Hydrogels for filling soft tissue

Abstract

The present disclosure relates to a method for preparing a hydrogel comprising cross-linked polysaccharides, in particular a method for preparing an injectable hydrogel comprising cross-linked hyaluronic acid. The present disclosure also relates to hydrogels, preferably injectable, obtainable by the method, compositions comprising the hydrogels, and uses of the hydrogels.

Description

[Technical field]

[0001] The present invention relates to a method for preparing a hydrogel comprising crosslinked polysaccharides, in particular a method for preparing an injectable hydrogel comprising crosslinked hyaluronic acid. The present disclosure also relates to a hydrogel, preferably an injectable one, obtainable by the method, a composition comprising the hydrogel, and the use of the hydrogel. [Background technology]

[0002] Polysaccharide-based hydrogels, especially hyaluronic acid-based hydrogels, are widely used to fill or increase the volume of soft tissues, especially skin, for example, where there is a volume defect (e.g. wrinkles, scars). Hydrogels suitable for filling soft tissues are typically crosslinked hydrogels, i.e. the polysaccharides are crosslinked by using one or more crosslinking agents. This crosslinking makes it possible to obtain hydrogels with the mechanical properties desired for filling soft tissues.

[0003] Biodegradable and biocompatible hyaluronic acid-based hydrogels can cause adverse reactions, although the incidence is low, occurring 6-24 months after application of the filler, particularly migration of the filler to areas distant from its application area in or on the body (Chae SY, Lee KC, Jang YH, Lee SJ, Kim DW, Lee WJ. A Case of the Migration of Hyaluronic Acid Filler from Nose to Forehead Occurring as Two Sequential Soft Lumps. Ann Dermatol. 2016;28(5):645-647 and Mosleh, Rasha&Mukari, Abed&Krausz, Judit&Hartstein, Morris&Hamed Azzam, Shirin. (2019). Orbit mass secondary to migration of dermal hyaluronic acid filler. JAAD Case Reports. 5.488-490). Several reasons have been proposed to explain this phenomenon, such as poor injection technique, too large injection volume, physical activity after injection (muscular activity, exercise), slippage induced by pressure (e.g. massage), intravascular injection, lymphatic infiltration or gravity, etc. To reduce the risk of migration of polysaccharide-based gels, several methods have already been described, in particular the application of a repulsive electric field to at least part of the periphery of the treatment site (WO2020 / 172371). Summary of the Invention

[0004] Nevertheless, there remains a need to provide cross-linked polysaccharide-based hydrogels that have suitable mechanical properties for filling soft tissues and that do not migrate within the tissue after application, in particular cross-linked hyaluronic acid-based hydrogels in which the hyaluronic acid chains are preserved.

[0005] The present invention relates to a method for preparing a hydrogel, preferably an injectable one, comprising the following steps: a) providing at least one polysaccharide; b) providing at least one crosslinking agent, said crosslinking agent comprising at least two identical or different functional groups Z selected from isocyanate, amino, epoxide, carboxyl, N-succinimidyloxycarbonyl, N-sulfosuccinimidyloxycarbonyl, halocarbonyl, isothiocyanate, vinyl, formyl, hydroxyl, sulfhydryl, hydrazino, acylhydrazino, aminooxy, carbodiimide, and acid anhydride residues; c) preparing a crosslinking reaction medium comprising a polysaccharide, a crosslinking agent and a solvent, the total amount of the crosslinking agent being in the range of 0.001 to less than 0.02 moles per mole of repeating unit of the polysaccharide, and the duration of the reaction medium preparation step is not more than 5 hours; d) placing the reaction medium obtained at the end of step c) at a pressure P equal to or less than atmospheric pressure and at a temperature T above the eutectic temperature of the reaction medium measured at pressure P and below the freezing point temperature of the reaction medium measured at pressure P, for a period ranging from 2 weeks to 17 weeks; The present invention relates to a method comprising the steps of:

[0006] The present invention also relates to hydrogels obtainable by the methods described herein, as well as cosmetic or pharmaceutical compositions comprising such hydrogels, and their use in tissue filling and / or replacement, for preventing and / or treating alterations in the viscoelastic or biomechanical properties of the skin; for filling volume defects in the skin, in particular for filling wrinkles, fine lines and scars; for example for reducing nasolabial and glabella folds, for increasing the volume of the cheekbones, chin or lips, for restoring volume to the face, in particular the cheeks, temples, facial contours and the area around the eyes; or for reducing the appearance of wrinkles and fine lines; or for stimulating, regenerating, moisturizing, firming or restoring radiance to the skin, in particular by mesotherapy.

[0007] Other aspects of the invention are described below and in the claims. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 shows the adhesion of hydrogels according to the invention on metal (FIG. 1A), glass (FIG. 1B), plastic (FIG. 1C) and collagen (FIG. 1D) surfaces in comparison with conventional hydrogels. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] definition The term "gel" refers to a polymer network that is swollen with a fluid throughout its volume. This means that a gel consists of two media dispersed in each other, one solid and the other liquid. The so-called solid medium is composed of long molecules of polymers that are bound together by weak bonds (e.g. hydrogen bonds) or covalent bonds (crosslinks). The liquid medium is composed of the solvent. Gels generally correspond to products that have a phase angle δ equal to or less than 45°, advantageously in the range of 2° to 45° or in the range of 20° to 45°, at 1 Hz or 1 Pa pressure for a deformation of 0.1%.

[0010] The term "hydrogel" refers to a gel as defined above in which the solvent constituting the liquid medium is predominantly water (e.g. at least 90% by weight, in particular at least 95% by weight, in particular at least 99% by weight of the liquid medium). Preferably, the liquid medium comprises, in particular consists of, a buffer solution, in particular phosphate buffered saline, advantageously allowing a pH of the liquid medium comprised between 6.8 and 7.8.

[0011] The term "injectable gel" refers to a gel that can be flowed and manually injected by using a syringe equipped with a needle with a diameter in the range of 0.1 to 0.5 mm, for example a 30G, 27G, 26G or 25G hypodermic needle. Preferentially, an injectable gel is a gel that has an average extrusion force of less than or equal to 25 N, preferably in the range of 5 to 25 N, more preferably in the range of 8 to 15 N, when measured at room temperature with a syringe with an outer diameter of less than or equal to 6.3 mm, with a needle with a diameter of less than or equal to 0.4 mm (27G) and a length of half an inch, using a dynamometer at a fixed speed of approximately 12.5 mm / min.

[0012] The extensibility of a product refers to its ability to be stretched between the two surfaces to which it is attached. Extensibility can be determined using a texture analyzer, a sensory analysis carried out by a panel, or rheological and mechanical measurements, including in particular the measurement of the phase angle (δ) or tensile tests.

[0013] In particular, this characteristic can be measured as described by P. Michaels et al. (Micheels et al., Comparison of two Swiss-designed hyaluronic acid gels: Six-month clinical follow-up, Journal of Drugs in Dermatology, 2017, 16:154-161, "Resistance to stretching") or by performing a tack test and measuring the length of the gel thread upon traction.

[0014] The stickiness of a product refers to the ability of the product to adhere to a surface. Stickiness can be determined qualitatively using sensory analysis carried out using a panel or by moving a mass over a surface. Stickiness can also be determined quantitatively by measuring the adhesion force to a surface by a drag machine or mechanical analysis.

[0015] The term "polysaccharide" refers to a polymer composed of monosaccharides (preferentially the D enantiomer) linked together by glycosidic bonds.

[0016] The term "monosaccharide" refers to an unmodified or modified monosaccharide.

[0017] An "unmodified monosaccharide" is a sugar of the formula H-(CHOH) x -CO-(CHOH) y -H (x and y are independently integers between 0 and 5, with the proviso that 2≦x+y≦5), the monosaccharides may exist in the linear form represented by the above formula, or in a cyclized form by reacting a CO function (aldehyde or ketone) with one of the OH groups to form a hemiacetal or hemiketal group. Preferably, the monosaccharides are in the cyclized form. Two types of monosaccharides exist: aldoses, which carry an aldehyde function (when x or y is 0), and ketoses, which carry a ketone function (when neither x nor y is 0).

[0018] Monosaccharides are classified according to the number of carbon atoms. For example, a monosaccharide with six carbons (x+y=5) has the formula CH 12 It is an O6 hexose and can be allose, altrose, glucose, mannose, gulose, idose, galactose or talose.

[0019] A monosaccharide with 5 carbons (x+y=4) has the formula CH 10 The monosaccharide is a pentose of O5, which may be ribose, arabinose, xylose, or lyxose. Preferably, the monosaccharide is a hexose, i.e., x+y=5. The monosaccharide further contains x+y asymmetric carbons, thus 2 (x+y-1) Each pair of enantiomers is designated by a different name, and enantiomers of the same pair are called D and L enantiomers, respectively.

[0020] "Modified monosaccharides" include, for example, One or more of the -OH functional groups may be substituted with another functional group, e.g. (i) OR groups, where R represents a (C1-C6) alkyl group, such as methyl or ethyl; a hydroxy-(C1-C6) alkyl group, such as hydroxyethyl (-CH2CH2OH) or hydroxypropyl (-CH2-CH(OH)-CH3); a carboxy-(C1-C6) alkyl group, such as carboxymethyl (-CH2COOH); or a CO-(C1-C6) alkyl group, such as acetyl; and / or (ii) an NR'R" group, where R' and R" independently represent H, (C1-C6)alkyl or CO-(C1-C6)alkyl, e.g., acetyl; and / or (iii) OSO3H group and / or - the terminal CHOH function(s) have been replaced by a COOH or CHO group and the -CH(OH)-CH(OH)- bond has been oxidized to give two -terminal CHO (aldehyde) groups in place of this bond; and / or - a terminal CH2OH function is condensed with an OH function to form an -O-CH2- chain, Refers to an unmodified monosaccharide as defined above.

[0021] The expression "repeating unit" of a polysaccharide refers to a structural unit composed of one or more (usually one or two) monosaccharides, the repetition of which produces a complete polysaccharide chain.

[0022] Some or all of the monosaccharides may be in modified form.

[0023] The monosaccharides, if modified, can be in different modified forms.

[0024] The term "physiologically acceptable" generally refers to something that is safe, non-toxic, biologically or otherwise desirable, and acceptable for cosmetic (i.e., non-therapeutic) or therapeutic human or veterinary use, including use by injection into the human or animal body or topical application to the skin.

[0025] Salts useful in the context of the present invention are preferably physiologically acceptable salts. The term "physiologically acceptable salts" refers in particular to: (1) Pharmaceutically acceptable acid addition salts formed with pharmaceutically acceptable inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, or with pharmaceutically acceptable organic acids, such as formic acid, acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxynaphthoic acid, 2-hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, muconic acid, 2-naphthalenesulfonic acid, propionic acid, salicylic acid, succinic acid, dibenzoyl-L-tartaric acid, tartaric acid, p-toluenesulfonic acid, trimethylacetic acid, trifluoroacetic acid, and the like; and (2) Pharmaceutically acceptable base addition salts formed when an acidic proton present in the parent compound is either replaced by a metal ion, such as an alkali metal ion (e.g., Na, K), alkaline earth metal ion (e.g., Ca, Mg), zinc ion, silver ion or aluminum ion, or is coordinated with a pharma-ceutically acceptable organic base, such as diethanolamine, ethanolamine, N-methylglucamine, triethanolamine, tromethamine, etc., or a pharma-ceutically acceptable inorganic base, such as aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, etc.

[0026] The "degree of modification" (MOD) of a polysaccharide, such as hyaluronic acid, is the molar amount of crosslinker attached to the polysaccharide by one or more of its termini, expressed per 100 moles of repeating units of the polysaccharide. The degree of modification can be determined by methods known to those skilled in the art, such as by nuclear magnetic resonance spectroscopy (NMR).

[0027] "Molar crosslinking ratio" (CR), expressed as a percentage, refers to the molar ratio of the amount of crosslinker introduced into the crosslinking reaction medium to the amount of polysaccharide repeating units, expressed per 100 moles of polysaccharide repeating units in the crosslinking medium.

[0028] The expression "therapeutically active ingredient" refers to a substance for curing or alleviating symptoms and / or preventing disease; a substance having curative or preventive properties with respect to a human or animal disease, as well as any substance that can be used in or administered to humans or animals for the purpose of establishing a medical diagnosis or for the purpose of repairing, correcting or modifying their physiological functions by exerting a pharmacological, immunological or metabolic effect.

[0029] The expression "cosmetic active ingredient" refers to any non-therapeutic substance specifically intended to be brought into contact with various surfaces of the human body, such as epidermis, hair, nails, lips, breasts and teeth, exclusively or primarily for the purpose of cleaning, protecting, perfumed, maintaining in good condition or modifying the appearance or odor thereof.

[0030] The term "approximately" means that the value concerned can be 10%, in particular 5%, in particular 1% lower or higher than the specified value.

[0031] The expression "spacer group" refers to a fragment containing at least one atom intended to link together two chemical groups in the same molecule. Preferentially, the spacer group contains at least one carbon atom.

[0032] The term "halogen" refers to a fluorine, chlorine, bromine or iodine atom.

[0033] An "epoxide" group is an ethylene oxide residue attached to the remainder of the molecule by one of its carbon atoms.

[0034] An "N-succinimidyloxycarbonyl" group is a group of the following formula GR1:

[0035] [ka]

[0036] An "N-sulfosuccinimidyloxycarbonyl" group is a group of the following formula GR2.

[0037] [ka]

[0038] A "halocarbonyl" group is a group of the formula --CO--Hal, where Hal represents a halogen, for example, Cl or Br.

[0039] A "carbodiimide" group is a group that contains a -N=C=N- unit, more specifically a group of the formula -N=C=NR a is a group of R a represents an aliphatic hydrocarbon group having 1 to 20 carbon atoms, preferably a (C1-C6) alkyl group, of which one or more carbon atoms are optionally replaced by heteroatoms selected from O, S and N, in particular N.

[0040] An "acid anhydride residue" is a group comprising a -C(O)-OC(O)- unit, more particularly a monovalent cyclic group comprising a -C(O)-OC(O)- unit, for example a monocyclic monovalent saturated hydrocarbon group comprising 5 to 10, in particular 5 or 6, carbon atoms (of which 3 consecutive carbon atoms are replaced by -C(O)-OC(O)-), optionally with one or more, in particular one further carbon atom (preferably not consecutive to the 3 carbon atoms replaced by -C(O)-OC(O)-), each replaced by a heteroatom, for example N, O or S, in particular N. The acid anhydride residue may in particular correspond to the following chemical formula GR3:

[0041] [ka]

[0042] The acid anhydride residues may also be selected from maleic anhydride or succinic anhydride residues.

[0043] The expressions "aliphatic hydrocarbon chain" or "aliphatic hydrocarbon group" refer to linear, branched and / or cyclic, saturated or unsaturated, but non-aromatic hydrocarbon groups, advantageously containing 1 to 50, in particular 1 to 20, for example 1 to 12 or 1 to 6 carbon atoms. These are in particular alkyl groups.

[0044] The expression "branched aliphatic hydrocarbon chain" refers in particular to a primary aliphatic hydrocarbon chain which contains at least one secondary aliphatic hydrocarbon chain.

[0045] The expression "star-shaped aliphatic hydrocarbon chain" refers to a branched aliphatic hydrocarbon chain that includes several secondary aliphatic hydrocarbon chains that all originate from a single branch point.

[0046] The terms "C1-Cx alkyl" or "(C1-Cx)alkyl" or alternatively "alkyl having 1 to x carbon atoms" refer to a saturated linear or branched monovalent hydrocarbon radical having 1 to x carbon atoms, where x is an integer, such as methyl, ethyl, isopropyl, tert-butyl, n-pentyl, cyclopropyl, cyclohexyl, and the like.

[0047] The expression "(C1-Cx)alkylene" refers to a saturated linear or branched divalent hydrocarbon radical having 1 to x carbon atoms (x being an integer), such as methane-1,1-diyl, ethane-1,1-diyl, ethane-1,2-diyl, propane-1,3-diyl, butane-1,4-diyl, butane-1,3-diyl, butane-1,2-diyl, pentane-1,5-diyl, hexane-1,6-diyl, hexane-1,5-diyl, heptane-1,7-diyl, octane-1,8-diyl, nonane-1,9-diyl, decane-1,10-diyl, etc. In particular, the methane-1,1-diyl or propane-1,3-diyl radical.

[0048] The term "hydroxy-(C1-Cx)alkyl" refers to a (C1-Cx)alkyl group as defined above that is substituted by a hydroxyl (OH) group, such as hydroxyethyl (-CH2CH2OH) or hydroxypropyl (e.g. -CH2-CH(OH)-CH3).

[0049] The term "carboxy-(C1-Cx)alkyl" refers to a (C1-Cx)alkyl group as defined above that is substituted by a carboxyl group (COOH), such as carboxymethyl (-CH2COOH).

[0050] The term "aryl" refers to a monovalent aromatic hydrocarbon radical, preferably having from 6 to 10 carbon atoms and containing one or more rings, such as a phenyl or naphthyl radical.

[0051] The term "arylene" refers to a divalent aromatic hydrocarbon-based radical, preferably having from 6 to 10 carbon atoms and containing one or more rings, such as a phenylene radical.

[0052] The expression "aryl-(C1-Cx)alkyl" refers to an aryl group as defined above, linked to the remainder of the molecule via a (C1-Cx)alkyl chain as defined above, where x is an integer, such as a benzyl or phenylethyl group.

[0053] The expression "polyvalent group" refers to a group that can form several covalent bonds with other groups of the same compound or two different compounds. The bonds to the other groups can be formed from the same atom of the polyvalent group or from different atoms of the polyvalent group, preferably from different atoms of the polyvalent group. In particular, the polyvalent group is a divalent group and therefore can form two covalent bonds with two other groups of the same compound or two different compounds. The number of covalent bonds that can be formed refers to the valence of the polyvalent group.

[0054] Detailed Description of the Invention The inventors have developed a method that makes it possible to prepare hydrogels that meet the needs expressed. The hydrogels obtained have a cohesive property that allows them to adhere to the tissue and therefore not to migrate in the tissue after their application / injection.

[0055] Currently available fillers produce volume to fill soft tissues, but do not resist tissue sagging. However, aging of the skin over time is associated with changes in skin structure due to the appearance of wrinkles, as well as sagging of fat areas. Interestingly, in certain embodiments, the hydrogel according to the present invention, especially when administered and arranged in mesh form, can be used to hold tissue due to its extensibility and prevent aging-related tissue sagging. The hydrogel according to the present invention is therefore particularly useful for filling and / or replacing soft tissues.

[0056] Moreover, the obtained hydrogel is highly biocompatible. In fact, the preparation method uses a very small amount of crosslinker (less than 0.001-0.02 moles of crosslinker per mole of polysaccharide repeating unit). Therefore, the obtained hydrogel is weakly crosslinked.

[0057] In addition, the resulting hydrogels are highly mucoadhesive, thereby allowing attachment to the mucosa and therefore good persistence of the hydrogel at the administration site(s), as visible by qualitative ex vivo or in vitro tests following the labeled components.

[0058] The method for preparing a hydrogel, preferably injectable, developed by the inventors comprises the following steps: a) providing at least one polysaccharide; b) providing at least one crosslinking agent, said crosslinking agent comprising at least two identical or different functional groups Z selected from isocyanate, amino, epoxide, carboxyl, N-succinimidyloxycarbonyl, N-sulfosuccinimidyloxycarbonyl, halocarbonyl, isothiocyanate, vinyl, formyl, hydroxyl, sulfhydryl, hydrazino, acylhydrazino, aminooxy, carbodiimide, and acid anhydride residues; c) preparing a crosslinking reaction medium comprising a polysaccharide, a crosslinking agent and a solvent, the total amount of the crosslinking agent being in the range of 0.001 to less than 0.02 moles per mole of repeating unit of the polysaccharide, and the duration of the reaction medium preparation step is not more than 5 hours; d) placing the reaction medium obtained at the end of step c) at a pressure P equal to or less than atmospheric pressure and at a temperature T above the eutectic temperature of the reaction medium measured at pressure P and below the freezing point temperature of the reaction medium measured at pressure P, for a period ranging from 2 weeks to 17 weeks; Includes.

[0059] In the above methods, the polysaccharide and / or the cross-linking agent may be in the form of a salt.

[0060] It will be readily apparent that in the method of the invention, cross-linking of the polysaccharides occurs primarily during step d), ie when the reaction medium is in the frozen state.

[0061] When it comes to the question of optimal crosslinking temperature, the properties of the hydrogel obtained by the method of the present invention are especially the most surprising, since the prior art recommends not to go below 10°C and does not mention the use of temperatures below 0°C. (Jaeuk Baek, Yingfang Fan, Seol-Ha Jeong, Ho-Yong Lee, Hyun-do Jung, Hyoun-Ee Kim, Sukwha Kim, Tae-Sik Jang.Facile strategy involving low-temperature chemical cross-linking to enhance the physical and biological properties of hyaluronic acid hydrogel, Carbohydrate Polymers 202(2018)545~553).

[0062] Moreover, when it came to the issue of cryogelation, the amount of crosslinker used was still very high (Anna Strom, Anette Larsson, Oguz Okay, Preparation and physical properties of hyaluronic acid-based cryogels, J.Appl.Polym.Sci.2015, 42194; WO201029344). Otherwise the products prepared could not withstand the heat sterilization conditions (autoclave sterilization) commonly applied to this type of product. These products, unstable to sterilization, ultimately have mainly viscous components and / or a large proportion of low weight average molecular weight polymers, which is undesirable for applications filling soft tissues. However, for obvious safety reasons, it is always desirable to propose products that contain as few chemical modifications as possible.

[0063] The hydrogels of the invention differ from those prepared with the same constituents but without freezing in their macroporous structure, i.e. they have interconnected macropores, the size of which varies from less than one micron to several hundred microns, depending on the synthesis conditions, i.e. as a function of the nature and amounts of the reactants, as well as the temperature and the freezing rate. These macropores are visible during morphological analysis by scanning electron microscopy in the frozen state or by confocal laser scanning microscopy after staining the gels, for example with rhodamine B.

[0064] A hydrogel that has been returned to room temperature after a crosslinking step in the frozen state may be designated by the term "cryogel".

[0065] Moreover, while cryogelation was known to yield only a small number of hyaluronic acid fragments, it remains surprising that the method of the present invention preserves the structure of high weight average molecular weight polysaccharides, and in particular the structure of high weight average molecular weight hyaluronic acid.

[0066] The process according to the invention makes it possible to prepare crosslinked polysaccharide gels without degradation into fragments of low weight average molecular weight.

[0067] A polysaccharide with a high weight-average molecular weight refers to a polysaccharide whose weight-average molecular weight corresponds to that of a naturally (biologically) synthesized polysaccharide. For example, a hyaluronic acid polysaccharide with a high weight-average molecular weight refers to a polysaccharide having a weight-average molecular weight equal to or greater than 1 MDa, preferentially in the range of 1 MDa to 5 MDa, for example 1 MDa to 3 MDa or 1.5 MDa to 3 MDa, a chondroitin sulfate or chondroitin polysaccharide with a high weight-average molecular weight refers to a polysaccharide having a weight-average molecular weight equal to or greater than 30 kDa, preferentially in the range of 30 to 150 kDa, and a chitosan polysaccharide with a high weight-average molecular weight refers to a polysaccharide having an average molecular weight equal to or greater than 100 kDa, preferentially in the range of 100 kDa to 250 kDa. The polysaccharide provided in step a) of the method is typically a polysaccharide with a high weight-average molecular weight.

[0068] A low weight average molecular weight polysaccharide refers to a polysaccharide having an average molecular weight significantly lower than the average molecular weight of a biologically synthesized polysaccharide, more specifically, a low weight average molecular weight polysaccharide refers to a polysaccharide having a weight average molecular weight equal to or less than one third of the weight of the high molecular weight polysaccharide provided in step a) of preparing the hydrogel.

[0069] For example, a low weight-average molecular weight hyaluronic acid polysaccharide refers to a polysaccharide having a weight-average molecular weight in the range of 0.04 to 0.3 MDa, preferentially 0.08 to 0.20 MDa, more preferentially 0.08 to 0.15 MDa; a low weight-average molecular weight chondroitin sulfate or chondroitin polysaccharide refers to a polysaccharide having a weight-average molecular weight equal to or less than 50 kDa; and a low weight-average molecular weight chitosan polysaccharide refers to a polysaccharide having a weight-average molecular weight equal to or less than 30 kDa.

[0070] The absence of low molecular weight fragments is advantageous.Indeed, hyaluronic acid is known to be biocompatible, but it has already been reported in the literature that hyaluronic acid with low weight average molecular weight can cause long-term adverse effects after injection in patients (Cyphert JM, Trempus CS, Garantziotis S.Size Matters:Molecular Weight Specificity of Hyaluronan Effects in Cell Biology.International Journal of Cell Biology 2015;2015:563818).

[0071] Components used in the preparation of hydrogels polysaccharide A polysaccharide can be any macromolecule made up of monosaccharides linked together by glycosidic bonds.

[0072] Preferably, the polysaccharide is selected from pectin and pectin 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.

[0073] Pectic substances, including pectin, are polysaccharides composed of a backbone of D-galacturonic acid in acid form, which may be esterified with methanol, and L-rhamnose, which can form branches with other monosaccharides.

[0074] Chitosan and chitin are polysaccharides composed of repeating D-glucosamine units linked together by β-(1,4) bonds, some of which are N-acetylated. More specifically, chitosan has a degree of acetylation of less than 50%, while chitin has a degree of acetylation of more than 50%.

[0075] Cellulose is a polysaccharide composed of linear chains of D-glucose molecules.

[0076] Cellulose derivatives include methyl cellulose, ethyl cellulose, ethyl methyl cellulose, hydroxypropyl methyl cellulose (HPMC), hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC) and carboxymethyl cellulose (CMC).

[0077] Agarose is a polysaccharide containing the disaccharides D-galactose and 3,6-anhydro-L-galactopyranose as repeating units.

[0078] Glycosaminoglycans are linear polysaccharides composed of repeating disaccharide units, which contain a hexosamine (glucosamine (GlcN) or galactosamine (GalN)) and another monosaccharide (glucuronic acid (GlcA), iduronic acid (IdoA) or galactose (Gal)). The hexosamine and other monosaccharides may optionally be sulfated and / or acetylated. The glycosaminoglycan may be, inter alia, hyaluronic acid, heparosan, dermatan sulfate, keratan sulfate, chondroitin or chondroitin sulfate.

[0079] Hyaluronic acid is a glycosaminoglycan whose repeating units are disaccharides composed of D-glucuronic acid and N-acetyl-D-glucosamine linked together by alternating glycosidic β-(1,4) and β-(1,3) bonds. When hyaluronic acid is in the form of a salt, it is also called hyaluronate or hyaluronan.

[0080] In the context of the present invention, hyaluronic acid can have a weight average molecular weight in the range of 0.05-10 MDa, preferably in the range of 0.5-5 MDa, and even more preferentially greater than 0.05 MDa, for example in the range of 0.07-10 MDa or 0.07-5 MDa, or 0.5-5 MDa or 1-5 MDa or 2-4 MDa. Hyaluronic acid can be present in salt form, in particular in physiologically acceptable salt form, such as sodium salt, potassium salt, zinc salt, calcium salt, magnesium salt, silver salt, calcium salt and mixtures thereof. More specifically, hyaluronic acid is present in acid form or in sodium salt form (NaHA).

[0081] Heparosan is a glycosaminoglycan whose repeating unit is a disaccharide composed of glucuronic acid (GlcA) linked to N-acetylglucosamine (GlcNAc) by an α-(1,4) bond. Each disaccharide repeating unit is linked to the next unit by a β-(1,4) bond.

[0082] Chondroitin sulfate is a glycosaminoglycan whose repeating unit is a disaccharide composed of glucuronic acid linked by a β-(1,3) bond to a sulfated N-acetylgalactosamine, i.e., it contains at least one sulfate substituent. Each disaccharide repeating unit is linked to the next unit by a β-(1,4) bond.

[0083] Dermatan sulfate is a glycosaminoglycan whose repeating units are sulfated disaccharides, i.e., L-iduronic acid and N-acetyl-galactosamine linked by α-(1,3) bonds, containing at least one sulfate substituent. Advantageously, the disaccharide is sulfated at the C4 position of N-acetylgalactosamine, the C6 position of N-acetylgalactosamine, the C2 position of L-iduronic acid, or a combination of these positions. Each disaccharide repeating unit is linked to the next unit by a β-(1,4) bond.

[0084] Keratan sulfate is a glycosaminoglycan whose repeating unit is a sulfated disaccharide, i.e., composed of D-galactose and N-acetylglucosamine linked by alternating β-(1,4) and β-(1,3) bonds, containing at least one sulfate substituent.

[0085] The polysaccharides may be present in salt form, in particular in physiologically acceptable salt form, such as sodium, potassium, zinc, calcium, magnesium, silver salts and mixtures thereof, more particularly in the form of the sodium or potassium salt.

[0086] Advantageously, the polysaccharide is a glycosaminoglycan or a salt thereof, preferentially hyaluronic acid or a salt thereof, more preferentially hyaluronic acid or one of its physiologically acceptable salts, such as the sodium, potassium, zinc, silver salts and mixtures thereof, and even more preferentially hyaluronic acid or its sodium salt.

[0087] Polysaccharides generally have a weight average molecular weight in the range of 0.03 to 10 MDa.

[0088] Preferably, when the polysaccharide is hyaluronic acid, it has a weight average molecular weight (MW) in the range of 0.05 to 10 MDa, preferentially in the range of 0.5 to 5 MDa and even more preferentially above 0.05 MDa, for example in the range of 0.07 to 10 MDa or 0.07 to 5 MDa, or 0.5 to 5 MDa or 1 to 5 MDa or 2 to 4 MDa.

[0089] Crosslinking Agent A cross-linking agent is a compound containing at least two functional groups capable of covalently bonding with functional groups present on a polysaccharide, such as OH, CHO, NH2 or COOH groups carried by the polysaccharide, thus inducing bonds (cross-links) between polysaccharide chains and / or bonds on the same polysaccharide chain.

[0090] Crosslinkers useful in the context of the present invention preferably contain at least two, preferably two to eight, especially two functional groups (designated "Z groups") independently selected from isocyanate (-N=C=O), amino (-NH2), epoxide, carboxyl (-COOH), N-succinimidyloxycarbonyl, N-sulfosuccinimidyloxycarbonyl, halocarbonyl, isothiocyanate (-N=C=S), vinyl (-CH=CH2), formyl (-CH=O), hydroxyl (-OH), sulfhydryl (-SH), hydrazino (-NH-NH2), acylhydrazino (-CO-NH-NH2), aminooxy (-O-NH2), carbodiimide groups, and acid anhydride residues. Preferably, the functional groups are identical.

[0091] Isocyanate groups can react with OH or NH2 groups of polysaccharides to form carbamate or urea functional groups. Amino groups can react with COOH groups of polysaccharides to form amide functional groups. Epoxide groups can react with OH or COOH groups of polysaccharides to form ether or ester functional groups. Carboxyl groups can react with OH or NH2 groups of polysaccharides to form ester or amide functional groups. N-succinimidyloxycarbonyl and N-sulfosuccinimidyloxycarbonyl groups can react with OH or NH2 groups of polysaccharides to form ester or amide functional groups.

[0092] Halocarbonyl groups can react with OH or NH2 groups of polysaccharides to form ester or amide functional groups. Isothiocyanate groups can react with OH or NH2 groups of polysaccharides to form thiocarbamate or thiourea functional groups. Vinyl groups can react with OH groups of polysaccharides to form ether functional groups. Formyl groups can react with OH or NH2 groups of polysaccharides to form hemiacetal or hemiaminal functional groups. Hydroxy groups can react with COOH groups of polysaccharides to form ester functional groups. Sulfhydryl groups can react with COOH groups of polysaccharides to form thioester functional groups. Hydrazino (-NH-NH2) groups can react with CHO groups of polysaccharides to form hydrazone functional groups. Acylhydrazino groups can react with CHO groups of polysaccharides to form carbonylated hydrazone =NNHC(O)- functional groups. The aminooxy group can react with the CHO group of the polysaccharide to form an oxime = NO- functional group. The carbodiimide group can react with the COOH group of the polysaccharide to form a CO-NR a -CO-NH functional groups can be obtained and the anhydride residues can react with OH or NH2 groups of the polysaccharide to form ester or amide functional groups.

[0093] Preferably, the functional groups Z are identical and represent an epoxide or vinyl group, more preferentially an epoxide group.

[0094] According to another advantageous embodiment, the functional groups Z are identical and are selected from amino, vinyl, formyl and carbodiimide groups, preferably amino groups.

[0095] In particular, the crosslinkers are hexamethylene diisocyanate, diphenylmethylene 4,4'-diisocyanate, four-arm PEG20K-isocyanate, spermine (or 1,12-diamino-5,9-diazadodecane), spermidine (or 1,8-diamino-5-azaoctane), cadaverine (or 1,5-diaminopentane), putrescine (or 1,4-diaminobutane), poly(ethylene glycol) diamine, ethylenediamine, 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxy-octane, poly(ethylene glycol) diglycidyl ether (PEGDGE), 1,2-bis(2,3-epoxypropoxy) ether, ... The poly(ethylene glycol) diacid is selected from the group consisting of dimethylsiloxane (EGDGE), 1,3-bis(3-glycidyloxypropyl)tetramethyldisiloxane, poly(dimethylsiloxane) terminated at each end with a diglycidyl ether (CAS number: 130167-23-6), poly(ethylene glycol) diacid, disuccinimidyl suberate, bis(sulfosuccinimidyl) suberate, sebacoyl chloride, 1,4-butanediisothiocyanate, divinyl sulfone (DVS), glutaraldehyde, polyethylene glycol, 1,5-pentanedithiol, adipic acid dihydrazide, bis-aminooxy-poly(ethylene glycol), diethylenetriaminepentaacetic acid dianhydride, and mixtures thereof.

[0096] When the functional group Z is an epoxide group, the crosslinker is preferably selected from 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxy-octane, poly(ethylene glycol) diglycidyl ether (PEGDGE), 1,2-bis(2,3-epoxypropoxy)ethane (EGDGE), 1,3-bis(3-glycidyloxypropyl)tetramethyldisiloxane, poly(dimethylsiloxane) terminated at each end with a diglycidyl ether (CAS number: 130167-23-6), hydroxyapatite beads modified to carry epoxy groups, and mixtures thereof.

[0097] More preferentially, the crosslinking agent is selected from 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxy-octane, poly(ethylene glycol) diglycidyl ether (PEGDGE), 1,2-bis(2,3-epoxypropoxy)ethane (EGDGE), and mixtures thereof.

[0098] When the functional group Z is an amino group, the crosslinker is preferably a polyamine selected from spermine (or 1,12-diamino-5,9-diazadodecane), spermidine (or 1,8-diamino-5-azaoctane), cadaverine (or 1,5-diaminopentane), putrescine (or 1,4-diaminobutane), their salts or mixtures thereof, and more preferentially the crosslinker is a polyamine selected from spermine, spermidine and mixtures thereof.

[0099] The cross-linking agent may be selected from hydroxyapatite beads modified to carry epoxy groups, compounds of formula I as described below, and mixtures thereof.

[0100] Preferably, the crosslinker is a compound of formula I Y-(Z) n in which the identical or different functional groups Z are as defined above, n is an integer equal to or greater than 2, in particular ranging from 2 to 8, preferably equal to 2, Y is a polyvalent hydrocarbon group having a valence n and 1 to 150 carbon atoms, in particular an aliphatic group; - 1 or 2 or more (e.g., 1 to 150, or 1 to 50, or 1 to 15, or 1 or 2) CH2 units are selected from arylene, -O-, -S-, -S(O)-, -C(=O)-, -SO2-, -N(R 1 )-, and -[SiR 2 R 3 O] m -SiR 2 R 3 -, optionally replaced by one or more divalent units selected from R 1 represents a hydrogen atom, an aliphatic hydrocarbon group having 1 to 6 carbon atoms, or an aryl-(C1-C6)alkyl; m is an integer from 1 to 20, inclusive; Same or different R 2 and R 3 is a hydrogen atom; a halogen atom; -OR 11 Group(R 11 represents a hydrogen atom, an aryl group, or an aliphatic hydrocarbon group having 1 to 6 carbon atoms; aryl; or an aliphatic hydrocarbon group having 1 to 6 carbon atoms which may be substituted by one or more groups selected from a halogen atom, an aryl, or a hydroxyl; - the polyvalent group is unsubstituted or substituted by one or more monovalent groups selected from halogen atoms, hydroxyl, and aryl-(C1-C6)alkyl, preferably unsubstituted. In particular, n is an integer between 2 and 8, preferably n represents 2, 3 or 4 and even more preferentially n is equal to 2.

[0101] Advantageously, R 1 represents a hydrogen atom or a (C1-C6) alkyl group.

[0102] In particular, the same or different R 2and R 3 represents an aliphatic hydrocarbon group having 1 to 6 carbon atoms, more specifically a (C1-C6) alkyl group.

[0103] Preferably, in the definition of Y, the polyvalent hydrocarbon group has a valence n and may be an aliphatic or aromatic polyvalent hydrocarbon group having 1 to 150 carbon atoms, preferentially 1 to 50 carbon atoms, more preferentially 1 to 20 carbon atoms, even more preferentially 2 to 20 carbon atoms, preferably aliphatic and in particular saturated.

[0104] In particular, in the definition of Y, the polyvalent hydrocarbon radical is a saturated, in particular a linear, aliphatic polyvalent hydrocarbon radical.

[0105] Preferably, Y is a group in which one or more CH2 units are -O-, -SO2-, -[SiR 2 R 3 O] m -SiR 2 R 3 - and -NH-(R 2 , R 3 and m is as defined above), optionally replaced by one or more divalent units selected from

[0106] In particular, Y is a polyvalent hydrocarbon group as defined above, preferably aliphatic, saturated, in particular linear, branched or star-shaped, and optionally at least two CH2 units are replaced by -O-, in particular by 1 to 50 CH2 units, more particularly by 1 to 15 CH2 units, or at least one, preferably one or two, CH2 units are replaced by an -NH- unit, or at least one, preferably one, CH2 unit is replaced by a -SO2- unit, or at least two, preferably two, CH2 units are replaced by -O- and at least one, preferably one, CH2 unit is replaced by -[SiR 2R 3 O] m -SiR 2 R 3 -Units (R 2 , R 3 and m is as previously described.

[0107] More specifically, when one or more CH2 units are replaced by -O-, the replaced one or more units are replaced such that Y comprises one or more -CH2-CH2-O- units. In particular, Y comprises 1 to 50 -CH2-CH2-O- units, advantageously 2 to 25 -CH2-CH2-O- units, more advantageously 2 to 15 -CH2-CH2-O- units. Y may comprise only -CH2-CH2-O- units.

[0108] More preferentially, Y is an alkyl group, preferably linear, containing 1 to 150, in particular 1 to 50, in particular 1 to 20, for example 1 to 12, in particular 1 to 6 carbon atoms, optionally one or more CH2 units are replaced by one or more divalent units selected from -O- and -NH-, more particularly by 1 to 50, in particular 1 to 15, for example 1 or 2 divalent units selected from -O- and -NH-.

[0109] According to a first embodiment, identical or different R 2 and R 3 -OR 11 represents a group, R 11 is as mentioned above. In particular, R 11 represents an aliphatic hydrocarbon group having 1 to 6 carbon atoms, more specifically a (C1-C6) alkyl group.

[0110] According to a second embodiment, R 2 and R 3represents an aliphatic hydrocarbon group having 1 to 6 carbon atoms, which may be substituted (preferably unsubstituted) by one or more groups selected from halogen atoms, aryl or hydroxyl, more preferentially an unsubstituted (C1-C6) alkyl group, such as methyl or ethyl.

[0111] Advantageously, the crosslinking agent is a compound of formula Ia: Z 1 -Y 1 -Z 2 [wherein, identical or different Z 1 and Z 2 the groups are selected from isocyanate, amino, epoxide, carboxyl, N-succinimidyloxycarbonyl, N-sulfosuccinimidyloxycarbonyl, halocarbonyl, isothiocyanate, vinyl, formyl, hydroxyl, sulfhydryl, hydrazino, acylhydrazino, aminooxy, carbodiimide, and acid anhydride residues; Y 1 represents a divalent hydrocarbon chain, especially aliphatic, having 1 to 50 carbon atoms, - one or more (e.g., 1 to 15, or 1 or 2) CH2 units are arylene, -O-, -S-, -S(O)-, -C(=O)-, -SO2-, -N(R 1 )-, and -[SiR 2 R 3 O]m-SiR 2 R 3 -(R 1 is optionally replaced by one or more divalent units selected from a hydrogen atom, an aliphatic hydrocarbon group having 1 to 6 carbon atoms, or an aryl-(C1-C6)alkyl; m is an integer from 1 to 20, inclusive; Same or different R 2 and R 3 is a hydrogen atom; a halogen atom; -OR 11 Group(R 11represents a hydrogen atom, an aryl group, or an aliphatic hydrocarbon group having 1 to 6 carbon atoms; aryl; or an aliphatic hydrocarbon group having 1 to 6 carbon atoms which may be substituted by one or more groups selected from a halogen atom, an aryl, or a hydroxyl; - the chain is unsubstituted or substituted by one or more monovalent groups selected from halogen atoms, hydroxyl or aryl-(C1-C6)alkyl.

[0112] Z 1 and Z 2 The group has the same definition as the Z group defined above.

[0113] Y 1 has the same definition as Y defined above, but with valence n equal to 2.

[0114] In particular, Y 1 may contain only -CH2-CH2-O- units, as defined above.

[0115] Preferably, the crosslinker of formula I or Ia is -[SiR 2 R 3 O] m -SiR 2 R 3 -Does not include units.

[0116] Other components Further components may be lubricants; cosmetic active ingredients such as antioxidants, coenzymes, amino acids, vitamins, minerals, and nucleic acids; therapeutic active ingredients such as anesthetics, antibiotics, antifungals, and adrenaline and its derivatives, and mixtures thereof.

[0117] Polysaccharides, in particular non-crosslinked hyaluronic acid or non-crosslinked heparosan, may be mentioned as examples of lubricants.

[0118] Examples of anesthetic agents include, but are not limited to, ambucaine, amoxecaine, amylocaine, aprindine, aptocaine, articaine, benzocaine, betoxycaine, bupivacaine, butacaine, butamben, butanilicaine, chlorobutanol, chloroprocaine, cinchocaine, clodacaine, cocaine, cryofluoran, cyclomethycaine, dexivacaine, diamocaine, diperodone, dyclonine, etidocaine, euprosin, febuverine, fomocaine, guafecainol, heptacaine, hexylcaine, hydroxyprocaine, hydroxytetracaine, isobutamben, leucine, Nocaine, levobupivacaine, levoxadrol, lidamidine, lidocaine, rotucaine, menglitate, mepivacaine, meprylcaine, myrtecaine, octacaine, octodrine, oxetacaine, oxybuprocaine, paretoxycaine, palidocaine, phenacaine, piperocaine, pyridocaine, polidocanol, pramocaine, prilocaine, procaine, propanocaine, propipocaine, propoxycaine, proxymetacaine, pyrrocaine, catacaine, quinisocaine, lysocaine, rhodocaine, ropivacaine, tetracaine, tricaine, trimecaine, salts thereof, in particular the hydrochlorides, or mixtures thereof.

[0119] Examples of antioxidants include, but are not limited to, glutathione, reduced glutathione, ellagic acid, spermine, resveratrol, retinol, L-carnitine, polyols, polyphenols, flavanols, theaflavins, catechins, caffeine, ubiquinol, ubiquinone, alpha-lipoic acid and derivatives thereof, and mixtures thereof.

[0120] Exemplary amino acids include, but are not limited to, arginine (e.g., L-arginine), isoleucine (e.g., L-isoleucine), leucine (e.g., L-leucine), lysine (e.g., L-lysine or L-lysine monohydrate), glycine, valine (e.g., L-valine), threonine (e.g., L-threonine), proline (e.g., L-proline), methionine, histidine, phenylalanine, tryptophan, cysteine, derivatives thereof (e.g., N-acetylated derivatives, such as N-acetyl-L-cysteine), and mixtures thereof.

[0121] Examples of vitamins and their salts include, but are not limited to, vitamins E, A, C, B, in particular vitamins B6, B8, B4, B5, B9, B7, B12, and better still pyridoxine and its derivatives and / or salts, preferably pyridoxine hydrochloride.

[0122] Examples of minerals include, but are not limited to, zinc salts (e.g., zinc acetate, especially anhydrous), magnesium salts, calcium salts (e.g., hydroxyapatite, especially in beaded form), potassium salts, manganese salts, sodium salts, copper salts (e.g., copper sulfate, especially the pentahydrate), optionally in hydrated form, and mixtures thereof.

[0123] Examples of nucleic acids include, but are not limited to, adenosine, cytidine, guanosine, thymidine, cytosine, derivatives thereof and mixtures thereof.

[0124] Coenzymes include coenzyme Q10, CoA, NAD, NADP, and mixtures thereof.

[0125] Adrenaline derivatives include adrenaline, noradrenaline and mixtures thereof.

[0126] The amount of additional compound will obviously depend on the nature of said compound, the desired effect, and the goal of the compositions described herein.

[0127] method The steps of the method of the present invention may be as described below.

[0128] Providing at least one polysaccharide or a salt thereof (step a) Step a) of the method according to the invention comprises providing at least one polysaccharide or a salt thereof, in particular a physiologically acceptable salt thereof.

[0129] The polysaccharide is as described above. Preferably, the polysaccharide is hyaluronic acid or a salt of hyaluronic acid, preferably the sodium salt.

[0130] The polysaccharide may be provided wholly or partially in hydrated form or in dry form, e.g. in powder or fiber form. More specifically, in step a) the polysaccharide is provided in dry form, e.g. in powder or fiber form.

[0131] When the polysaccharide is provided in a hydrated form, the polysaccharide is in the form of a non-crosslinked gel or solution.

[0132] In particular, when the polysaccharide is in a hydrated form, it is an aqueous non-crosslinked gel or solution. More specifically, the polysaccharide is mixed with water, optionally with the addition of phosphate buffer or supplemented phosphate buffer, i.e., may contain further components, as defined in the further steps. It should therefore be understood that the aqueous non-crosslinked gel or aqueous polysaccharide solution does not contain sodium hydroxide.

[0133] Providing at least one cross-linking agent or a salt thereof (step b) Step b) of the method according to the invention comprises providing at least one cross-linking agent or a salt thereof, in particular a physiologically acceptable salt thereof.

[0134] The crosslinking agent is as described above.

[0135] Prepare the crosslinking reaction medium (step c) Step c) of the method according to the invention comprises preparing a cross-linking reaction medium, the reaction medium comprising a polysaccharide, a cross-linking agent and a solvent.

[0136] The solvent is typically water or a mixture containing water and an organic solvent (typically a mixture containing at least 90% by weight water, or at least 95% by weight, or at least 99% by weight water, based on the total weight of the solvent). For example, an organic solvent, such as an alcohol, in particular ethanol, or DMSO, can be used to solubilize the crosslinker prior to its addition to the aqueous reaction medium, for example when the crosslinker is a poly(dimethylsiloxane) terminated at each end with a diglycidyl ether (CAS number: 130167-23-6).

[0137] The reaction medium may also contain salts, pH adjusters, such as Bronsted bases, more preferentially hydroxide salts, such as sodium hydroxide or potassium hydroxide, further components as described above, and mixtures thereof. The addition of a Bronsted base is particularly effective in activating the functional groups Z of the crosslinker, such as Z 1 or Z 2 may be particularly necessary when they represent epoxide or vinyl groups. In these cases, crosslinking occurs at a pH equal to or greater than 10, more advantageously equal to or greater than 12, which requires the addition of a Brönsted base to the reaction medium (e.g. sodium hydroxide) at a concentration typically comprised between 0.10 M and 0.30 M.

[0138] The reaction medium is typically prepared from one or more polysaccharides in dry form. If the reaction medium is prepared from one or more polysaccharides in hydrated form, the aqueous non-crosslinked gel or aqueous polysaccharide solution used for the preparation of the reaction medium does not contain sodium hydroxide. Thus, the maximum contact time of the polysaccharide with sodium hydroxide before the start of step d) is 5 hours, for example 15 minutes to 4 hours or 30 minutes to 2 hours, whether the polysaccharide is provided in dry or hydrated form.

[0139] The total amount of crosslinker in the reaction medium varies from 0.001 to less than 0.02 moles per mole of polysaccharide repeating unit, for example from 0.001 to 0.015 moles per mole of polysaccharide repeating unit, preferably from 0.001 to 0.01 moles per mole of polysaccharide repeating unit, even more preferably from 0.001 to 0.008 moles or from 0.001 to 0.005 moles per mole of polysaccharide repeating unit. When the polysaccharide is a glycosaminoglycan, for example hyaluronic acid, the repeating unit is a disaccharide unit.

[0140] The mass concentration of polysaccharide or polysaccharide salt in the reaction medium advantageously ranges from 50 to 300 mg, preferably from 100 to 200 mg, per g of solvent.

[0141] Step c) of the process according to the invention typically comprises homogenizing the reaction medium, which is usually carried out by three-dimensional stirring, stirring with a mixer, stirring with a blade or stirring with a spatula.

[0142] Step c) is typically carried out at a temperature in the range of 4 to 35°C, preferably 15 to 25°C.

[0143] The duration of the step of preparing the reaction medium does not exceed 5 hours. It generally varies between 15 minutes and 4 hours, preferably between 30 minutes and 2 hours. The reaction medium obtained at the end of step c) is advantageously directly subjected to the conditions of step d) according to the invention.

[0144] Crosslinking at temperature T and pressure P (step d) The reaction medium obtained at the end of step c) is then placed at a pressure P equal to or less than atmospheric pressure and at a temperature T above the eutectic point temperature of the reaction medium (i.e. of the mixture comprising the polysaccharide, the crosslinker, the solvent and optional salts, pH adjusters and further components) measured at pressure P and below the freezing point temperature of the reaction medium measured at pressure P, for a period ranging from 2 weeks to 17 weeks.

[0145] The freezing point temperature of a reaction medium refers to the temperature at which the mixture of components of the reaction medium solidifies, i.e. becomes non-fluid, on a macroscopic scale. Below the freezing point, the mixture is in a frozen state, which is characterized by the coexistence of components in solid and liquid forms. The frozen state is maintained up to the freezing point temperature of the reaction medium.

[0146] The eutectic temperature of a reaction medium is the temperature below which a mixture of the components of the reaction medium changes from a frozen state (liquid and solid phases coexisting) to a completely solid state, i.e., all components of the mixture are in solid form.

[0147] The freezing point and eutectic point of a mixture vary depending on the pressure to which the mixture is subjected, and therefore the freezing point and eutectic point are measured at pressure P.

[0148] The freezing and eutectic points can be determined by differential scanning calorimetry, a method which makes it possible to determine phase transitions.

[0149] To do this, the product to be studied is gradually cooled until its phase transition is observed.

[0150] The temperature T is preferably greater than or equal to -55°C and less than or equal to -5°C, preferably in the range from -35°C to -10°C. Even more preferably, the temperature T is approximately -20°C.

[0151] The pressure P is preferably atmospheric pressure. Atmospheric pressure is the pressure exerted by the air constituting the atmosphere on any surface with which it is in contact. Atmospheric pressure 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 equal to or greater than -55°C and equal to or less than -5°C, preferably T varies between -35°C and -10°C and is approximately -20°C.

[0152] Preferably, the reaction medium obtained at the end of step c) is subjected to said pressure P and said temperature T for a period ranging from 2 weeks to 15 weeks, preferably from 2 weeks to 13 weeks, preferably from 2 weeks to 11 weeks, preferably from 2 weeks to 9 weeks, more preferably from 3 weeks to 6 weeks, or approximately from 4 weeks to 5 weeks, or approximately 30 days.

[0153] As indicated above, the crosslinking of the polysaccharides mainly occurs during step d) (it can nevertheless start from step c). This step therefore makes it possible to crosslink the polysaccharide chains with one another. The functional groups of the crosslinking agent react with the functional groups present on the polysaccharides to link the polysaccharide chains with one another and crosslink them by forming intramolecular 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 -OH or -COOH or -CHO groups present on the polysaccharide, for example hyaluronic acid. Thus, a crosslinked polysaccharide is obtained, which comprises at least one crosslinking link between two polysaccharide chains, which is the residue of the crosslinking agent. In particular, after step d), the crosslinked polysaccharide comprises at least one crosslinking bond between two polysaccharide chains, said crosslinking bond being more particularly a polyvalent group Y as described above, preferably a divalent group Y as described above. 1 Certain functional groups Z of the crosslinker (e.g., Z 1 and Z 2 ) does not react with the polysaccharide chain. In particular, the crosslinker has two functional groups Z 1 and Z 2 When the functional group Z 1 one of which is capable of reacting with the polysaccharide, and the other functional group Z 2 does not react with either polysaccharide. The pendant linkages are then formed.

[0154] The crosslinking may 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 may be added simultaneously or separately over time to the reaction medium. Step b) may therefore comprise repeated crosslinking steps. The crosslinking is then carried out in the presence of a crosslinking agent in a total amount ranging from 0.1 to less than 2 moles, preferably ranging from 0.1 to 1.5 moles or 0.1 to 1 moles or 0.1 to 0.8 moles or 0.1 to 0.5 moles of crosslinking agent (or their salts) per 100 moles of repeating units of the polysaccharide. The crosslinking conditions, in particular the content, duration and temperature of the crosslinking agent, as well as the weight-average molecular weight (Mw) of the polysaccharide used, are mutually dependent.

[0155] More specifically, the higher the temperature of the cross-linking reaction, the shorter the reaction time may be to obtain the same degree of modification of the polysaccharide with the cross-linking agent.

[0156] The lower the content of crosslinker, the longer the reaction time must be to obtain similar mechanical properties of the resulting gel. In other words, the lower the molar percentage of crosslinker, the fewer reactive functional groups are present in the reaction medium, and the less likely two groups are to associate and react together, so the longer the reaction time must be to make the functional groups react with each other, form crosslinks, and thus obtain a gel with the desired properties.

[0157] Conversely, too much crosslinker and too long a crosslinking time will result in a gel that is brittle and too stiff, and therefore not interesting for cosmetic applications.

[0158] The higher the weight average molecular weight (Mw) of the polysaccharide, the lower the degree of modification required to obtain a gel with given mechanical properties.

[0159] For the same molar percentage of crosslinker, the lower the weight average molecular weight (Mw) of the polysaccharide, the longer the reaction time to obtain a gel with given mechanical properties.

[0160] When the functional group Z of the crosslinking agent is an amino group, the crosslinking reaction with the polysaccharide is advantageously carried out in the presence of at least one activating agent and, where appropriate, combined with at least one coupling auxiliary.

[0161] In this regard, the activating agent may be selected from water-soluble carbodiimides, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 1-ethyl-3-[3-(trimethylamino)propyl]carbodiimide hydrochloride (ETC), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMC), their salts, and mixtures thereof, and may be preferably represented by EDC.

[0162] Concerning the coupling auxiliary, if present, the coupling auxiliary may be selected from N-hydroxysuccinimide (NHS), N-hydroxybenzotriazole (HOBt), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazole (HOOBt), 1-hydroxy-7-azabenzotriazole (HAt) and N-hydroxysulfosuccinimide (sulfoNHS), and mixtures thereof, and may be preferably represented by HOBt.

[0163] Further steps The process according to the invention may comprise one or more further steps, such as the addition of one or more further components, purification, sterilization, sieving, swelling and / or packaging.

[0164] The method according to the present invention may comprise the step of adding at least one further component.The further component may be selected from lubricants; cosmetic active ingredients, such as antioxidants, coenzymes, amino acids, vitamins, minerals and nucleic acids; therapeutic active ingredients, such as anesthetics, antibiotics, antifungals and adrenaline and its derivatives, and mixtures thereof.The further component may be as described above.

[0165] The process according to the invention may comprise at least one purification step.

[0166] Purification may be carried out by dialysis.

[0167] The method according to the invention may include the step of sterilizing the hydrogel.

[0168] Sterilization is preferably carried out by heating. Sterilization is generally carried out by raising the temperature of the sterilization medium to a temperature called the plateau temperature and maintaining that temperature for a specified period of time called the plateau time. Sterilization is preferably carried out at a plateau temperature ranging from 121°C to 135°C, for a plateau time ranging from 1 minute to 20 minutes, with an F0≧15. The sterilization value F0 corresponds to the time required to inactivate 90% of the microbial population present in the product to be sterilized at 121°C in a few minutes. Alternatively, sterilization can be carried out by gamma radiation, by UV radiation or by using ethylene oxide.

[0169] The method may include a step of sieving the hydrogel, more particularly with a sieve having a porosity comprised between 50 and 2000 μm. This sieving step makes it possible to obtain a more homogeneous hydrogel, using an extrusion force that is as constant as possible, i.e. as regular as possible. The skilled person knows how to select a sieve with a pore size adapted to the mechanical properties of the hydrogel to be prepared.

[0170] The method may include a step of swelling the hydrogel, during which the polysaccharide concentration of the hydrogel is adjusted.

[0171] In particular, a solvent is added, such as water, a phosphate buffer or water for injection. More particularly, the solvent added has a pH around the physiological pH (6.8-7.8). The polysaccharide concentration obtained after the swelling step advantageously varies between 1 mg per gram of gel and 50 mg per gram of hydrogel, more advantageously between 5 mg per gram of hydrogel and 35 mg per gram of hydrogel, and even more advantageously between 10 mg per gram of hydrogel and 30 mg per gram of hydrogel.

[0172] The method may include packaging the hydrogel.

[0173] If applicable, the step of adding one or more further components is preferentially carried out after the purification step.

[0174] If applicable, the step of adding one or more further components is preferentially carried out before the sterilization step.

[0175] In particular, the step of adding one or more further components may also comprise the addition of at least one therapeutic active ingredient, or at least one cosmetic active ingredient, or a mixture thereof. If at least one therapeutic active ingredient and / or at least one cosmetic active ingredient is added, the step of adding one or more further components is preferably carried out after step d).

[0176] Where applicable, a purification step is preferentially carried out after the step of adding one or more further components.

[0177] Where applicable, a purification step is preferentially carried out before the sterilization step.

[0178] Where applicable, a purification step is preferentially carried out before the sieving step.

[0179] A sterilization step is preferably performed after steps a) to d) and any further steps, in particular the hydrogel is sterilized after being packaged in its injection device, the gel being packaged after all steps of the method and before sterilization.

[0180] Hydrogels Another object of the invention is a hydrogel which may be obtained by the method of the invention. Such a hydrogel may also be designated by the term cryogel.

[0181] The hydrogel comprises one or more polysaccharides as defined above.

[0182] The polysaccharide is cross-linked. The molar degree of cross-linking of the polysaccharide is greater than 0% and less than 2%, preferably less than or equal to 1.5% or less than or equal to 1%, even more preferentially less than or equal to 0.8% or less than or equal to 0.5%, in particular between 0.1% and 0.8% or between 0.1 and 0.5% (moles of cross-linking agent per 100 moles of repeating units of the polysaccharide).

[0183] The one or more cross-linking agents are as defined above.

[0184] The crosslinking agent is preferably a crosslinking agent whose functional group is an epoxide group.Thus, the crosslinking agent is preferably 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxy-octane, poly(ethylene glycol) diglycidyl ether (PEGDGE), 1,2-bis(2,3-epoxypropoxy)ethane (EGDGE), and mixtures thereof.

[0185] The hydrogels of the present invention have adhesive characteristics. In some embodiments, the hydrogels of the present invention have extensible characteristics.

[0186] The hydrogel is preferably an injectable hydrogel.

[0187] The hydrogel is preferably sterilized, in particular heat sterilized at a plateau temperature of 121° C. to 135° C., preferably for a plateau time in the range of 1 min to 20 min, with an F0≧15.

[0188] The hydrogel is preferably homogeneous.

[0189] The hydrogel may comprise one or more further components as described above, in particular one or more further components selected from lubricating substances, anesthetic agents, antioxidants, amino acids, vitamins, minerals, nucleic acids and mixtures thereof.

[0190] Preferentially, hydrogels according to the invention that are acceptable for the therapeutic and / or cosmetic applications targeted by the invention have a crossover stress (or stress at the intersection of the G' and G'' moduli) equal to or greater than 50 Pa, preferentially between 50 and 5000 Pa, even more preferentially between 100 and 1000 Pa, preferably greater than 150 Pa, and an elastic modulus G' equal to or greater than 20 Pa, preferentially between 100 Pa and 2000 Pa, even more preferentially between 110 Pa and 1000 Pa.

[0191] Preferentially, hydrogels according to the invention that are acceptable for the therapeutic and / or cosmetic applications targeted by the invention have a cohesive force between 1N and 30N, preferably greater than 4N, for example in the range of 6-20N or in the range of 6-15N. This cohesive force is measured using a rheometer by mechanical compression. For this, the gel is deposited on a Peltier plate with an initial gap of 2.60 mm, then compressed at 25°C and at a constant speed of 100 μm / s to 70% of the initial gap, and finally, at the end of the compression process, the cohesive force of the gel is measured. The more easily the gel is cohesive, i.e. the higher the value of the cohesive force, the more it is able to withstand stresses, for example those that it may be subjected to after administration to a subject.

[0192] Preferentially, hydrogels according to the invention that are acceptable for the therapeutic and / or cosmetic applications targeted by the invention have a tack test result in the range of 1.5 to 15 cm, preferentially 1.5 to 10 cm and even more preferentially 2 to 7 cm.

[0193] Preferentially, the hydrogels according to the invention that are acceptable for the therapeutic and / or cosmetic applications targeted by the invention have adhesive characteristics and adhere to surfaces such as glass, metal, human tissue, collagen or plastics, and in addition, they maintain a cohesive structure when moving on these surfaces.

[0194] composition Another object of the invention is a composition comprising a hydrogel according to the invention. This composition is preferably a cosmetic or pharmaceutical composition. This composition can also comprise a physiologically acceptable excipient.

[0195] The hydrogel according to the present invention comprises a cross-linked polysaccharide. The composition may further comprise a non-cross-linked polysaccharide.

[0196] Thus, the composition according to the invention may contain from 0.1 to 5% by weight, preferably from 1 to 3% by weight, of polysaccharides (for example hyaluronic acid), relative to the total weight of said composition, the polysaccharides (for example hyaluronic acid) being present in crosslinked form, optionally in non-crosslinked form. In particular, the content of non-crosslinked polysaccharides (for example hyaluronic acid) varies from 0 to 40% by weight, preferentially from 1 to 40% by weight, more preferentially from 5 to 30% by weight, relative to the total weight of polysaccharides (for example hyaluronic acid) present in the composition.

[0197] The polysaccharide of the hydrogel is as defined above.

[0198] The composition according to the invention is preferably a sterilized composition, in particular a heat sterilized composition at a plateau temperature comprised between 121° C. and 135° C., preferably over a plateau duration of 1 min to 20 min, with an F0≧15.

[0199] The compounds according to the invention are preferably injectable compositions.The compositions according to the invention then preferably comprise a physiologically acceptable medium, preferably a physiologically acceptable aqueous medium.

[0200] The physiologically acceptable aqueous medium can comprise a physiologically acceptable solvent or solvent mixture, and preferably comprises water.

[0201] The physiologically acceptable medium may also contain isotonic agents, such as saccharides, sodium chloride and mixtures thereof.

[0202] The physiologically acceptable medium may also include at least one isotonic physiologically acceptable saline solution.

[0203] Preferably, the balanced saline solution is a phosphate buffered saline solution, in particular a KH2PO4 / K2HPO4 saline solution buffer.

[0204] The composition according to the invention may also contain at least one additional compound selected from lubricants, anesthetics, antioxidants, amino acids, vitamins, minerals, nucleic acids, coenzymes, adrenaline derivatives, and mixtures thereof. The additional compound may be as described above.

[0205] Purpose The hydrogels or compositions according to the invention may have therapeutic and / or cosmetic applications.

[0206] The present invention therefore also relates to a hydrogel or a composition according to the invention for use in filling and / or replacing tissue, in particular soft tissue, by injecting the hydrogel or composition into the tissue.

[0207] In particular, the hydrogels or compositions according to the invention are used in oral care, more particularly in the treatment of gingival recession or for filling periodontal pockets, more particularly for treating defects in the gingival structure that may occur with tooth loss, with aging, with periodontal disease and disorders, or after placing a dental implant, crown or bridge.

[0208] The hydrogels or compositions according to the invention may also be used in ophthalmology, more particularly to protect eye structures during eye surgery, such as anterior or posterior ocular surgery, cataract extraction possibly involving the implantation of an intraocular lens, corneal transplant surgery, glaucoma filtration surgery, or secondary lens implantation, etc. In this case, the hydrogels or compositions according to the invention are more particularly injected into the eye.

[0209] The hydrogels or compositions according to the invention may also be used in orthopedics or rheumatology, for example by injection into the synovial cavity. The hydrogels or compositions according to the invention are then used as viscosupplementation therapy.

[0210] The hydrogels or compositions according to the invention may also be used in the treatment of lipodystrophy.

[0211] The hydrogels or compositions according to the invention may be used in cosmetic surgery, in particular for gynecoplasty and / or penoplasty.

[0212] The hydrogel or composition according to the invention is more particularly administered by injection.

[0213] Advantageously, the hydrogel or composition according to the invention may be used for its mucoadhesive properties which are useful in the treatment of gum pain.

[0214] The present invention also relates to a method for treating the pathologies indicated above, comprising the step of administering an effective dose of the hydrogel or composition to an individual in need thereof.

[0215] The effective dose of the hydrogel or composition will vary according to a number of parameters, such as the chosen route of administration, the weight, age, sex, the progression of the pathology being treated, and the susceptibility of the individual to be treated.

[0216] An object of the invention is preferably the cosmetic, and therefore non-therapeutic, use of a hydrogel or a composition according to the invention for preventing and / or treating alterations in the viscoelastic or biomechanical properties of the skin, in particular by mesotherapy, in particular for stimulating, regenerating, moisturizing, firming or restoring radiance to the skin; for filling volume defects in the skin, in particular for filling wrinkles, fine lines or scars (in particular cavity scars); or for reducing the appearance of wrinkles and fine lines.

[0217] For example, the invention relates to the cosmetic use of a hydrogel or a composition according to the invention for reducing nasolabial folds and frown lines; for increasing the volume of the cheekbones, chin or lips; for restoring volume to the face, in particular the cheeks, temples, facial contours and around the eyes; or for stimulating, regenerating, moisturizing, firming or restoring radiance to the skin, in particular by mesotherapy.

[0218] In particular, the hydrogel or composition according to the invention is an anti-aging hydrogel or composition.The hydrogel or composition according to the invention is more particularly administered by injection.

[0219] The present invention also relates to a cosmetic, preferably anti-aging, treatment method for keratinous materials, in particular for the skin, which comprises the step of administering at least a hydrogel or a composition according to the invention onto or through said keratinous materials, more particularly by injection.

[0220] Administration can be by injection, in particular intraepidermal and / or intradermal and / or subcutaneous injection. Administration by intraepidermal and / or intradermal and / or subcutaneous injection according to the invention aims to inject the hydrogel or composition of the invention into the epidermis, dermis epidermis and / or skin area. The hydrogel or composition according to the invention can also be administered by supraperiosteal injection.

[0221] The hydrogel or composition according to the present invention can be injected using any method known to those skilled in the art.In particular, the hydrogel or composition according to the present invention can be administered by using an injection device suitable for intraepidermal and / or intradermal and / or subcutaneous and / or supraperiosteal injection.The injection device can be selected from, in particular, a syringe, a set of microsyringes, a wire, a laser or hydraulic device, an injection gun, a needleless injection device, or a microneedle roller.

[0222] The injection device may comprise any conventionally used injection means suitable for intraepidermal and / or intradermal and / or subcutaneous and / or supraperiosteal injection. Preferably, such means may be a hypodermic needle or cannula or a set of microneedles.

[0223] The needle or cannula according to the invention may have a diameter in the range of 18 to 34 G, preferably 25 to 32 G, and a length in the range of 4 to 70 mm, preferably 4 to 25 mm. The needle or cannula is advantageously for single use.

[0224] Advantageously, the needle or cannula is associated with a syringe or any other device making it possible to deliver said hydrogel or said injectable composition through the needle or cannula.

[0225] According to an alternative embodiment, the catheter may be inserted between the needle / cannula and the syringe, which may be actuated manually by a specialist or by a syringe holder, e.g. a gun, in a known manner.

[0226] Preferably, the injection device is a syringe.

[0227] In an alternative embodiment, the injection device may be adapted for mesotherapy techniques.

[0228] Mesotherapy is a treatment technique by intraepidermal and / or intradermal and / or subcutaneous injection of a composition or hydrogel. According to this technique, the composition or hydrogel is administered by injecting in the form of multiple small droplets into the epidermis, into the epidermal-dermal junction and / or into the dermis, in particular to reach the subcutaneous coating. The technique of mesotherapy is described in particular in the book "Traite de mesotherapie [Treatise on mesotherapy]" by Jacques Le Coz, Masson edition, 2004. Mesotherapy performed on the face is also called mesolift or mesoglow.

[0229] Administration may also be topical.

[0230] Preferably, administration is by topical application onto the surface of the skin, more particularly onto epithelia, and even more particularly onto facial epithelia.

[0231] The present invention therefore also relates to an injection device as described above comprising a hydrogel or a composition according to the invention.

[0232] The following examples are offered for illustrative purposes and should not be construed as limiting the invention in any way. EXAMPLES

[0233] 1.Material - non-crosslinked sodium hyaluronate (HTL) of 1.5 MDa and 4 MDa, - BDDE (Sigma Aldrich), - DVS (Fisher Scientific), - 0.25M NaOH, - 1M HCl, - phosphate buffer (Braun), - Lidocaine hydrochloride, - Three-dimensional mixing device, - DHR-2 rheometer, - Dynamometers and test benches, - Paddle mill homogenizer, - Sterile polyethylene bags.

[0234] 2. Method 2.1 Measurement of viscoelastic properties The viscoelastic properties of the resulting hydrogels were measured using a rheometer (DHR-2) with a stainless steel cone (1°-40 mm) and anodized aluminum Peltier plate (42 mm) (air gap 24 μm) in a cone-plane configuration.

[0235] 0.5 g of sterile hydrogel is deposited between the Peltier plate and the cone. A stress scan is then performed at 1 Hz and 25° C. The elastic modulus G′, viscous modulus G″ and phase angle δ are reported for a stress of 5 Pa.

[0236] The stress τ at the intersection of G' and G'' is determined at the intersection of the curves of the G' and G'' moduli and is expressed in Pascals.

[0237] 2.2 Measurement of pushing force The extrusion force (Newtons) of the gel packaged in the syringe was measured at room temperature through a 27G half-inch needle at a constant speed of 12.5 mm / min by using a test bench equipped with a dynamometer. The extrusion force results are the average of the extrusion forces of at least two samples.

[0238] 2.3 Determination of extensibility (tack test) The tack test is carried out on a DHR-2 rheometer (TA instruments) equipped with a crude steel structure (40 mm diameter) and a crude steel Peltier plate (40 mm diameter). 1 g of product is deposited between the two structures at 25° C. and the superstructure is rapidly approached to the Peltier plate at a speed of 2 mm / s up to a gap of 2 mm. The superstructure then compresses the gel at an approach speed of 0.1 mm / s up to a gap of 0.1 mm. The superstructure is then raised at 0.1 mm / s and the distance at which the gel maintains its extensibility characteristics between the two structures is reported. The extensibility determined by the length unit is therefore expressed in cm.

[0239] 2.4 Measurement of cohesive strength To measure the cohesive strength (or mechanical strength, expressed in Newtons), the gel is deposited on a Peltier plate with an initial gap of 2.60 mm. The gel is then compressed at a constant speed of 100 μm / s to 70% of the initial gap at 25° C. The cohesive strength of the gel is measured at the end of the compression process.

[0240] 2.5 Analysis of soluble hyaluronic acid Soluble hyaluronic acid, i.e., sHA, is analyzed using high performance liquid chromatography coupled with a multi-angle light scattering detector and a refractive index detector (HPLC-SEC-MALS-RI, ASTRA software (Wyatt Technology Corp.)). Samples are diluted approximately 10-fold according to their initial concentration of hyaluronic acid in the SEC mobile phase, filtered sodium nitrate solution 150 mM, pH 7.2. The diluted mixture consists of compact insoluble and soluble hyaluronic acid. The soluble hyaluronic acid portion of the samples was allowed to release under shaking on an orbital shaker for 5 days to ensure that soluble hyaluronic acid was not artificially generated.

[0241] The soluble portion is separated from the insoluble portion by gentle filtration methods (syringe with a 0.45 μm filter) and then subjected to SEC analysis.

[0242] To prevent and avoid overloading the SEC column, test various injection volumes to achieve a background free signal of at least 5 to 1.

[0243] The HPLC-SEC system uses a column duo for a wide range of hyaluronic acid ranging from 500 Da to 20 MDa for optimal peak resolution in the chromatograph.

[0244] To perform optimal molecular weight analysis, a refractive index increment value or dn / dc (change in refractive index / polysaccharide concentration in the analytical solvent) of 0.165 mL / g was determined based on an internal sample analyzed under the same conditions as the sample.

[0245] The chromatograms obtained by SEC are analyzed to quantify the molecular weight, distribution and percentage of soluble hyaluronic acid in each sample.

[0246] The weight average molecular weight of the sample is the data provided by the HPLC-SEC software. The soluble hyaluronic acid fraction percentage (sHA%) is also output directly from the HPLC-SEC software after inputting the mass concentration (mg / mL) of the total hyaluronic acid of the analyzed sample.

[0247] Said percentage may vary from composition to composition, depending on its preparation technique and the dispersion (w / d) (weight molar mass / number molar mass) of the sample.

[0248] The %sHA for several molecular weight limits (<250 kDa, <100 kDa, <50 kDa, <30 kDa and <20 kDa) is also output directly from the HPLC software, however this does not take into account the amount of hyaluronic acid actually released from the gel.

[0249] As a result, normalized sHA% was calculated for multiple molecular weight limits to normalize the values ​​provided by the software to take into account the amount of hyaluronic acid actually released from the analyzed samples.

[0250] A direct comparison of the results is then possible.

[0251] 3. Working Example Example 1: Hyaluronic acid gels crosslinked with BDDE either below or above the freezing point of the reaction medium Prototypes Nos. 1-9 were prepared in the following manner with the BDDE contents (BDDE moles CR) listed in Table 1.

[0252] BDDE and sodium hyaluronate (1.5 MDa, 120 mg / g) were dissolved in 0.25 M aqueous sodium hydroxide in a sterile bag. The mixture was then homogenized in a paddle mill at 210 rpm for three cycles of 15 min at room temperature.

[0253] The mixture was then maintained at atmospheric pressure and subjected to the temperatures and durations shown in Table 1. The pH of the mixture was approximately 13.

[0254] If the process was carried out at a temperature below the freezing point of the reaction medium (BDDE, sodium hyaluronate, aqueous sodium hydroxide), the mixture was thawed before continuing the preparation.

[0255] 1N HCl solution was then added to the sterile bag to obtain a pH of 7.3±0.5. The mixture was diluted with phosphate buffered saline (PBS) to a concentration of 23 mg hyaluronic acid per gram of product. The mixture was homogenized for 24 hours using a three-dimensional stirring device. The mixture was dialyzed.

[0256] Sodium hyaluronate (4 MDa) was added as a lubricating substance.

[0257] Aqueous lidocaine hydrochloride solution was added to give 0.3% by weight lidocaine hydrochloride based on the weight of the resulting product.

[0258] The product thus obtained was sieved and then packaged into syringes.

[0259] Finally, the product was sterilized by autoclaving (plateau temperature of 121°C to 135°C, F0 > 15).

[0260] After sterilization, the prototypes were analyzed. The elastic modulus G', the phase angle δ and the stress τ at the intersection of G' and G'' were determined. The results are presented in Table 1 below.

[0261] [Table 1]

[0262] For reference, the properties of Restylane Lyft, a commercial product with a degree of modification of 1% (the lowest value known for crosslinked hyaluronic acid gels for aesthetic applications), are presented in Table 2.

[0263] [Table 2]

[0264] The commercial product, Restylane Lyft, has a very low cohesive strength (2.2 N) compared to the gel obtained by using the method of the present invention.

[0265] While a low molar crosslinking rate of 2.2% did not allow the preparation of a gel even after 3 days of crosslinking at 21 °C (Prototype 1 with δ > 45°), the method of the present invention made it possible to prepare a hydrogel (Prototype 6) with desirable mechanical properties with a molar crosslinking rate of only 0.4%.

[0266] Although extending the crosslinking time increases the elastic modulus, the gels have a lower τ, in other words, the gels are potentially more brittle (see prototypes 5 vs. 4 and 7 vs. 6).

[0267] Surprisingly, the gels obtained by the method of the invention have rheological properties and cohesive strengths that are at least as interesting or even higher than those of the gels obtained by conventional methods at room temperature with a significantly higher degree of crosslinking (Prototype 2) (otherwise the HA concentration and preparation method being identical).

[0268] Example 2: Method according to the invention for cross-linking hyaluronic acid to DVS Prototypes X1 and X2 were prepared in the following manner with the DVS contents (DVS mole CR) listed in Table 3.

[0269] Sodium hyaluronate (1.5 MDa, 120 mg / g) is dissolved in a 0.25 M aqueous solution of DVS and sodium hydroxide in a sterile bag. The mixture is homogenized in a paddle mill at room temperature at 210 rpm for three cycles of 15 minutes. The mixture is placed and maintained at the temperature and duration shown in Table 3. The pH of the mixture was approximately 13.

[0270] The mixture is thawed before continuing with the preparation.

[0271] 1N HCl solution is added to the sterile bag until a pH of 7±0.5 is obtained. The six mixtures are diluted with phosphate buffer PBS to a concentration of 23 mg of hyaluronic acid per gram of product.

[0272] The mixture is homogenized for 24 hours by using a three-dimensional stirring device.

[0273] The mixture is then dialyzed and a fixed amount of sodium hyaluronate (4 MDa) is added as a lubricating substance.

[0274] Aqueous lidocaine hydrochloride solution is added to obtain 0.3% by weight lidocaine hydrochloride based on the weight of the resulting product.

[0275] The product thus obtained was sieved and then packaged into syringes.

[0276] Finally, the product was sterilized by autoclaving (plateau temperature of 121°C to 135°C, F0 > 15).

[0277] Prototype X0 (comparison) was prepared similarly to prototype X1, with only the cross-linking step being changed, i.e. cross-linking at 21° C. for 0.75 days.

[0278] [Table 3]

[0279] After sterilization, the prototypes were analyzed. The elastic modulus G', the phase angle δ and the stress τ at the intersection of G' and G'' were determined. The results are presented in Table 4 below.

[0280] [Table 4]

[0281] Prototypes X1 and X2 of hyaluronic acid cross-linked to DVS are gels (phase angle less than 45° C.) and have properties suitable for the therapeutic, cosmetic and aesthetic applications targeted by the present invention (stress at the intersection of G′ and G″ is greater than 50 Pa, G′ is greater than 20 Pa).

[0282] Prototype X0 was produced with the same degree of cross-linking as X1, but does not appear to have a gel morphology (δ>45°) when cross-linked conventionally (cross-linked at room temperature).

[0283] The method of the present invention appears to be well suited to crosslinkers having chemical functional groups other than epoxides.

[0284] Example 3: Stability study of cryogels according to the invention The prototypes presented in Table 5 were placed in an accelerated forced decomposition oven at 40° C. to monitor the evolution of the rheological properties over a period of six months. These conditions model the equivalent of two years of storage at room temperature. Each prototype was produced according to the synthesis protocol described in Example 1 under the conditions presented in Table 5.

[0285] [Table 5]

[0286] The change in phase angle was measured over time and is presented in Table 6 as phase angle reduction: % Phase Angle Reduction = (phase angle at 6 months at 40°C - initial phase angle) / initial phase angle x 100.

[0287] [Table 6]

[0288] The prototypes Y1, Y2 and Y3 with low crosslinking rates obtained by the method of the invention show a slight decrease in their viscoelastic properties after an accelerated aging study mimicking 2 years of storage at 25° C. under adverse conditions. This decrease is acceptable and is similar to that in the conventional gel (Y4) with a higher molar crosslinking rate obtained by the conventional method (crosslinking at room temperature).

[0289] Example 4: Analysis of soluble hyaluronic acid in cryogels The soluble hyaluronic acid of the gel in Table 7 (gel prepared according to Example 1) was analyzed by HPLC-SEC equipped with MALS and RI detector. The gel after sterilization was diluted at 37°C with gentle stirring to extract the soluble hyaluronic acid. The results are presented in Table 8. The weight average molecular weight (MW) and amount of soluble hyaluronic acid with MW less than 50 kDa are listed.

[0290] [Table 7]

[0291] [Table 8]

[0292] All these prototypes are formulated using the same hyaluronic acid raw material with a MW of >1 MDa, therefore the generation of fragments below 50 kDa indicates some degree of degradation of the hyaluronic acid during the process.

[0293] It is therefore evident that the two prototypes obtained by the conventional method (K1 and K2) are the only ones that contain traces of soluble hyaluronic acid with a MW of less than 50 kDa. All prototypes obtained by the method of the invention, otherwise with all other parameters equal, show a better preservation of the integrity of the hyaluronic acid chains and therefore do not show any traces of sHA less than 50 kDa.

[0294] Example 5: Comparison of the integrity of soluble hyaluronic acid chains in cryogels obtained by the method according to the invention or by the freezing method according to the prior art The size distribution of hyaluronic acid chains in hydrogels prepared according to the method described in Oschlaeger C.; Bossler, F.; Willenbacher, N. Synthesis, structural and micro-mechanical properties of 3D hyaluronic acid-based cryogel scaffolds (Biomacromolecules 2016) and in hydrogels prepared according to the present invention were compared to assess the respective ability of each method to preserve hyaluronic acid chains.

[0295] According to the prior art method, the first step of dissolving the hyaluronic acid fibers in sodium hydroxide is carried out for 24 hours at 4° C. Since the crosslinking process has already started during these 24 hours before cryogelation, only a 10 day cryogelation step is sufficient to obtain a gel with interesting properties close to those of the present invention.

[0296] In the method of the present invention, this step of dissolving the hyaluronic acid fibers (step a); preparing the reaction medium) is carried out at room temperature for about 1 hour, followed by freezing for a significantly longer period. The conditions for this comparison are summarized in Table 9.

[0297] To compare the effect of each method on the integrity of the hyaluronic acid chains, hyaluronic acid fibers were subjected to both methods without the use of crosslinkers. Thus, after each crucial step of the method (dilution of the fibers, cryogelation), it is possible to know the MW and the hyaluronic acid chain distribution by HPLC-SEC. A decrease in MW and an increase in the presence of small hyaluronic acid fragments are then synonymous with the degradation of hyaluronic acid by the method. For each HPLC-SEC analysis (system as described above), samples were neutralized to physiological pH and diluted to approximately 0.5 mg / mL.

[0298] [Table 9]

[0299] Starting from the same raw material with MW of 859 kDa, using the same aqueous solutions for dissolving, neutralizing and diluting, and using the same equipment and the same conditions for analyzing hyaluronic acid chains, it can be observed that the method according to the prior art gives a reduction of about 100 kDa in the MW of hyaluronic acid compared to the method of the present invention.This is also reflected in the presence of 0.5% hyaluronic acid with MW less than 50 kDa, while the method of the present invention does not show any trace of these hyaluronic acid fragments.

[0300] It is interesting to note that the freezing step of the two methods does not increase the degradation of hyaluronic acid, since only little degradation was observed during the fiber dissolution step over 24 hours at 4°C. Therefore, only this step has an effect on the MW of hyaluronic acid. The control sample (dissolved for 1 hour at 21°C according to the method of the present invention) showed no degradation compared to the initial raw material. The reduction in MW has a negative effect on the final viscoelastic properties of the gel, and the presence of small hyaluronic acid fragments may have an effect on the local biological inflammatory response after in vivo implantation of the gel.

[0301] Example 6: Evaluation of gel extensibility (tack test) The extensibility of the gels presented in Table 10 (prepared according to Example 1) was evaluated by a tack test, measuring the distance over which the gel can maintain bond / cohesion between two surfaces.

[0302] [Table 10]

[0303] Tack test data is presented in Table 11.

[0304] [Table 11]

[0305] Figure 1 shows the implementation of the tack test on prototypes Z2 and Z3.

[0306] After compression of the gel, the surfaces are separated.

[0307] Prototypes Z1 and Z3 have the highest extensibility, however Z1, according to its rheological properties, is not a viscoelastic gel (phase angle >45°) and therefore behaves as a viscous solution.

[0308] In contrast, Z3 is indeed a gel that has a greater extension range than the other prototypes.

[0309] Z2 and Z4 do not have extensibility characteristics: Z2 is a gel obtained by a conventional method (crosslinking at room temperature), whereas Z4 is a hydrogel obtained by the method of the present invention, which has a very high molar crosslinking rate (>2%), which makes the gel simultaneously elastic, brittle and non-extensible.

[0310] Thus, the prototype Z3, with its low degree of modification, obtained by the method of the invention, gives the best results in terms of obtaining a viscoelastic gel with the desired properties and a distensible gel (cohesion in traction).

[0311] Example 7: Evaluation of gel adhesion and affinity with surfaces The adhesion of gel J2 according to the invention (Prototype 6 in Example 1) and conventional gel J1 (Prototype 2 in Example 1) was qualitatively observed. The gels are presented in Table 12. The adhesion or non-adhesion of the tested gels to the model surfaces is observed after depositing a mass of 1 g of gel on said surface. The gel is pushed 10 cm and the state of the mass and the behavior of the gel are observed. The four model surfaces are a metal surface (Figure 1A), a glass surface (Figure 1B), a plastic surface (Figure 1C) and a gelatin surface (Figure 1D).

[0312] [Table 12]

[0313] Prototype J1, obtained by conventional crosslinking techniques, tends to roll on the model surfaces when moved. In contrast, prototype J2, obtained by cryogelation and with a G' relatively close to that of J1, tends to adhere systematically to these four model surfaces.

[0314] The force required to move J2 across the four model surfaces is also greater due to the adhesion of the gel to the motion. Note also that the prototype J2 maintains its cohesion (single block of gel) and does not crumble along its motion across the surface, despite adhesion to the path of its motion. This could be an important advantage in vivo, as gels tend to be less mobile due to their adhesion to surfaces.

Claims

1. A method for preparing a hydrogel, preferably an injectable one, comprising the following steps: a) providing at least one polysaccharide; b) providing at least one crosslinking agent, said crosslinking agent comprising at least two identical or different functional groups Z selected from isocyanate, amino, epoxide, carboxyl, N-succinimidyloxycarbonyl, N-sulfosuccinimidyloxycarbonyl, halocarbonyl, isothiocyanate, vinyl, formyl, hydroxyl, sulfhydryl, hydrazino, acylhydrazino, aminooxy, carbodiimide, and acid anhydride residues; c) preparing a crosslinking reaction medium comprising a polysaccharide, a crosslinking agent and a solvent, the total amount of the crosslinking agent being in the range of 0.001 to less than 0.02 moles per mole of repeating unit of the polysaccharide, and the duration of the step of preparing the reaction medium is not more than 5 hours; d) placing the reaction medium obtained at the end of step c) at a pressure P equal to or less than atmospheric pressure and at a temperature T above the eutectic temperature of the reaction medium measured at pressure P and below the freezing point temperature of the reaction medium measured at pressure P, for a period ranging from 2 weeks to 17 weeks; A method comprising:

2. The method according to claim 1, wherein the polysaccharide is a glycosaminoglycan, preferably the polysaccharide is hyaluronic acid or a salt thereof.

3. 3. The method according to claim 1 or 2, wherein the functional group Z of the crosslinking agent is an epoxide group, preferably the crosslinking agent is selected from 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxy-octane, poly(ethylene glycol) diglycidyl ether (PEGDG), 1,2-bis(2,3-epoxypropoxy)ethane (EGDGE), and mixtures thereof.

4. 4. The method according to any one of claims 1 to 3, wherein the total amount of cross-linking agent is in the range of 0.001 to 0.01 mole per mole of polysaccharide repeat unit, preferably 0.001 to 0.005 mole per mole of polysaccharide repeat unit.

5. 5. The method according to any one of claims 1 to 4, wherein the temperature T is greater than or equal to -55°C and less than or equal to -5°C, preferably around -20°C.

6. The method according to any one of claims 1 to 5, wherein the pressure P is equal to atmospheric pressure.

7. The method according to any one of claims 1 to 6, further comprising a sterilization step.

8. A hydrogel obtainable by the method according to any one of claims 1 to 7.

9. A cosmetic or pharmaceutical composition comprising the hydrogel of claim 8.

10. 10. A hydrogel according to claim 8 or a composition according to claim 9 for use in filling and / or replacing tissue.

11. 10. Cosmetic use of a hydrogel according to claim 8 or a composition according to claim 9 for preventing and / or treating changes in the viscoelastic or biomechanical properties of the skin; for filling volume defects in the skin, in particular for filling wrinkles, fine lines and scars; for reducing nasolabial folds and glabella, for increasing the volume of the cheekbones, chin or lips, for restoring volume to the face, in particular the cheeks, temples, facial contours and the area around the eyes; or for reducing the appearance of wrinkles and fine lines; or for stimulating, regenerating, moisturizing, firming or restoring radiance to the skin, in particular by mesotherapy.

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