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

Abstract

The disclosure provides a method for preparing a sterile hydrogel comprising a cross-linked polysaccharide, a non-cross-linked polysaccharide, or a mixture thereof, the method comprising the steps of: (1) preparing a hydrogel comprising a cross-linked polysaccharide, a non-cross-linked polysaccharide or a mixture thereof and further comprising at least 1 mM citrate ions; and (2) sterilizing, preferably heat sterilizing, the hydrogel comprising at least 1 mM citrate ions to obtain a sterile hydrogel comprising a cross-linked polyssaccharide, a non-cross-linked polysaccharide or a mixture thereof.

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 method for preparing 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.

[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] Furthermore, hydrogels based on crosslinked and / or non-crosslinked hyaluronic acid intended for filling soft tissues must be sterile. Thus, the processes for preparing hydrogels based on crosslinked and / or non-crosslinked hyaluronic acid intended for injection generally include a step of sterilization of the previously formed hydrogel. Sterilization is typically carried out by heat, for example in an autoclave. It has been observed that these sterilization conditions tend to degrade the crosslinked and / or non-crosslinked hyaluronic acid, leading to degradations in the rheological properties of the hydrogels.

[0008] Thus, a need remains for the provision of a process for preparing sterile hydrogels comprising a crosslinked (e.g.: crosslinked hyaluronic acid) and / or non-crosslinked (e.g.: non-crosslinked hyaluronic acid) polysaccharide which is as respectful as possible of the properties of the hydrogels, i.e. which causes the least possible degradation of the rheological properties of the hydrogels during sterilization, for example by heat, as well as over time. BRIEF DESCRIPTION OF THE INVENTION

[0009] The present invention relates to a method for preparing a sterile hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide, or a mixture thereof, the method comprising the following steps:

[0010] (1) preparing a hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide or a mixture thereof and further comprising at least 1 mM citrate ions; and

[0011] (2) sterilizing, preferably by heat, the hydrogel to obtain a sterile hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide or a mixture thereof.

[0012] The invention also relates to a hydrogel obtained by the method of the invention.

[0013] Finally, the invention relates to the use of citrate ions for protecting a hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide or their mixture, in particular a crosslinked, non-crosslinked hyaluronic acid or their mixture, and optionally an anesthetic agent, from the degradation of its rheological properties during its sterilization, preferably by heat and to the use of citrate ions for preserving the stability over time of hydrogels comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide or their mixture, in particular a crosslinked, non-crosslinked hyaluronic acid or their mixture, and optionally an anesthetic agent.

[0014] Other aspects of the invention are as described below and in the claims.

[0015] DETAILED DESCRIPTION OF THE INVENTION

[0016] Definitions

[0017] 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°.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.

[0018] 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 32 G, 30 G, 27 G, 26 G, 25 G hypodermic needle. Preferably, an “injectable hydrogel” is a hydrogel having an average extrusion force of less than or equal to 25 N, preferably ranging from 5 to 25 N, more preferably ranging from 8 to 15 N, when measured with a dynamometer, at a fixed speed of approximately 12.5 mm / min, in syringes with an external diameter greater than or equal to 6.3 mm, with a needle with an external diameter less than or equal to 0.4 mm (27 G) and a length ! ”, at room temperature.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] A "crosslinked polysaccharide" refers to a polysaccharide that has been modified during a crosslinking reaction.

[0023] 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.

[0024] The term "crosslinking agent" refers to any compound capable of introducing crosslinking between different polysaccharide chains.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] Process

[0031] Unexpectedly, the inventors discovered that the addition of citrate ions during the preparation of hydrogels comprising a crosslinked and / or non-crosslinked polysaccharide, in particular a crosslinked and / or non-crosslinked hyaluronic acid, makes it possible to effectively protect the hydrogel from degradation of its rheological properties during sterilization, in particular during heat sterilization. The hydrogels obtained by the process of the present invention thus exhibit lesser changes in their rheological properties compared to hydrogels prepared by an equivalent process without the addition of citrate ions. The hydrogels obtained by the process of the present invention also exhibit better preservation of their rheological properties over time.

[0032] The present invention thus relates to a process for preparing a sterile hydrogel comprising a crosslinked and / or non-crosslinked polysaccharide, the process comprising the following steps:

[0033] (1) preparation of a hydrogel comprising a crosslinked and / or non-crosslinked polysaccharide and further comprising at least 1 mM citrate ions; and

[0034] (2) sterilization, preferably by heat, of the hydrogel to obtain a sterile hydrogel comprising a crosslinked and / or non-crosslinked polysaccharide.

[0035] The hydrogel comprising a crosslinked and / or non-crosslinked polysaccharide and further comprising at least 1 mM of citrate ions according to step (1) can be prepared according to two alternative methods:

[0036] - method 1: by adding citrate ions in powder form or in solution form when preparing a hydrogel from a previously crosslinked and / or non-crosslinked polysaccharide; or

[0037] - method 2: when the hydrogel comprises a crosslinked polysaccharide, by carrying out the crosslinking of the polysaccharide in a reaction medium comprising citrate ions and then preparing the hydrogel from the crosslinked polysaccharide obtained.

[0038] METHOD 1

[0039] When the method of the present invention implements method 1, step (1) of preparing the hydrogel comprises a step of adding, to the crosslinked polysaccharide or to the non-crosslinked polysaccharide or to their mixture, a solution comprising citrate ions in an amount sufficient to reach a citrate ion concentration of at least 1 mM in the hydrogel.

[0040] In a variant, when the method of the present invention implements method 1, step (1) of preparing a hydrogel comprises a step of adding, to the crosslinked polysaccharide or to the non-crosslinked polysaccharide or to their mixture, citrate ions in powder form in an amount sufficient to achieve a citrate ion concentration of at least 1 mM in the hydrogel.

[0041] In some embodiments, preparing the hydrogel comprises adding citrate ions in powder form and in solution form, preferably the powder and solution being added at different stages of preparing the hydrogel.

[0042] Crosslinked and / or non-crosslinked polysaccharide

[0043] 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.

[0044] 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.

[0045] 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.

[0046] A crosslinked polysaccharide may be prepared by any method known to those skilled in the art.

[0047] 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.

[0048] For example, the crosslinked polysaccharide can be prepared as described in WO2010131175A1 and WO201277054A1.

[0049] The method of the present invention may thus comprise, before the step of preparing the hydrogel, a step of preparing a crosslinked polysaccharide.

[0050] 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).

[0051] 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.

[0052] 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, bisalkoxyamine, dithiols such as polyethylene glycol dithiol and their mixtures.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.

[0053] 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.

[0054] 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%.

[0055] The crosslinked polysaccharide may in particular be prepared by a process comprising the following steps:

[0056] (a1) preparing a crosslinking reaction medium comprising one or more polysaccharide(s), one or more crosslinking agent(s) and a solvent; and

[0057] (a2) reacting the reaction medium to obtain a crosslinked polysaccharide.

[0058] The polysaccharide is as described above.

[0059] 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.

[0060] The crosslinking agent is as described above.

[0061] 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.

[0062] 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.

[0063] The total amount of crosslinking agent in the reaction medium typically varies from 0.001 to 0.10 moles per 1 mole of polysaccharide repeating unit, preferably from 0.001 to 0.08 moles or from 0.001 to 0.06 moles per 1 mole of polysaccharide repeating unit, preferably from 0.001 to 0.04 moles per 1 mole of polysaccharide repeating unit, preferably from 0.001 to 0.03 moles per 1 mole of polysaccharide repeating unit, preferably 0.001 to 0.02 moles per 1 mole of polysaccharide repeating unit, more preferably from 0.001 to 0.01 moles per 1 mole of polysaccharide repeating unit, even more preferably from 0.001 to 0.005 moles per 1 mole of polysaccharide repeating unit. When the polysaccharide is a glycosaminoglycan such as hyaluronic acid, the repeating unit is a disaccharide unit.

[0064] 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.

[0065] Step (a1) typically comprises a step of homogenizing the reaction medium. Homogenization is generally carried out by three-dimensional stirring, stirring with a mixer, stirring with blades or stirring with a spatula.

[0066] 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.

[0067] Step (a2) consists of reacting the reaction medium to obtain a crosslinked polysaccharide. Advantageously, step (a2) is carried out directly after step (a1).

[0068] 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.

[0069] Crosslinking can be carried out in the presence of several crosslinking agents. When crosslinking is carried out in the presence of several crosslinking agents, the crosslinking agents can be added simultaneously or separately over time to the reaction medium. Step (a2) can thus comprise repeated crosslinking steps, advantageously step (a2) comprises a single crosslinking step. Crosslinking is then carried out in the presence of a total amount 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.

[0070] 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.

[0071] 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 greater than or equal to 0°C and less than or equal to 30°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.

[0072] In certain embodiments, step (a2) can be carried out by placing the reaction medium 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.

[0073] 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.

[0074] 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 moles per 1 mole of repeating unit of the polysaccharide.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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 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.

[0081] 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).

[0082] The crosslinked and / or non-crosslinked polysaccharides described above are useful for implementing the method 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.

[0083] The method of the present invention according to Method 1 comprises preparing a hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide or a mixture thereof and further comprising at least 1 mM citrate ions, preferably from 1 to 12 mM citrate ions.

[0084] The preparation of the hydrogel comprises at least one step of adding citrate ions to the crosslinked and / or non-crosslinked polysaccharide. The amount of citrate ions added in this step makes it possible to achieve a citrate ion concentration in the prepared hydrogel of at least 1 mM, preferably ranging from 1 to 12 mM.

[0085] The preparation of the hydrogel advantageously includes a step of adjustment to physiological pH, in particular ranging from 6.8 to 7.8.

[0086] In one variant, the citrate ions are added in powder form to the crosslinked and / or non-crosslinked polysaccharide. The amount of citrate ions in powder form added at this step makes it possible to achieve a citrate ion concentration in the prepared hydrogel of at least 1 mM, preferably ranging from 1 to 12 mM. Typically when the addition of citrate ions in powder form is carried out, a neutralization of the effect of the citrate ions on the pH of the hydrogel is carried out.

[0087] In another variant, the citrate ions are added in the form of a solution (solution comprising citrate ions) to the crosslinked and / or non-crosslinked polysaccharide. The amount of the solution comprising citrate ions added in this step makes it possible to achieve a citrate ion concentration in the prepared hydrogel of at least 1 mM. Preferably, the citrate ion concentration in the hydrogel varies from 1 to 150 mM or from 1 to 100 mM or from 1 to 50 mM or from 1 to 20 mM or from 1 to 12 mM.

[0088] In some embodiments, the amount of citrate ions added (as a solution or powder) is such that a citrate ion concentration in the hydrogel of at least 1.5 mM, or at least 2 mM, or at least 2.5 mM, or at least 3 mM, or at least 3.5 mM is achieved. The maximum concentration of citrate ions in the hydrogel is generally 12 mM. In some embodiments, the amount of citrate ions added (as a solution or powder) is such that a citrate ion concentration in the hydrogel of from 2 to 12 mM, or from 3 to 11 mM, or from 3 to 9 mM, or from 3 to 8 mM or from 4 to 8 mM or from 3 to 5 mM is achieved.

[0089] In some embodiments, the amount of citrate ions added (in solution or powder form) achieves a citrate ion concentration in the hydrogel ranging from 5 to 12 mM.

[0090] A solution comprising citrate ions means a solution whose pH allows citrate ions to be present in solution in that solution or means a solution capable of releasing citrate ions once added during the preparation of the hydrogel. The solution comprising citrate ions is preferably prepared from citric acid or sodium citrate, calcium citrate, potassium citrate or magnesium citrate.

[0091] The solution comprising added citrate ions typically has a pH ranging from 6.8 to 7.8 (physiological pH). If the solution does not have such a pH, the pH is adjusted, if necessary, during the preparation of the hydrogel so that the final hydrogel has such a pH.

[0092] The concentration of citrate ions in the solution is chosen to limit the dilution effect that can be caused by adding the solution during the preparation of the hydrogel, such a dilution effect of the hydrogel being undesirable. The maximum concentration of citrate ions that can be added to the hydrogel is then limited by the adjustment of the pH. Indeed, adjusting the pH of the solution to reach a physiological pH is less easy beyond a certain concentration of citrate ions. The solution comprising citrate ions is typically prepared so that the solution is concentrated in citrate ions, for example 100 times more concentrated compared to the final concentration of citrate ions in the hydrogel.

[0093] The solution comprising citrate ions is typically prepared in water or in a physiologically acceptable buffer, preferably by adding citric acid or sodium citrate, or calcium citrate, or potassium citrate or magnesium citrate in water or a physiologically acceptable buffer. Examples of buffers include, but are not limited to, N-carbamoylmethyl taurine (CAS No: 7365-82-4), 3-[N,N-bis(hydroxyethyl)amino]-2-hydroxypropane sulfonic acid sodium salt (CAS No: 102783-62-0), 3-morpholino-2-hydroxypropane sulfonic acid (CAS No: 68399-77-9), 1,4-piperazinediethane sulfonic acid (CAS No: 5625-37-6), 1,4-piperazine-N,N'-bispropane sulfonic acid (CAS No: 5625-56-9), 2-hydroxy-3-[tris(hydroxymethyl)methylamino]-1-propane sulfonic acid (CAS No: 68399-81-5), 2-[(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid (CAS No: 7365-44-8),N- tris(hydroxymethyl)methylglycine (CAS No: 5704-04-1 ), 3-(N-morpholino)propanesulfonic acid (CAS No: 1132-61-2), tris(hydroxymethyl)aminomethane (CAS No: 77-86-1 ), bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (CAS No: 6976-37-0), N,N-bis(2-hydroxyethyl)taurine (CAS No: 10191-18-1 ), 4-(2-Hydroxyethyl)piperazine-1 -ethanesulfonic acid (CAS No: 7365-45-9), 1,4-Piperazinediethanesulfonic acid (CAS No: 5625-37-6), 4- (2-hydroxyethyl)piperazine-1-(2-hydroxypropane-3-sulfonic acid) (CAS No: 68399-78-0), phosphate buffers such as PBS with a pH around physiological pH (CAS No: 7647-14-5, 7447-40-7).,

[0094] Preferably, the buffer is selected from 3-(N-morpholino)propane sulfonic acid (CAS No: 1132-61-2), tris(hydroxymethyl)aminomethane (CAS No: 77-86-1), bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (CAS No: 6976-37-0), N,N-bis(2-hydroxyethyl)taurine (CAS No: 10191-18-1), 4-(2-Hydroxyethyl)piperazine-1-ethane sulfonic acid (CAS No: 7365-45-9) and phosphate buffers such as PBS with a pH around physiological pH (CAS No: 7647-14-5, 7447-40-7).

[0095] Preferably the buffer is a phosphate buffer, particularly a saline buffer of NaH2PO4 / Na2HPO4 or KH2PO4 / K2HPO4. The pH of the solution comprising citrate ions is typically adjusted by means of addition of acid or base.

[0096] Thus, in some embodiments, the solution comprising citrate ions is a solution of citric acid in a phosphate buffer, the pH of which varies from 6.8 to 7.8. In some embodiments, the solution comprising citrate ions is a solution of sodium citrate in a phosphate buffer, the pH of which varies from 6.8 to 7.8.

[0097] In preferred embodiments, the citrate ions are added as a solution comprising citrate ions, the solution being as described above. Preferably, the solution is a citric acid solution or a sodium citrate or calcium citrate solution, or potassium citrate or magnesium citrate solution. The solution is preferably a citric acid solution or sodium citrate or calcium citrate or potassium citrate or magnesium citrate solution in a physiologically acceptable buffer, such as a phosphate buffer.

[0098] The preparation of a hydrogel from a crosslinked and / or non-crosslinked polysaccharide may be carried out in a conventional manner, except that citrate ions are added during the preparation of the hydrogel. Thus, the preparation of a hydrogel comprising a crosslinked and / or non-crosslinked polysaccharide may comprise one or more of the following conventional steps:

[0099] pH adjustment (1);

[0100] Dilution (2);

[0101] Purification (3);

[0102] Addition of at least one additional component (4);

[0103] Extrusion (5).

[0104] These steps, well known to those skilled in the art, may be as described below. They may be at least partly concomitant.

[0105] 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.

[0106] 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.

[0107] Citrate ions (in powder form or in solution) may be added at the time of, before or after one of these conventional steps. Alternatively, the citrate ions are added before the extrusion step (5) so as to obtain a homogeneous gel.

[0108] When a purification step (3) is implemented, the citrate ions can be added before or after the purification step (3), advantageously the citrate ions are added after the purification step (3). The addition of the citrate ions after the purification step ensures better control of the citrate ion concentration in the prepared hydrogel.

[0109] Preferably, citrate ions are added between the purification (3) and extrusion (5) steps.

[0110] The addition of citrate 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.

[0111] Preferably, the citrate ions 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 solution comprising citrate ions is concomitant with the step of adding at least one additional component (4). In particular, in certain embodiments, the addition of the solution comprising citrate ions is concomitant with the addition of an anesthetic solution. In particular, in certain embodiments, the addition of the solution comprising citrate ions is concomitant with the addition of a lubricating agent.In some embodiments, the added citrate ion-comprising solution may comprise other components, particularly a lubricating agent, e.g., uncrosslinked hyaluronic acid, uncrosslinked heparosan, or a mixture thereof.

[0112] The steps of dilution (2), addition of at least one additional component (4) and addition of citrate ions may be concomitant.

[0113] Citrate ions can be added after the pH adjustment step (1). Citrate ions can be added between the pH adjustment (1) and extrusion (5) steps when both steps are implemented. pH adjustment (1)

[0114] 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).

[0115] Dilution (2)

[0116] 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 polysaccharide concentration 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 salts. 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.

[0117] Purification (3)

[0118] The process for preparing the hydrogel may comprise at least one purification step.

[0119] The purification step aims to remove any undesirable impurities. These impurities may result from the crosslinking of the polysaccharide, for example resulting from step (a2) described above. Such impurities may include, 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. The purification step may therefore be particularly implemented when the hydrogel comprises a crosslinked polysaccharide.

[0120] Purification can be carried out by dialysis or by filtration, for example by dynamic tangential filtration (“DCF” for Dynamic Cross-flow Filtration).

[0121] Addition of additional components (4)

[0122] The method for preparing the hydrogel may comprise one or more steps 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.

[0123] 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.

[0124] 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.,

[0125] 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.

[0126] Examples of amino acids include, but are not limited to, arginine (eg, L-arginine), isoleucine (eg, L-isoleucine), leucine (eg, L-leucine), lysine (eg, L-lysine or L-lysine monohydrate), glycine, valine (eg, L-valine), threonine (eg, L-threonine), proline (eg, L-proline), methionine, histidine, phenylalanine, tryptophan, cysteine, their derivatives (eg, N-acetylated derivatives such as N-acetyl-L-cysteine), and a mixture thereof. Examples of vitamins and their salts include, but are not limited to, vitamins E, A, C, B, especially vitamins B6, B8, B4, B5, B9, B7, B12, and more preferably pyridoxine and its derivatives and / or salts, preferably pyridoxine hydrochloride.

[0127] Examples of minerals include, but are not limited to, zinc salts (e.g., zinc acetate, in particular dehydrated, or zinc citrate; zinc citrate will preferably be chosen), 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. Zinc citrate will preferably be chosen as an additional component.

[0128] 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.

[0129] Adrenaline derivatives include adrenaline, noradrenaline and a mixture of these.

[0130] Extrusion (5)

[0131] 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 perforations have 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.

[0132] METHOD 2

[0133] When the method of the present invention implements method 2, step (1) of preparing the hydrogel comprises the following steps:

[0134] (a) 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 citrate ions in an amount sufficient to allow the preparation of a hydrogel comprising a crosslinked polysaccharide and further comprising at least 1 mM of citrate ions; (b) preparation of a hydrogel from the crosslinked polysaccharide obtained at the end of step (a) and optionally from a non-crosslinked polysaccharide.

[0135] The crosslinked polysaccharide may in particular be prepared by a process comprising the following steps:

[0136] (a1) preparing a crosslinking reaction medium comprising one or more polysaccharide(s), one or more crosslinking agent(s), a solvent and citrate ions in an amount sufficient to allow the preparation of a hydrogel based on a crosslinked polysaccharide comprising at least 1 mM of citrate ions; and

[0137] (a2) reacting the reaction medium to obtain a crosslinked polysaccharide.

[0138] Steps (a), (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 reaction medium also comprises citrate ions.

[0139] Citrate ions are typically present in the reaction medium in an amount to achieve a citrate ion concentration in the hydrogel of at least 1.5 mM, or at least 2 mM, or at least 2.5 mM, or at least 3 mM or at least 3.5 mM. The maximum concentration of citrate ions in the hydrogel is typically 20 mM or 12 mM.

[0140] In some embodiments, the amount of citrate ions present in the reaction medium makes it possible to achieve a citrate ion concentration in the hydrogel ranging from 2 to 20 mM, or 2 to 12 mM, or 3 to 11 mM, or 3 to 9 mM, or 3 to 8 mM or 4 to 8 mM or 3 to 5 mM.

[0141] In certain embodiments, the amount of citrate ions present in the reaction medium makes it possible to achieve a concentration of citrate ions in the hydrogel ranging from 5 to 12 mM.

[0142] The citrate ions present in the reaction medium may result from the addition of citric acid or an aqueous solution of citric acid to the reaction medium.

[0143] In some embodiments, the citrate ions present in the reaction medium result from the addition of sodium citrate or an aqueous solution of sodium citrate to the reaction medium.

[0144] In certain embodiments, the citrate ions present in the reaction medium result from the addition of calcium citrate, or potassium citrate, or magnesium citrate or one of their solutions to the reaction medium. At the end of step (a2), the crosslinked polysaccharide is typically in the form of a gel comprising citrate ions. This gel is generally directly used in the remainder of the process of the invention (step (b)). No covalent bond is formed between the polysaccharide and the citrate ions.

[0145] The preparation of a hydrogel (step (b)) from the crosslinked polysaccharide obtained at the end of step (a) 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 (a) or (a2) typically comprises one or more of the following conventional steps:

[0146] pH adjustment (1);

[0147] Dilution (2);

[0148] Purification (3);

[0149] Addition of at least one additional component (4);

[0150] Extrusion (5).

[0151] 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.

[0152] Sterilization of the hydrogel (step (2))

[0153] 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 FO > 15. The sterilizing value FO corresponds to the time required, in minutes, at 121°C, to inactivate 90% of the population of microorganisms present in the product to be sterilized. Alternatively, sterilization can be carried out in particular by gamma ray, UV radiation or by means of ethylene oxide.

[0154] The hydrogel obtained at the end of the process according to the invention typically has a pH ranging from 6.8 to 7.8 (physiological pH). METHOD 1 OR 2: Optional step

[0155] The method of the present invention (method 1 or 2) 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 (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.

[0156] Sterile Hvdroqel

[0157] The sterile hydrogel obtained by the method of the present invention (method 1 or 2) is a hydrogel based on a crosslinked polysaccharide or a non-crosslinked polysaccharide or a mixture thereof. The sterile hydrogel obtained by the method of the present invention (method 1 or 2) therefore comprises a crosslinked polysaccharide, or a non-crosslinked polysaccharide, or a mixture of a crosslinked polysaccharide and a non-crosslinked polysaccharide. It is understood that the crosslinked polysaccharide may be a mixture of crosslinked polysaccharides.

[0158] The sterile hydrogel obtained by the method of the present invention (method 1 or 2) has a physiological pH, i.e., 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.

[0159] The sterile hydrogel obtained by the method of the present invention (method 1 or 2) 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°.

[0160] The hydrogel obtained by the method of the present invention is preferably an injectable hydrogel, that is to say 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.

[0161] The hydrogel obtained 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.

[0162] 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.

[0163] 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).

[0164] 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%.

[0165] 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.

[0166] The sterile hydrogels prepared according to the process 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.

[0167] 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 intra-epidermal and / or intradermal and / or subcutaneous and / or supra-periosteal injection. The injection device can in particular be chosen from a syringe, a set of micro-syringes, a thread, a laser or hydraulic device, an injection gun, a needle-free injection device, or a micro-needle roller.

[0168] The sterile hydrogels prepared according to the method of the invention are preferably injected subcutaneously.

[0169] They can involve deep applications, mid-line applications and / or superficial applications.

[0170] They may have therapeutic and / or cosmetic and / or cosmeceutical applications.

[0171] In the cosmetic field, hydrogels can be particularly useful for compensating for tissue volume losses due to aging.

[0172] They can be used in the prevention and / or cosmetic treatment of an alteration of 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 folds; to increase the volume of the cheekbones, chin or lips; to restore the volumes of the face, in particular the cheeks, temples, the oval of the face, and the contour of the eye; to reduce the appearance of wrinkles and fine lines. The process for preparing sterile hydrogels of the present invention is respectful of the properties of the hydrogels, that is to say that it results in lesser modifications of the rheological properties of the hydrogels during sterilization.Indeed, a better conservation of the rheological properties of the hydrogels was observed after sterilization (better conservation of the elastic modulus G', better conservation of the phase angle) compared to hydrogels prepared by a process without the addition of citrate ions.

[0173] The process of the present invention allows the preparation of sterile hydrogels whose decrease in elastic modulus G' after sterilization does not exceed 50%, 45%, 40%, 35% or 30% of the value of the elastic modulus G' before sterilization.

[0174] Since the step of adding citrate ions according to method 1, particularly in the form of a solution, has the effect of slightly diluting the hydrogel, it could be expected that the rheological properties of the hydrogel would be negatively impacted by this addition. Unexpectedly, it was observed that the addition of a solution comprising citrate ions has a favorable effect on the properties of the hydrogel during sterilization.

[0175] Furthermore, the addition of citrate ions, particularly in the form of a solution, allows the properties of the hydrogel to be preserved over time. Indeed, a better conservation of the rheological properties of the hydrogels over time has been observed (better conservation of the elastic modulus G', better conservation of the phase angle) compared to hydrogels prepared by a process without the addition of citrate ions and which tend to see their rheological properties decrease more significantly over the months.

[0176] The use of citrate ions, in particular in the form of a solution, in a process for preparing a hydrogel therefore makes it possible to protect a hydrogel comprising a crosslinked and / or non-crosslinked polysaccharide, in particular comprising at least one crosslinked polysaccharide from the degradation of its rheological properties during sterilization, preferably by heat. The use of citrate ions, in particular in the form of a solution, in a process for preparing a hydrogel comprising a crosslinked and / or non-crosslinked polysaccharide, in particular comprising at least one crosslinked polysaccharide, also makes it possible to preserve the length of the crosslinked and / or non-crosslinked polysaccharide chains.

[0177] The use of citrate ions, in particular in the form of a solution, in a process for preparing a hydrogel comprising a crosslinked and / or non-crosslinked polysaccharide, in particular comprising at least one crosslinked polysaccharide, also makes it possible to preserve the stability of the hydrogels, in particular after sterilization, over time, in particular to increase the stability of the hydrogels, in particular after sterilization, over time compared with identical hydrogels not comprising citrate ions. In other words, the hydrogels obtained according to the invention maintain their rheological properties more effectively over time after sterilization.

[0178] Furthermore, it is known that the additional presence of an anesthetic agent in a hydrogel comprising a crosslinked and / or non-crosslinked polysaccharide leads to increased degradation of the rheological properties of the hydrogels during sterilization, preferably by heat. The addition of citrate 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 citrate ions.

[0179] It should also be observed that a process for preparing a hydrogel according to method 2, in which step (a2) is carried out at a pressure P less than or equal to atmospheric pressure and at a temperature T greater than the temperature of the eutectic point of the reaction medium as measured at pressure P and less than the temperature of the freezing point of the reaction medium as measured at pressure P, results in very good preservation of the rheological properties of the hydrogels, the hydrogels prepared under these conditions being able to be more sensitive to sterilization.

[0180] The following examples are given for illustrative purposes, but should in no way be considered as limiting the present invention.

[0181] EXAMPLES

[0182] 1. Materials

[0183] - Non-crosslinked sodium hyaluronate

[0184] - BDDE (Sigma Aldrich)

[0185] - NaOH 0.25M

[0186] - 1 M HCl

[0187] - Citric acid (Sigma Aldrich) (CAS No: 5949-29-1)

[0188] - Divinylsulfone (Sigma Aldrich)

[0189] - Phosphate Buffer (BBraun),

[0190] - Lidocaine Hydrochloride - Three-dimensional agitator

[0191] - DHR-2 Rheometer

[0192] - Dynamometer and test bench

[0193] - Homogenizer Paddle mill

[0194] - Sterile polyethylene bag

[0195] 2. Methods

[0196] Measurement of viscoelastic properties

[0197] 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).

[0198] 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.

[0199] 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.

[0200] 3. Examples

[0201] 3.1 Example 1

[0202] Two cross-linked hyaluronic acid hydrogels were prepared from a high molecular weight hyaluronic acid 3 MDa and BDDE in a 0.25M aqueous sodium hydroxide solution (cross-linked for 1 month at -20°C). The cross-linked polysaccharides had a molar cross-linking rate of 0.2%. PBS phosphate buffer and 1 N HCl solution were then added to the cross-linked polysaccharides until a pH of 7.3 ± 0.5 was obtained. The resulting hydrogels were homogenized using a three-dimensional stirrer. The mixtures were dialyzed. The resulting hydrogels had either a concentration of 15 mg of hyaluronic acid per gram of product (hydrogel A) or a concentration of 23 mg of hyaluronic acid per gram of product (hydrogel B).

[0203] To the hydrogels obtained, a solution of non-crosslinked high molecular weight sodium hyaluronate is then added as a lubricant (same quantity of high molecular weight sodium hyaluronate in the different mixtures) including, or not, citrate ions.

[0204] The solution comprising citrate ions and high molecular weight sodium hyaluronate is prepared as follows. Citric acid (in powder form) is dissolved in phosphate buffer, and the pH is then adjusted with 5M NaOH to reach a physiological pH (pH = 6.8 - 7.8), finally high molecular weight sodium hyaluronate is added as a lubricant. The concentration of citrate ions in the solution is adapted by taking into account the dilution effect following the addition of this solution into the cross-linked hyaluronic acid hydrogel. Indeed, the concentration of citrate ions indicated in Table 1 corresponds to the final concentration in the hydrogel.

[0205] The prepared solution comprising citric acid and high molecular weight sodium hyaluronate or the solution comprising high molecular weight sodium hyaluronate alone is then mixed with the crosslinked hyaluronic acid hydrogel in a stirring tank.

[0206] The products obtained (hydrogels A, B) were sieved and then packaged in a syringe.

[0207] Finally, the products were sterilized in an autoclave (plate temperature between 121°C and 135°C with FO > 15).

[0208] Before and after sterilization, the prototypes were analyzed. The elastic modulus G' and the phase angle 5 were determined. The results are shown in Table 1 below.

[0209] The prototypes have a molar crosslinking rate of 0.2%.

[0210] Table 1

[0211] 1AG' (%) = (G' after sterilization - G' before sterilization) / (G' before sterilization) * 100

[0212] 2 A 5 (%) = (5 after sterilization - 5 before sterilization) / (5 before sterilization) *100

[0213] * 1 mL of hydrogel was considered to weigh one gram.

[0214] It is observed that the hydrogels prepared from a process according to the invention comprising a step of adding a citric acid solution (hydrogels A2, B2) exhibit less degradation of their rheological properties after sterilization compared to hydrogels prepared by an equivalent process without addition of a citric acid solution (hydrogels A1, B1). Indeed, it has been observed that hydrogels A2 and B2 exhibit a higher elastic modulus (G') after sterilization than hydrogels A1 and B1 after sterilization. The decrease in the elastic modulus (G') is therefore lower after sterilization for hydrogels A2 and B2. It has also been observed that hydrogels A2 and B2 exhibit a lower phase angle (5) after sterilization than hydrogels A1 and B1 after sterilization.

[0215] 3.2 Example 2

[0216] 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. The cross-linked polysaccharide has a molar cross-linking rate of 2%. PBS phosphate buffer and 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 mixture is dialyzed. The resulting hydrogels have a concentration of 15 mg of hyaluronic acid per gram of product.

[0217] The following are then added to the hydrogels obtained, depending on the case:

[0218] - a solution of high molecular weight sodium hyaluronate as a lubricant (same quantity in the different mixtures) including, or not, citrate ions;

[0219] - an aqueous solution of lidocaine hydrochloride to obtain 0.3% by weight of lidocaine hydrochloride relative to the weight of the final hydrogel;

[0220] - a citric acid solution. Hydrogels C

[0221] For C hydrogels, only a solution of uncrosslinked high molecular weight sodium hyaluronate with or without citrate ions is added.

[0222] The solution comprising citrate ions and high molecular weight sodium hyaluronate is prepared as follows. Citric acid (in powder form) is dissolved in phosphate buffer, the pH is then adjusted with 5M NaOH to reach a physiological pH (pH = 6.8-7.6), finally high molecular weight sodium hyaluronate is added as a lubricant. The concentration of citrate ions in the solution is adapted by taking into account the dilution effect following the addition of this solution to the mixture comprising crosslinked hyaluronic acid. Indeed, the concentration of citrate ions indicated in Table 2 corresponds to the final concentration in the hydrogel.

[0223] The prepared solution comprising citric acid and high molecular weight sodium hyaluronate is then mixed with the mixture comprising crosslinked hyaluronic acid in a stirring tank.

[0224] Hydrogels D

[0225] For D hydrogels, a high molecular weight sodium hyaluronate solution, an anesthetic solution and optionally a citric acid solution are added.

[0226] The citric acid solution is added at the same time as the anesthetic solution, with the citric acid solution and anesthetic solution being added after the addition of the high molecular weight sodium hyaluronate solution.

[0227] A citric acid solution is prepared. Citric acid (in powder form) is first dissolved in phosphate buffer, then 5M NaOH is added to adjust the pH to a physiological level. The goal is to make a concentrated solution 100 times the actual concentration desired in the final hydrogel. This is to avoid excessive dilution of the hydrogel due to the addition of the citric acid solution.

[0228] The products obtained (hydrogels C and D) were sieved to the order of microns and then packaged in a syringe.

[0229] Finally, the products were sterilized in an autoclave (plate temperature between 121°C and 135°C with FO > 15). Before and after sterilization, the prototypes were analyzed. The elastic modulus G' and the phase angle 5 were determined. The results are presented in Table 2 below. Table 2

[0230] 1 AG' (%) = (G' after sterilization - G' before sterilization) / (G' before sterilization) * 100

[0231] 2 A 5 (%) = (5 after sterilization - 5 before sterilization) / (5 before sterilization) *100

[0232] * it was considered that 1 mL of hydrogel weighs one gram. It is observed that the hydrogels prepared from a process according to the invention comprising a step of addition of citrate ions (addition of a solution of non-crosslinked high molecular weight sodium hyaluronate comprising citrate ions or addition of a citric acid solution) (hydrogels C2, D2 to D6) exhibit lesser changes in their rheological properties after sterilization compared to hydrogels prepared by an equivalent process without addition of citrate ions (hydrogels C1 and D1). Indeed, it was observed that hydrogels C2 and D2 to D6 exhibit a higher elastic modulus (G') after sterilization than hydrogels C1 and D1. The decrease in the elastic modulus (G') is therefore lower after sterilization for hydrogels C2 and D2 to D6. It was also observed that hydrogels C2 and D2 to D6 exhibit a lower phase angle (5) after sterilization than hydrogels C1 and D1.

[0233] After 1 month at 40°C, the hydrogel C2 prepared from a process according to the invention comprising a step of adding a solution comprising citrate ions does not show any changes in its rheological properties compared to the hydrogel C1 prepared by an equivalent process without addition of such a solution.

[0234] 3.3 Example 3

[0235] A crosslinked hyaluronic acid hydrogel is prepared from a high molecular weight hyaluronic acid 4MDa and BDDE in a 0.25M aqueous sodium hydroxide solution. The crosslinked polysaccharide has a crosslinking rate of 2%. 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 resulting hydrogel is homogenized using a three-dimensional stirrer. The mixture is dialyzed. The resulting hydrogels have a concentration of 15 mg of hyaluronic acid per gram of product. The following are then added to the resulting hydrogels:

[0236] - a solution of high molecular weight sodium hyaluronate as a lubricant (same quantity in the different mixtures);

[0237] - an aqueous solution of lidocaine hydrochloride to obtain 0.3% by weight of lidocaine hydrochloride relative to the weight of the final hydrogel;

[0238] - possibly a citric acid solution.

[0239] For hydrogels E2 and E3, the citric acid solution is added at the same time as the anesthetic solution, with the citric acid solution and anesthetic solution being added after the addition of the high molecular weight sodium hyaluronate solution.

[0240] A citric acid solution is prepared. Citric acid (in powder form) is first dissolved in phosphate buffer and then 5M NaOH is added to adjust the pH to a physiological level. The objective is to make a concentrated solution 100 times 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 citric acid solution. The products obtained (hydrogels E1, E2 and E3) were sieved to the order of one micron and then packaged in a syringe.

[0241] Finally, the products were sterilized in an autoclave (plate temperature between 121°C and 135°C with FO > 15).

[0242] Before and after sterilization, hydrogels E1-E3 were analyzed. The elastic modulus G' and phase angle 5 were determined. The results are shown in Table 3 below.

[0243] Table 3

[0244] 1 AG' (%)= (G' T 2 months - G' T0) / (G' TO) *100

[0245] After 2 months at 40°C, hydrogels E2 and E3 prepared from a process according to the invention comprising a step of adding a solution comprising citrate ions exhibit a minor modification of their rheological properties compared to hydrogel E1 prepared by an equivalent process without addition of such a solution.

[0246] 3.4 Example 4

[0247] A hydrogel is prepared from a high molecular weight hyaluronic acid 1.5 MDa and divinylsulfone in a 0.25M aqueous sodium hydroxide solution previously mixed with 0.1M sodium citrate (crosslinking for 4 hours at 21°C). The crosslinked polysaccharide has a crosslinking rate of 0.5%. 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 obtained hydrogel is homogenized using a three-dimensional stirrer. The obtained hydrogel has a concentration of 23 mg of hyaluronic acid per gram of product (hydrogel F1). To the obtained hydrogel, a high molecular weight sodium hyaluronate solution is then added as a lubricant;

[0248] The hydrogel obtained was sieved and then packaged in a syringe.

[0249] Finally, the obtained hydrogel was sterilized in an autoclave (plateau temperature between 121°C and 135°C with FO > 15).

[0250] After sterilization, hydrogel F1 was analyzed. The elastic modulus G' and phase angle 5 were determined. The results are shown in Table 4 below.

[0251] Table 4

[0252] 1 AG' (%) = (G' after sterilization - G' before sterilization) / (G' before sterilization) * 100

[0253] 2 A 5 (%) = (5 after sterilization - 5 before sterilization) / (5 before sterilization) *100

[0254] * 1 mL of hydrogel was considered to weigh one gram.

[0255] It is observed that the hydrogel prepared from a process according to the invention has good rheological properties. 3.5 Example 5

[0256] Three hydrogels are prepared from a high molecular weight hyaluronic acid

[0257] 1.5 MDa and BDDE in a 0.25M aqueous sodium hydroxide solution (crosslinking for 72 hours at 21°C).

[0258] For hydrogels G2 and G3, 0.1 M sodium citrate was previously mixed with the 0.25 M aqueous sodium hydroxide solution. The crosslinked polysaccharide has a crosslinking rate of 2.3%. Phosphate buffer and 1 N HCl solution are then added to the crosslinked polysaccharide until a pH of 7.3 ± 0.5 is obtained. The hydrogels obtained are homogenized using a three-dimensional stirrer. The hydrogels obtained have a concentration of 23 mg of hyaluronic acid per gram of product (hydrogel G1, hydrogel G2, hydrogel G3).

[0259] To the hydrogels obtained, a solution of high molecular weight sodium hyaluronate is then added as a lubricant (same quantity in the different mixtures).

[0260] The hydrogels obtained were sieved and then packaged in a syringe.

[0261] Finally, the obtained hydrogels were sterilized in an autoclave (plateau temperature between 121°C and 135°C with FO > 15).

[0262] After sterilization, hydrogels G1, G2 and G3 were analyzed. The elastic modulus G' and phase angle 5 were determined. The results are shown in Table 5 below.

[0263]

[0264] Table 5

[0265] 1 AG' (%) = (G' after sterilization - G' before sterilization) / (G' before sterilization) * 100

[0266] 2 A 5 (%) = (5 after sterilization - 5 before sterilization) / (5 before sterilization) *100

[0267] * 1 mL of hydrogel was considered to weigh one gram.

[0268] It is observed that hydrogels prepared from a process according to the invention comprising citrate ions in the crosslinking reaction medium exhibit lesser modifications in their rheological properties after sterilization compared to hydrogels prepared by an equivalent process without the addition of citrate ions.

Claims

CLAIMS 1. A method of preparing a sterile hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide, or a mixture thereof, the method comprising the following steps: (1) preparing a hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide, or a mixture thereof, and further comprising at least 1 mM citrate ions; and (2) sterilizing, preferably by heat, the hydrogel comprising at least 1 mM citrate ions to obtain a sterile hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide or a mixture thereof.

2. The method of claim 1 wherein step (1) comprises adding, to the crosslinked polysaccharide or the non-crosslinked polysaccharide or to their mixture, a solution comprising citrate ions in an amount sufficient to achieve a citrate ion concentration of at least 1 mM in the hydrogel.

3. The method of claim 1 wherein step (1) comprises adding, to the crosslinked polysaccharide or the non-crosslinked polysaccharide or to their mixture, citrate ions in powder form in an amount sufficient to achieve a citrate ion concentration of at least 1 mM in the hydrogel.

4. Method according to claim 2 wherein the solution comprising citrate ions has a pH ranging from 6.8 to 7.

8.

5. Method according to claim 2 or 4 wherein the solution comprising citrate ions is a solution of citric acid or sodium citrate, preferably a solution of citric acid or sodium citrate in a physiologically acceptable buffer.

6. Method according to claim 5 in which the physiologically acceptable buffer is a phosphate buffer.

7. Method according to any one of claims 2 to 6 in which the quantity of citrate ions added to the hydrogel makes it possible to achieve a concentration of citrate ions ranging from 1 to 12 mM in the hydrogel.

8. Method according to any one of claims 2 to 7 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.

9. Method according to claim 8 comprising a dilution step and / or a step of adding at least one additional component, in which the citrate ions are added during the dilution step and / or during the step of adding at least one additional component.

10. Method according to claim 1 in which step (1) comprises the following steps: (a) preparing a crosslinked polysaccharide from a crosslinking reaction medium comprising one or more polysaccharide(s), one or more crosslinking agent(s), a solvent and citrate ions in an amount sufficient to allow the preparation of a hydrogel based on a crosslinked polysaccharide comprising at least 1 mM of citrate ions; (b) preparation of a hydrogel from the crosslinked polysaccharide obtained at the end of step (a) and optionally from a non-crosslinked polysaccharide.

11. Method according to any one of claims 1 to 10 in which the polysaccharide is a hyaluronic acid.

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

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

14. Sterile hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide or their mixture, in particular a crosslinked hyaluronic acid, a non-crosslinked hyaluronic acid or their mixture, obtained by the method according to one of claims 1 to 13.

15. Use of citrate ions for protecting a hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide or a mixture thereof, in particular a crosslinked, non-crosslinked hyaluronic acid or a mixture thereof, and optionally an anesthetic agent, from the degradation of its rheological properties during its sterilization, preferably by heat.

16. Use of citrate ions for preserving the stability over time of a hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide or a mixture thereof, in particular a crosslinked, non-crosslinked hyaluronic acid or a mixture thereof, and optionally an anesthetic agent.

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