyaluronate as a lubricant (same quantity in the different mixtures);

[0326] - an aqueous solution of lidocaine hydrochloride to obtain 0.3% by weight of lidocaine hydrochloride relative to the weight of the final hydrogel;

[0327] - possibly a solution comprising zinc and citrate ions.

[0328] The solution comprising zinc and citrate ions is prepared as follows. Citric acid (in powder form) is first dissolved in phosphate buffer, then zinc chloride is dissolved (ZnCh powder), and finally 5M NaOH is added to adjust the pH to a physiological level. The objective is to make a solution concentrated 100 times (in zinc and citrate ions) compared to the actual concentration desired in the final hydrogel. This is to avoid too strong a dilution effect of the hydrogel due to the addition of the zinc and citric acid solution.

[0329] The solution containing zinc and citrate ions is added at the same time as the anesthetic solution, after the addition of the high molecular weight sodium hyaluronate solution.

[0330] The hydrogels obtained were sieved and then packaged in a syringe.

[0331] Finally, the obtained hydrogels were sterilized in an autoclave (plateau temperature between 121°C and 135°C with F0 > 15). After sterilization, hydrogels D1 and D2 were analyzed. No hydrogel showed precipitation. The elastic modulus G' and phase angle 5 were determined. The results are shown in Table 4 below.

[0332] The hydrogels have a molar crosslinking rate of 0.5%.

[0333] Table 4

[0334] 1 AG' (%) = (G' after sterilization - G' before sterilization) / (G' before sterilization) * 100

[0335] 2 A 5 (%) = (5 after sterilization - 5 before sterilization) / (5 before sterilization) *100

[0336] * 1 mL of hydrogel was considered to weigh one gram.

[0337] It is observed that hydrogels prepared from a process according to the invention comprising a step of adding a solution comprising zinc and citrate ions exhibit lesser modifications in their rheological properties after sterilization compared to hydrogels prepared by an equivalent process without addition of such a solution.

[0338] Furthermore, it was observed that the addition of a solution comprising zinc and citrate ions allows the structure of the hydrogels to be preserved from degradation over time (Table 5).

[0339] Table 5

[0340] 1 AG' (%)= (G' T6months - G' TO) / (G' TO) *100

[0341] After 6 months at 40°C, the hydrogel prepared from a process according to the invention comprising a step of adding a solution comprising zinc and citrate ions (D2) shows lesser changes in its rheological properties after sterilization compared to the hydrogel prepared by an equivalent process without addition of such a solution (D1). For the hydrogel D2 prepared from a process according to the invention comprising a step of adding a solution comprising zinc and citrate ions, no precipitation is observed after 6 months.

[0342] 3.6 Example 5

[0343] Two 20 mg / g uncrosslinked hyaluronic acid hydrogels were prepared from a high molecular weight hyaluronic acid 1.5 MDa in a buffer solution.

[0344] The solution comprising zinc added to the E1 hydrogel is prepared as follows. Zinc chloride is dissolved (ZnCh powder) in phosphate buffer and finally 5M NaOH is added to adjust the pH to a physiological level.

[0345] The solution comprising zinc and citrate ions added into the E2 hydrogel is prepared as follows. Citric acid (in powder form) is first dissolved in phosphate buffer, then zinc chloride is dissolved (ZnCh powder), and finally 5M NaOH is added to adjust the pH to a physiological level. The objective is to make a solution concentrated 100 times (in zinc and citrate ions) compared to the actual concentration desired in the final hydrogel. This is to avoid too strong a dilution effect of the hydrogel due to the addition of the zinc and citric acid solution.

[0346] Finally, the obtained hydrogels were sterilized in an autoclave (plateau temperature between 121°C and 135°C with F0 > 15).

[0347] Table 6

[0348] * 1 mL of hydrogel was considered to weigh one gram.

[0349] It is observed that the hydrogel prepared from a process according to the invention comprising a step of adding a solution comprising zinc and citrate ions does not exhibit precipitation after sterilization unlike the hydrogel prepared by an equivalent process without addition of citrate ions.

[0350] 3.7 Example 6

[0351] Three cross-linked hyaluronic acid hydrogels were prepared from a high molecular weight hyaluronic acid (1.5 MDa) and BDDE in a 0.25M aqueous sodium hydroxide solution (cross-linked for 3 hours at 52°C). The cross-linked polysaccharide had a cross-linking rate of 8.7%. Phosphate buffer and a 1 N HCl solution were then added to the cross-linked polysaccharide until a pH of 7.3 ± 0.5 was obtained. The resulting hydrogel was homogenized using a three-dimensional stirrer. The hydrogel was dialyzed. The resulting hydrogels had a concentration of 23 mg of hyaluronic acid per gram of hydrogel.

[0352] The following are then added to the hydrogels obtained:

[0353] - a solution of high molecular weight sodium hyaluronate as a lubricant (same quantity in the different mixtures);

[0354] - an aqueous solution of lidocaine hydrochloride to obtain 0.3% by weight of lidocaine hydrochloride relative to the weight of the final hydrogel;

[0355] - possibly a solution containing zinc and sodium citrate.

[0356] - optionally a solution comprising zinc and citric acid. The solution comprising zinc and citric acid is prepared as follows. Citric acid (in powder form) is first dissolved in phosphate buffer, then zinc chloride is dissolved (ZnCh powder) and finally 5M NaOH is added to adjust the pH to a physiological level. The objective is to make a solution concentrated 100 times (in zinc and citrate ions) compared to the actual concentration desired in the final hydrogel. This avoids too strong a dilution effect of the hydrogel due to the addition of the zinc and citric acid solution.

[0357] The solution comprising zinc and sodium citrate is prepared as follows. Sodium citrate (in powder form) is first dissolved in phosphate buffer and then zinc chloride is dissolved (ZnCh powder).

[0358] The solution containing zinc and citrate ions (from citric acid or sodium citrate) is added at the same time as the anesthetic solution, after the addition of the high molecular weight sodium hyaluronate solution.

[0359] The hydrogels obtained were sieved and then packaged in a syringe.

[0360] Finally, the obtained hydrogels were sterilized in an autoclave (plateau temperature between 121°C and 135°C with FO > 15).

[0361] After sterilization, hydrogels F1 - F3 were analyzed. No hydrogel showed precipitation. The elastic modulus G' and phase angle 5 were determined. The results are shown in Table 7 below.

[0362] Table 7

[0363] 1 AG' (%) = (G' after sterilization - G' before sterilization) / (G' before sterilization) * 100

[0364] 2 A 5 (%) = (5 after sterilization - 5 before sterilization) / (5 before sterilization) *100

[0365] * 1mL of hydrogel was considered to weigh one gram.

[0366] It is observed that the hydrogels prepared from a process according to the invention comprising a step of adding a solution comprising zinc and citrate ions exhibit lesser modifications of their rheological properties after sterilization compared to hydrogels prepared by an equivalent process without addition of such a solution. In addition, the hydrogel F3 comprising a step of adding a solution comprising zinc and citrate ions from sodium citrate exhibits lesser modifications of its rheological properties after sterilization compared to hydrogels prepared by a process comprising zinc and citrate ions from citric acid.

Claims

CLAIMS 1. A method of preparing a sterile hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide or a mixture thereof and further comprising zinc ions, the method comprising the following steps: (1) preparing a hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide or a mixture thereof, the preparation of the hydrogel comprising the following steps: - bringing the crosslinked polysaccharide, the non-crosslinked polysaccharide, or their mixture into contact with a physiological saline solution, preferably buffered, the physiological saline solution, preferably buffered, comprising phosphate or carbonate or sulfate salts or their mixtures, - addition of citrate ions to the crosslinked polysaccharide, to the non-crosslinked polysaccharide, or to their mixture, in an amount sufficient to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel, - addition of zinc ions to the crosslinked polysaccharide, to the non-crosslinked polysaccharide, or to their mixture, in an amount sufficient to achieve a zinc ion concentration of at most 20 mM in the hydrogel, the addition of the citrate ions and the zinc ions being carried out in a molar ratio [citrate ions] / [zinc ions] ranging from 1 to 20, and on the condition that the addition of the zinc ions is not carried out before the addition of the citrate ions when the contact with the physiological saline solution, preferably buffered, is carried out before the addition of the citrate ions; (2) sterilization, preferably by heat, of the hydrogel obtained at the end of step (1) to obtain a sterile hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide or their mixture and further comprising zinc ions.

2. Method according to claim 1 in which the addition of the citrate ions and the addition of the zinc ions are carried out after the step of contacting with the physiological saline solution, preferably buffered.

3. Method according to claim 1 or 2 in which the addition of the citrate ions and the addition of the zinc ions are carried out concomitantly, the citrate ions and the zinc ions being added as a solution comprising zinc ions and citrate ions.

4. Method according to claim 3 wherein the solution is a buffered physiological saline solution, preferably a phosphate buffer, comprising zinc ions and citrate ions.

5. Method according to any one of claims 1 to 4 in which step (1) of preparing a hydrogel comprises one or more of the following conventional steps: - pH adjustment; Dilution; Purification; - Addition of at least one additional component; Extrusion.

6. A method of preparing a sterile hydrogel comprising a crosslinked polysaccharide and optionally a non-crosslinked polysaccharide and further comprising zinc ions, the method comprising the following steps: (0) preparation of a crosslinked polysaccharide from a crosslinking reaction medium comprising one or more polysaccharide(s), one or more crosslinking agent(s), a solvent and zinc ions in an amount allowing the preparation of a hydrogel comprising at most 20 mM of zinc ions; (1) preparation of a hydrogel from the crosslinked polysaccharide obtained at the end of step (0) and optionally from a non-crosslinked polysaccharide, the preparation of the hydrogel comprising a step of bringing the crosslinked polysaccharide into contact with a physiological saline solution, preferably buffered, comprising phosphate or carbonate or sulfate salts or mixtures thereof; (2) sterilization, preferably by heat, of the hydrogel obtained at the end of step (1) to obtain a sterile hydrogel; in which: - the crosslinking reaction medium further comprises citrate ions in an amount sufficient to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel, the molar ratio [citrate ions present in the reaction medium] / [zinc ions present in the reaction medium] ranging from 1 to 20; or - step (1) further comprises, before the step of bringing the crosslinked polysaccharide into contact with the physiological saline solution, preferably buffered, a step of adding citrate ions in a quantity sufficient to reach a citrate ion concentration of at least 0.1 mM in the hydrogel, the molar ratio [citrate ions added] / [zinc ions present in the reaction medium] ranging from 1 to 20; or - the physiological saline solution, preferably buffered, further comprises citrate ions in a quantity sufficient to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel, the molar ratio [citrate ions present in the physiological saline solution, preferably buffered] / [zinc ions present in the reaction medium] ranging from 1 to 20.

7. A method of preparing a sterile hydrogel comprising a crosslinked polysaccharide and optionally a non-crosslinked polysaccharide and further comprising zinc ions, the method comprising the following steps: (0') preparation of a crosslinking reaction medium comprising: - one or more polysaccharide(s), - one or more crosslinking agent(s), - citrate ions in an amount sufficient to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel, - zinc ions in an amount allowing the preparation of a hydrogel comprising at most 20 mM of zinc ions, and - a physiological saline solution, preferably buffered, comprising phosphate or carbonate or sulfate salts or mixtures thereof; the molar ratio [citrate ions present in the reaction medium] / [zinc ions present in the reaction medium] ranging from 1 to 20, the preparation of the reaction medium being carried out by adding the citrate ions before any contact of the zinc ions with the physiological saline solution; (0) preparation of a crosslinked polysaccharide from the reaction medium obtained at the end of step (0'); (1) preparation of a hydrogel from the crosslinked polysaccharide obtained at the end of step (0) and optionally from a non-crosslinked polysaccharide; (2) sterilization, preferably by heat, of the hydrogel obtained at the end of step (1) to obtain a sterile hydrogel.

8. Method according to any one of claims 1 to 7 in which the polysaccharide is a hyaluronic acid.

9. A method according to any one of claims 1 to 8 wherein the physiological saline solution is a buffered physiological saline solution comprising phosphate salts, preferably a phosphate buffer.

10. A method according to any one of claims 1 to 9 wherein step (1) further comprises a step of adding an anesthetic agent.

11. Method according to any one of claims 1 to 10 further comprising a step of conditioning the hydrogel, preferably in an injection device, after step (1) and before step (2).

12. Method according to any one of claims 1 to 11 in which the sterilization is a heat sterilization, preferably carried out in an autoclave.

13. Sterile hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide or a mixture thereof, in particular a crosslinked hyaluronic acid, a non-crosslinked hyaluronic acid or a mixture thereof, and further comprising zinc and citrate ions obtained by the method according to one of claims 1 to 12.

14. The hydrogel of claim 13 further comprising an anesthetic agent.

15. Use of citrate ions to protect a hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide or their mixture, optionally an anesthetic agent, and in addition zinc ions, from the degradation of its rheological properties during its sterilization, preferably by heat.

16. Use of citrate ions to preserve the stability over time of the rheological properties of a hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide or their mixture, optionally an anesthetic agent, and in addition zinc ions.

17. Use of a solution comprising zinc ions and citrate ions to protect a hydrogel comprising a crosslinked and / or non-crosslinked polysaccharide, optionally an anesthetic agent, from degradation of its rheological properties during its sterilization, preferably by heat.

18. Use of a solution comprising zinc ions and citrate ions to preserve the stability over time of a hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide or their mixture and optionally an anesthetic agent.

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