EP3886929A1

EP3886929A1 - Biocompatible hydrogel, process for producing same, and use thereof in a mechanical viscosupplementation system

Info

Publication number: EP3886929A1
Application number: EP19809853.5A
Authority: EP
Inventors: Jean-Claude Giannotta; Charles CALLEJA
Original assignee: NVD
Current assignee: NVD
Priority date: 2018-11-30
Filing date: 2019-12-02
Publication date: 2021-10-06
Also published as: FR3089115A1; FR3089115B1; WO2020109628A1; US20220111124A1; CA3119242A1; US12102735B2

Abstract

The invention relates to a biocompatible hydrogel comprising between 0.3% and 30% by weight of dry matter of a copolymer formed at least from acrylamide, chitosan and N,N'-methylenebisacrylamide, as well as a diffusing agent.

Description

BIOCOMPATIBLE HYDROGEL, PREPARATION METHOD AND USE IN A VISCO-SUPPLEMENTATION SYSTEM

MECHANICAL

Technical field of the invention

The present invention relates to a biocompatible hydrogel. It further relates to a process for the preparation of such a hydrogel, to the use of the hydrogel and to a kit.

Prior art

The medical use of hydrogels containing polyacrylamide only was first disclosed in the patent document published under number EP0742022A, published on 1/13/1/1996.

Other patent documents published subsequently have concerned the use of biocompatible polyacrylamide hydrogels, in human health, as a filling agent, prostheses or visco-supplement (US7678146E3, EP308801 1 A).

For certain formulations of hydrogels, clinical publications mention a lasting biocompatibility of these hydrogels and their propensity to form crosslinked viscoelastic structures of interest for soft tissues in the case of external use, for example (wound care, Knapp et al., Clinical experiences with a new gel-like wound dressing after skin transplantation, Aktuelle Traumatologie, December 1984, pp. 275-281) or articular.

In vitro research has highlighted the possible capacity of crosslinked polyacrylamide hydrogels to form reservoirs for controlled and delayed release of biomolecules (Ferreira et al, Design of a Drug-Delivery System Based On Polyacrylamide Hydrogels, Evaluation of Structural Properties, Laboratories and Demonstrations, October 2000).

Numerous studies have shown the benefits of in situ supplementation of biomolecules carried in microcapsules, microspheres or vectors, contained in hydrogels and this, within joints for example, with chitosan microspheres. Janssen et al, Drugs and Polymers for Delivery Systems in OA Joints: Clinical Needs and Opportunities, Polymers 2014, 6, pp. 799-819. A difficulty to be resolved is to succeed in fixing, protecting and retaining in situ biocompatible microcapsules while avoiding aggregates and thus the initial excessive salting-out which can trigger possible inflammations.

Chitosan is a biopolymer, biocompatible and biodegradable which has multiple biomedical uses, in particular in cross-linked form in three dimensions. as a biomaterial (Croisier et al, Chitosan-based biomaterials for tissue engineering, European Polymer Journal, Volume 49, Issue 4, April 2013). The products of its degradation (glucosamine) are biocompatible, but the durability of the network is variable, in particular according to the mechanical and chemical constraints applied.

In this context, there is a growing need to design a mechanical support system for medical use that can meet the challenges of biocompatibility, biodegradability, visco-supplementation and carrying capacity in situ.

Summary of the invention

In view of the above, a technical problem which the invention proposes to solve is to produce for the health sector a new hydrogel which has improved properties compared to the hydrogels of chitosan or polyacrylamide of the art. anterior, forming effective support systems, having a carrying capacity in situ, and capable of allowing the release of a diffusing agent.

The solution of the invention to this problem first relates to a hydrogel comprising, on the one hand, between 0.3% and 30% by weight of dry matter of a copolymer formed at least of acrylamide, chitosan and N, N'-methylenebisacrylamide and, on the other hand, a diffusing agent.

Thus, the hydrogel has a viscoelastic matrix structure which allows prolonged mechanical hydration or visco-supplementation of the diffusing agent which is located, fixed, protected and released by the hydrogel during its degradation in situ. The hydrogel makes it possible to avoid the aggregates of diffusing agents and, consequently, the initial excessive releases.

Advantageously, the diffusing agent is chosen from inert ingredients having advantageous biomechanical properties or active agents, preferably substances of plant origin such as genepi extracts, substances of marine origin, such as extracts of green mussels. from New Zealand (Perna canaliculus), ortho-silicic acid, organic silicon, silanol, vitamins such as vitamins A, D3, E or C, metals such as gold or silver, pain relievers such as lidocaine, xylazine, detomidine, nonsteroidal anti inflammatory drugs, such as flunixin, ketoprofen, aspirin, corticosteroids such as prednisolone, triamcinolone, hyaluronic acid, glycosaminoglycans, chondroitin sulfate, methylsulfonyl of bromelain, arnica, collagen, antioxidants, fatty acids - the diffusing agent is included in a cargo ship chosen from microcapsules, microparticles and polymeric vehicles, preferably biodegradable microcapsules; - the hydrogel is substantially free of pyrogenic agents; - The mass ratio between acrylamide and chitosan is between 1/1 and 1/8; - The mass ratio between N, N'-methylenebisacrylamide and acrylamide is between 1/50 and 1/1000, preferably between 1/100 and 1/500; and the following ratio of the constituents in% by weight of the total weight of the hydrogel: acrylamide of between 0.3% and 20%; chitosan between 0.0375% and 10%; N, N'-methylenebisacrylamide between 0.004% and 0.4%; diffusing agent 0.001% and 30%; and H2O up to 100%.

The second object of the invention is a method of manufacturing a hydrogel as defined above, comprising the following steps: copolymerization of acrylamide and chitosan, in the presence of N, N'-methylenebisacrylamide and an initiator radical, in an aqueous medium to obtain a copolymer; washing the copolymer with water to obtain a washed copolymer; and adding a diffusing agent to obtain the hydrogel.

In a preferred embodiment, the process comprises the following stages: copolymerization of acrylamide and chitosan, in the presence of N, N'-methylenebisacrylamide, of a diffusing agent, and of a radical initiator, in an aqueous medium, to obtain a copolymer incorporating the diffusing agent then washing with water of the copolymer incorporating the diffusing agent to obtain the hydrogel.

Advantageously, the process comprises the following stages: copolymerization of acrylamide and chitosan introduced with a mass ratio of between 1/1 and 1/8, at a temperature between 20 and 60 ° C, preferably between 40 and 60 ° C , in the presence of N, N'-methylenebisacrylamide introduced with a mass ratio relative to the acrylamide of between 1/50 and 1/1000, preferably between 1/100 and 1/500, and of a radical initiator with a mass ratio with respect to acrylamide between 1/100 and 1/10 chosen from potassium persulfate or ammonium persulfate, optionally in combination with tetramethylethylenediamine with a mass ratio with respect to acrylamide between 1/2000 and 1/20, in an aqueous medium to obtain a copolymer; washing the copolymer with water to obtain a washed copolymer; and addition of the diffusing agent between 0.001% and 30% by weight of the total weight of the hydrogel, the diffusing agent being chosen from inert ingredients having biomechanical properties interesting or active ingredients, these agents preferably being substances of plant origin such as extracts of genepi, substances of marine origin such as extracts of New Zealand green mussels (Perna canaliculus), ortho-silicic acid , organic silicon, silanol, vitamins such as vitamins A, D3, E or C, metals such as gold or silver, pain relievers such as lidocaine, xylazine, detomidine, non-inflammatory drugs steroids, such as flunixin, ketoprofen, aspirin, corticosteroids such as prednisolone, triamcinolone, hyaluronic acid, glycosaminoglycans, chondroitin sulfate, methylsulfonylmethane, bromelain, arnica, collagen, antioxidants , fatty acids, possibly included in a cargo ship chosen from microcapsules, microparticles or polymeric vehicles, preferably biodegradable microcapsules, to obtain the hydrogel.

The third object of the invention is the use of a hydrogel as defined above, in a mechanical visco-supplementation system, for external or internal use.

Advantageously, the visco-supplementation system is a lubricant; - the visco-supplementation system is a cross-linked matrix with carrying or storage capacity; - the visco-supplementation system is a moisturizer; - the diffusing agent begins to diffuse between the 2nd and the 30th day after the administration, preferably between the 10th and the 20th day, in particular from the 15th day; and - the broadcasting agent is released over a period of between 2 weeks and 12 months, preferably between 1 month and 6 months.

A fourth object of the invention is a kit for external or internal mechanical visco-supplementation comprising a copolymer of acrylamide and of chitosan crosslinked with N, N'-methylenebisacrylamide, and a diffusing agent in solid phase or in suspension, said copolymer and said diffusing agent being premixed during manufacture in the form of a hydrogel or mixed extemporaneously to form a final hydrogel.

Brief description of the figures

The invention will be better understood on reading the following non-limiting description, drawn up with reference to the appended drawings, in which:

- Figure 1 shows schematically the grafting reaction of chitosan on polyacrylamide by intervention of a radical reaction (creation of free radicals using a radical initiator, noted I), and the crosslinking of the copolymer under the action N, N'- methylenebisacrylamide, according to the invention, o, p, q, r, n and m are the numbers of units of monomers;

FIG. 2A is a table which presents, after drying of the hydrogels (50 ° C. for more than 12 hours, and weighing of the dry matter, the mass percentage of dry matter. The V14 hydrogel is the copolymer of the invention ( 3.75% dry matter), the hydrogel V 5 is pure polyacrylamide and V20 is the copolymer with the diffusing agent.

FIG. 2B represents a comparison of the infrared spectra with Fourier transform (FT-IR), detailed in example 2, of the chitosanolyacrylamide-MBA (V14) copolymer (MBA for N, N'-methylenebisacrylamide) present in l hydrogel of the invention without diffusing agent, then V20 with diffusing agent, of a “pure” polyacrylamide gel (V 5) and “pure” chitosan (Chitosan);

FIG. 2C represents a comparison of the infrared spectra with Fourier transform (FT-IR), detailed in Example 2, of the chitosanolyacrylamide-MBA (V14) copolymer (MBA for N, N'-methylenebisacrylamide) present in l hydrogel of the invention, a “pure” polyacrylamide gel (V 5) and “pure” chitosan (Chitosan);

- The table in Figure 2D lists the majority FT-IR peaks of the chitosan-polyacrylamide-MBA copolymer;

- Figure 3A shows a dissolution test of the copolymer present in the hydrogel of the invention, in acetic acid, as described in Example 2 and Figure 3B illustrates the absence of dissolution of the copolymer in the acetic acid by filtration of the hydrogel after the dissolution test described in Example 2;

- Figure 4 shows the loss tangents following viscoelasticity measurements, in the different cases studied for the copolymer of the invention;

- Figure 5 is a diagram illustrating the copolymer washing step during which the residual monomers, represented by black circles, leave the three-dimensional network formed by the copolymer and represented in white, and are replaced by water molecules (gray circles) by an osmotic phenomenon;

- Figure 6 is a diagram which illustrates the washing of the copolymer in a dialysis membrane;

- Figure 7A is a photograph showing the placement of the dialysis membrane on the neck of an injection gun;

- Figure 7B is a photograph which shows the filling of the dialysis membrane using the injection gun; - Figure 7C is a photograph which shows the closure of the dialysis membranes by clips and nodes;

- Figure 8 compares the relative difference in mass gain, in percentage, of an unconstrained hydrogel in a tulle bag (black diamonds) and a constrained hydrogel in a dialysis membrane (gray triangles);

- Figure 9 shows the relative difference in the mass gain of two hydrogels, in percentage, as a function of time, in minutes, during the washing of the copolymer in a dialysis membrane;

FIG. 10 represents the relative difference in mass gain, in percentage, as a function of time in minutes of two hydrogels of 5% dry matter, of a copolymer as defined in the invention (squares), and a polymer of the prior art, a reference polyacrylamide-MBA (diamonds), in the presence of water;

- Figure 1 1 A represents the relative difference in weight gain, in percentage, as a function of time in minutes, of different copolymers as defined in the invention and N4 a (N ^* 1): Hydrogel 4.8 % PAAG-Chitosan copolymer,

N4 a (N ^* 2): 4.8% hydrogel PAAG-Chitosan copolymer,

N4 a (N ^* 3): 4.8% hydrogel PAAG-Chitosan copolymer,

N4 3a / 2 (N ^* 4): Hydrogel 4.8% PAAG-Chitosan copolymer, with + 50% of crosslinker MethylBisAcrylamide,

N4 a / 2 (N ^* 5): 4.8% hydrogel PAAG-Chitosan copolymer, with 50% of crosslinker MethylBisAcrylamide;

- Figure 1 1 B represents the relative difference in mass gain, in percentage, as a function of time in minutes, of 8 hydrogels of copolymer produced consecutively as defined in the invention;

- Figures 12A and 12B present tables from the XTT tests “In vitro Cytotoxicity Assay, Cell Growth Analysis via XTT-Staining and Grading Score Analysis”;

- Figure 13 illustrates the formula of chitosan, obtained by deacetylation of chitin. Chitosan contains glucosamine (group on the right in the figure);

- Figure 14 is a graph which corresponds to the IRFT spectrum of a sample referenced 1904-E0012452 of a 2.5% polyacrylamide hydrogel (in dark gray) and of a sample according to the invention referenced 1904-E0012453 of a hydrogel 5% polyacrylamide-chitosan copolymer (in light gray);

FIG. 15A is an electron scanning microscopy (SEM) image illustrating the crosslinked structure of the copolymer of the invention, after cryogenics and the FIG. 15B is a SEM image illustrating the crosslinked structure of the hydrogel of the invention, after cryogenics, in which a particle of the diffusing agent appears in a cell of the three-dimensional network;

- Figure 16 is a table which indicates the contents of residual acrylamide and methyl bis-acrylamide monomers, after dialysis. These values are below the measurement limits of 4ppm;

- Figure 17 illustrates the fact that the hydrogel copolymer according to the invention is formed by creating a macromolecular chain which is then organized in the form of a three-dimensional network thanks to the crosslinking agent (methyl bis-acrylamide);

FIGS. 18A, 18B and 18C are NMR curves of the 5% polyacrylamide-chitosan copolymer (FIG. 18A) which have several distinctive points, in particular specific for chitosan in 1% solution at 3.2, 3.6 and 4, 9 ppm (Fig. 18B) and in significant proportion compared to the NMR of the 2.5% polyacrylamide hydrogel alone (Fig. 18C).

Detailed description of the invention

According to the present invention, the terms "between ... and ..." used to define ranges of values must be understood as integrating the lower and upper limits of these ranges. The terms "% by weight" should be understood as "% by weight relative to the total weight of the hydrogel".

According to a first object, the invention relates on the one hand to a hydrogel for the health sector comprising between 0.3% and 30% by weight of dry matter of a copolymer formed at least of acrylamide, chitosan and N, N'-methylenebisacrylamide and, on the other hand, a diffusing agent.

A "hydrogel" is a gel, that is, a three-dimensional network of solids diluted in a fluid, the fluid of which is water (usually 80% or more by weight of the total weight of the hydrogel). The three-dimensional network of solids is generally a network of polymers. These are insoluble in water, but are able to swell substantially in the presence of a large amount of water.

Referring to Figure 1, the hydrogel of the invention comprises or consists of a three-dimensional molecular network trapping water molecules and carrying a diffusing agent. The three-dimensional molecular network is formed by a chitosan-polyacrylamide-MBA copolymer. Water is present in the hydrogel in an amount of 70 to 99.7% by weight, preferably in an amount of 90 to 96% by weight of water, excluding diffusing agent. The copolymer hydrogel according to the invention has the capacity to carry diffusing agents, in particular submicron capsules. The microstructure of the three-dimensional network of this hydrogel has cells the size of which is compatible with the particles carried away, which polyacrylamide gels produced according to the prior art do not offer. In addition, and as shown in the examples of the present description, the hydrogel according to the invention has a capacity 3 times greater in carrying water (hydrophilic swelling) compared to a hydrogel of the same concentration in polyacrylamide only. It therefore requires less substance with a copolymer hydrogel to provide as much hydration as with a polyacrylamide gel with equal initial dry matter concentrations.

The hydrogel of the invention is "biocompatible", that is to say that it does not degrade the biological medium in which it is used. This biocompatibility comes from the large amount of water absorbed by the hydrogel and from the non-toxic three-dimensional structure formed by a copolymer of chitosan and polyacrylamide crosslinked with N, N’-methylenebisacrylamide.

The hydrogel of the invention is "biodegradable" in the sense that it degrades to form entities which are not harmful to the environment in which it is found. In particular, one of the hydrogel degradation products of the invention, for example containing chitosan, is glucosamine, which is also produced naturally by the body from glucose and glutamine. Glucosamine plays a key role in maintaining the integrity of cartilage in all joints. It supports the lubricating action of synovial fluid, a natural lubricant for the joints.

The hydrogel of the invention is a chitosan-polyacrylamide-MBA hydrogel comprising a diffusing agent. This hydrogel therefore comprises a chitosan-polyacrylamide-MBA copolymer formed at least from acrylamide, chitosan and N, N’-methylenebisacrylamide.

Acrylamide is a monomer of synthetic origin. During polymerization, the monomers gather into a macromolecule which loses its toxicity. Also, it is very important to construct the three-dimensional network as best as possible and to wash the hydrogel after crosslinking so as to extract all of the residual potential monomers.

Chitosan is a material of natural and renewable origin. This biocompatible and biodegradable material, has no toxicity, is soluble in acetic acid and is capable of chemically grafting to other molecules. There are many different types of molecules reacting on chitosan. Mention may in particular be made of polyethylene glycol, polyvinyl alcohol, polyacrylic acid, hydroxycellulose, polyacrylates, polyacrylics, and polyacrylamide.

Chitosan is a polysaccharide composed of the random distribution of D-glucosamine linked in B- (1 -4) (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). It is produced by chemical deacetylation (in an alkaline medium) or enzymatic of chitin, the component of the exoskeleton of arthropods (crustaceans) or the endoskeleton of cephalopods (squid ...), sometimes also of plant origin (walls mushrooms) or of synthetic origin. This raw material is demineralized by treatment with hydrochloric acid, then deproteinized in the presence of soda or potassium hydroxide and finally discolored thanks to an oxidizing agent. The degree of acetylation (DA) is the percentage of acetylated units compared to the number of total units, it can be determined by Fourier transform infrared spectroscopy (IR-TF) or by a strong base titration. Chitosan is soluble in an acid medium unlike chitin which is insoluble. It is important to distinguish between the degree of acetylation (DA) and the degree of deacetylation (DD). One being the complement of the other, that is to say that chitosan having a DD of 85%, has 15% of acetyl groups and 85% of amine groups on its chains.

Chitosan is a macromolecule whose molar mass is between 50 kDa and 400 kDa (g / mole) which can also be viewed by a number of patterns from a few hundred to 2 or 3 thousand. The hydrogel comprises for example between 0.15% and 3% by weight of chitosan of the total weight of the hydrogel. In a more specific example, the hydrogel comprises between 0.30% and 2% by weight of chitosan of the total weight of the hydrogel.

N, N’-methylenebisacrylamide is a crosslinking agent. In the present invention, N, N’-methylenebisacrylamide makes it possible to obtain a three-dimensional structure, in particular by the formation of covalent bonds between the chitosan-polyacrylamide chains.

A constituent copolymer of the hydrogel according to the invention is formed of a succession of repeating units, called monomers, linked together by covalent bonds. It is understood by “copolymer”, within the meaning of the present invention, a polymer resulting from the copolymerization of at least two types of monomers, chemically different. Within the meaning of the invention, the copolymer is a material homogeneous with random, alternating and statistical sequence of the various monomers constituting it.

In one embodiment, the hydrogel of the invention comprises between 0.3% and 30% by weight, of the total weight of the hydrogel, of dry matter in copolymer. In another embodiment, it comprises between 0.3 and 30.4%, in particular between 0.3033 and 30.4%, more particularly between 0.3415 and 30.4% by weight, of the total weight of the hydrogel, from dry matter in copolymer. In another embodiment, the hydrogel of the invention comprises between 0.3033 and 30%, especially between 0.3415 and 30% by weight, of the total weight of the hydrogel, of dry matter in copolymer. Below 0.3% by weight of the total weight of the dry matter hydrogel in copolymer, the hydrogel may form more difficultly or not form all, that is to say that the mixture stays liquid. Above 30%, particularly beyond 30.4%, by weight of the total weight of the dry matter hydrogel in copolymer, the hydrogel may progressively become too hard and no longer be applicable or implantable or injectable. Advantageously, the proportion by weight of dry matter of the copolymer is equal to or greater than 2% by weight of the total weight of the hydrogel, or even 4%. Advantageously, this proportion by weight of dry matter of the copolymer is less than or equal to 15%, or even 10%.

In one embodiment, the hydrogel of the invention is substantially free of pyrogen. For the purposes of the invention, the terms "substantially free of pyrogen" are understood to mean a substantial exemption from substances inducing a rise in temperature. Various methods exist for identifying the presence of pyrogens and are well known to those skilled in the art. In particular, a method for identifying the presence of pyrogen consists in injecting a rabbit with 10 ml of solution per kg of body weight and measuring its temperature. If the rabbit's body temperature increases by 0.6 ° C, or if the total increase is more than 1.4 ° C in three rabbits, the solution is not substantially free of pyrogens. Another method for identifying the presence of pyrogen consists in using a MAT test ("Monocyte Activation Test" - marketed, for example, by Merck ™ under the names PyroDetect ™ and / or PyroMAT ™ introduced in the European Pharmacopoeia in 2010. This test was developed as an alternative to methods using animals and aims to offer the possibility of carrying out tests pyrogens in humans in an in vitro system. Within the meaning of the invention, water substantially free of pyrogen is said to be pyrogen-free.

In one embodiment, the hydrogel according to the invention also comprises a diffusing agent and / or another agent or ingredient.

For the purposes of the present invention, the term "diffusing agent" is understood to mean an ingredient or an active agent capable of diffusing apart from the hydrogel. The diffusing agent can be chosen from inert ingredients having interesting or active biomechanical properties. These agents can be substances of plant origin such as extracts of genepi, substances of marine origin such as extracts of New Zealand green mussels (Perna canaliculus), orthosilic acid, organic silicon, silanol, vitamins such as vitamins A, D3, E or C, metals such as gold or silver, pain relievers such as lidocaine, xylazine, detomidine, nonsteroidal anti-inflammatory drugs, such as flunixin , ketoprofen, aspirin, corticosteroids such as prednisolone, triamcinolone, hyaluronic acid, glycosaminoglycans, chondroitin sulfate, methylsulfonylmethane, bromelain, arnica, collagen, antioxidants, fatty acids.

In one embodiment, the diffusing agent of the invention can also be included in a cargo ship. This cargo is defined as a matrix capable of carrying the diffusing agent in the hydrogel. According to the invention, different cargo techniques can allow the diffusion agent to be taken on board, such as encapsulation or vectorization, the so-called dripping technology, the creation of emulsions or coatings. specific, polymeric grafting. In particular, the cargo can be chosen from microcapsules, microparticles or even polymeric vehicles. The size of the particles used is for example between 200 nm and 20,000 nm, which makes them particles larger than those of nanoparticles whose European standard describes a size less than 100 nm for more than 50% of between them.

The diffusing agent, included or not in a cargo ship, is retained in the hydrogel, either by physical retention, or by molecular interactions, covalent or not, examples of non-covalent interactions being ionic interactions, hydrogen bonding, or by any combination of these retention methods. In particular, the pore size, defined by the three-dimensional matrix structure of the hydrogel copolymer, can prevent the diffusing agent from being released until the hydrogel is degraded by one or more mechanisms. The diffusing agent can thus be released either by a change in pH or temperature, or under a mechanical action.

In one embodiment, the final hydrogel of the invention comprises between 0.0001% and 30% by mass of diffusing agent, out of the total mass. Preferably, the diffusing agent comprises approximately 1% and 25% by mass, relative to the total mass of the final hydrogel.

In a particular embodiment, the diffusing agent of the invention is an ortho-silicic acid, an organic silicon, or silanol.

In a particular embodiment, the diffusing agent is in the form of microparticles whose size (average diameter) is between 200 nm and 20,000 nm. These microparticles are therefore larger than those of nanoparticles whose European standard describes a size less than 100 nm for more than 50% of them.

In one embodiment, the mass ratio between acrylamide and chitosan is between 1/1 and 1/8. Preferably, it is between 1/2 and 1/6. In another embodiment, the mass ratio between chitosan and acrylamide is between 1/100 and 1/2. It is, in particular, between 1/8 and 1/2, in particular between 1/6 and 1/2.

In one embodiment, the mass ratio between N, N’-methylenebisacrylamide and acrylamide is between 1/50 and 1/1000. Preferably, the mass ratio between N, N’-methylenebisacrylamide and acrylamide is between 1/100 and 1/500.

In a particular embodiment, the hydrogel of the invention comprises the copolymer formed at least by acrylamide, chitosan and N, N'-methylenebisacrylamide, and the diffusing agent, according to the following ratio in% by weight of the total weight of the final hydrogel:

Acrylamide 0.3% and 20%

Chitosan 0.0375% and 10%

N, N’-methylenebisacrylamide 0.004% and 0.4%

diffusing agent 0.001% and 30%

H2O in a complementary manner up to 100%.

In another particular embodiment, the hydrogel of the invention comprises the copolymer formed at least by acrylamide, chitosan and N, N'- methylenebisacrylamide, and the diffusing agent, according to the following ratio in% by weight of the total weight of the final hydrogel:

Acrylamide 0.3% and 20%

Chitosan 0.003% and 10%

N, N’-methylenebisacrylamide 0.0003% and 0.4%

diffusing agent 0.001% and 30%

H2O in addition up to 100.

According to a second object, the invention relates to a process for manufacturing a hydrogel as defined above.

In a first embodiment, the method comprises the following steps: copolymerization of acrylamide and chitosan, in the presence of N, N’-methylenebisacrylamide and of a radical initiator, in an aqueous medium, to obtain a copolymer; washing the copolymer with water to obtain a washed copolymer; and adding the diffusing agent to obtain the hydrogel.

During the manufacturing process, chitosan copolymerizes with polyacrylamide by covalently bonding to it. Under the action of N, N’-methylènebisacrylamide (MBA), the system crosslinks in a three-dimensional network. The MBA makes it possible to covalently link different chains of chitosan-polyacrylamide to form a three-dimensional network.

The chitosan is added in an acidic aqueous solution, that is to say an aqueous solution whose pH is lower than 6. Preferably the acidic aqueous solution comprises an organic acid, in particular an organic acid whose pKa is between 4 and 6, such as a carboxylic acid. In particular, the acidic aqueous solution comprises acetic acid or hydrochloric acid.

The concentration of chitosan in the acidic aqueous solution is 0.1 to 5% by weight of the total weight of the solution.

The acidic aqueous solution of chitosan is mixed with the acrylamide at a temperature between 20 and 60 ° C. Preferably the temperature is between 40 and 60 ° C. The mass ratio between acrylamide and chitosan is between 1/1 and 1/8. In another embodiment, the mass ratio between the chitosan and the acrylamide is between 1/100 and 1/2, in particular between 1/8 and 1/2, in particular between 1/6 and 1/2.

The mass ratio between N, N’-methylenebisacrylamide and acrylamide is between 1/50 and 1/1000. Preferably, the mass ratio between N, N’-methylenebisacrylamide and acrylamide is between 1/100 and 1/500.

The copolymerization reaction is initiated by a radical initiator. In particular, the radical initiator is chosen from potassium persulfate or ammonium persulfate. The mass ratio between the radical initiator and the acrylamide is between 1/100 and 1/10. The radical initiator can optionally be used in combination with tetramethylethylenediamine (TEMED). TEMED is optional during copolymerization. Thus, the mass ratio between TEMED and acrylamide is between 1/2000 and 1/20. The absence of TEMED, a toxic catalyst, allows the formation of a hydrogel based on partially natural and biocompatible materials, and thus allows a more responsible bio-design.

Washing the hydrogel

The hydrogel is formed from a copolymerization of chitosan with acrylamide. The copolymer is crosslinked using N, N ’bis-acrylamide. The optimization of this crosslinking consists in making it as complete as possible so that, on the one hand, the three-dimensional network is the best formed and that, on the other hand, the monomers (acrylamide and N, N 'bis-acrylamic) toxic are as few as possible. However, caution requires "washing" the hydrogels formed so that any residual monomers in the hydrogel can be removed. The "washing" of the hydrogel therefore consists in immersing this hydrogel in water and using the phenomenon of osmosis to evacuate these monomers. According to the principle of osmosis, molecules move from areas with high concentrations to areas with lower concentrations. This displacement is described by Fick's law which expresses a linear relationship between the flow of matter and the concentration gradient thereof.

As shown in Figure 6A, at least two phenomena occur. The residual monomers trapped within the hydrogel (symbolized by the light gray color) will be discharged to the "washing" water (symbolized by the dark gray color). Conversely, the water outside the gel will enter the hydrogel. It follows from this phenomenon of washing by osmosis, a swelling of the hydrogel. Some preliminary tests of “washed” hydrogels show the variation in mass (in percent) as a function of time. As shown in Figure 9, this variation in the relative gain in mass increases linearly over time until almost 200% (swelling 3 times) in the case of the hydrogel N4a and around 300% (swelling 4 times), in the case of the N4a / 2 hydrogel for an immersion time of approximately 4 days.

It is clear that the significant expansion of hydrogels will result in an inversely proportional reduction in the amount of dry matter intrinsic to the gel.

Washing the copolymer with water makes it possible to remove contaminants and to ensure the absence of such contaminants within the hydrogel. Contaminants in the copolymer can be residual monomers, radical initiator residues, organic acids. In particular, repeated washing with water and analysis of the washing water, in particular by FT-IR, makes it possible to measure the level of residual monomers which have not participated in the copolymerization and / or crosslinking reaction and also ensures the absence of these residual monomers in the hydrogel. In particular, the hydrogel of the invention comprises less than 20 mg / ml of acrylamide and less than 20 U E / device of endotoxins.

The diffusing agent, possibly included in a cargo ship, can be added in the solid state or suspended in water. In particular, the diffusing agent can be added in suspension in pyrogenic water.

In a particular embodiment, the method of the invention comprises the following steps: copolymerization of acrylamide and chitosan introduced with a mass ratio of between 1/1 and 1/8, at a temperature between 20 and 60 ° C. , preferably between 40 and 60 ° C, in the presence of N, N'-methylenebisacrylamide introduced with a mass ratio with respect to the acrylamide of between 1/50 and 1/1000, preferably between 1/100 and 1 / 500, and of a radical initiator chosen from potassium persulfate or ammonium persulfate, optionally in combination with tetramethylethylenediamine, in an aqueous medium to obtain a copolymer; washing the copolymer with water over 3 to 15 washes for 48-240 hours to obtain a washed copolymer; and addition of the diffusing agent between 0.001% and 30%, to obtain the final hydrogel.

In a second embodiment, the method comprises the following stages: copolymerization of acrylamide and chitosan, in the presence of N, N'-methylenebisacrylamide, of a diffusing agent, and of a radical initiator, in a aqueous medium, to obtain a copolymer incorporating the diffusing agent; washing the copolymer incorporating the diffusing agent with water to obtain the hydrogel.

Advantageously, before the copolymerization reaction, the chitosan is dissolved in an aqueous solution at a pH of between 2 and 5, with magnetic or mechanical stirring, then neutralized and filtered under vacuum. The aqueous solution having a pH between 2 and 5 is ideally an aqueous solution of hydrochloric acid or acetic acid.

Advantageously, the copolymer incorporating the diffusing agent is extruded through specific pore grids before the washing step.

According to an advantageous embodiment, the washing step is carried out by dialysis, using dialysis membranes. In this case, a gel containment system is put in place so as to limit the swelling thereof while affecting as little as possible the movement of residual monomers towards the washing water. This is shown schematically in Figure 6B. To improve the washing efficiency, the water is changed regularly (once every 12 hours for example) and the agitation is facilitated by using magnetic agitation, mechanical agitation (blades), agitation by water pump, etc. . A first washing test was carried out to compare the effectiveness of a dialysis tube compared to a tube large enough not to limit swelling. Figures 7A, 7B and 7C illustrate the placement of the membrane on the neck, the filling of the membrane with a gun and the closure of the membranes with clips and knots. Figure 8 presents the results obtained in terms of relative deviation from weight gain versus time.

It will be noted that, in standard cases, the washing of the hydrogel lasts several days (5 to 6 days) so that most of the residual monomers can be removed. The residual monomer level decreases all the more quickly than the osmosis conditions are favorable, that is to say that the water is changed regularly and that the agitation is sufficient. The experimental results make it possible to define the minimum washing time of 2 days.

The washing step implemented during the preparation of the hydrogel is carried out by weighing the hydrogels each time the water in the tank is changed. These mass measurements make it possible to construct the curve for the evolution of the relative difference in mass gain as a function of time (Figures 1 1 A and 1 1 B). The curve shows a plateau corresponding to the confinement of the hydrogel within the membrane. We note that this plateau takes values understood between 50 and 70% much lower than the previous 200 to 300%, illustrating the good control of the expansion of the gel during washing.

According to a third object, the invention relates to the use of a hydrogel in a mechanical visco-supplementation system, for external or internal use.

The mechanical visco-supplementation according to the invention makes it possible to physically embed the diffusing agent in situ, to support the normal physiological and rheological conditions of wounds, joints or in the event of gastric ulcers, in particular in horses.

In one embodiment, the hydrogel of the invention is used for internal use in an implantable mechanical visco-supplementation system for supporting the soft tissues of mammals, see bones and cartilage according to the diffusing agent. Soft tissues are elements of the body, such as adipose tissue, connective tissue, synovial membrane of the joint capsule muscles, tendons, dermis or epidermis.

Mechanical visco-supplementation for internal use consists of locally implanting the hydrogel which acts as a support agent for the synovial membrane, synovial fluid or bone and cartilage as appropriate. The hydrogel thus allows the joint to support its mobility by a biomechanical action.

Advantageously, the mechanical visco-supplementation system for internal use is a lubricant.

In another embodiment, the hydrogel of the invention is used for external use in a mechanical visco-supplementation system. For external use, mechanical visco-supplementation consists in applying locally the hydrogel which acts as an agent to support healing. This support for healing results in the maintenance of wound humidity during healing thanks to the large amount of water present in the hydrogel.

Advantageously, the mechanical visco-supplementation system for external use is a moisturizer.

The use of the hydrogel according to the invention allows a delayed effect of the diffusing agent included in the hydrogel. In particular, the diffusing agent begins to diffuse between the 2nd and the 30th day after administration, preferably between the 10th and the 20th day, in particular from the 15th day.

The use of the hydrogel according to the invention also allows a prolonged effect of the diffusing agent included in the hydrogel. Preferably, the diffusing agent is released over a period between 2 weeks and 12 months, preferably between 1 month and 6 months.

Thus, the mechanical visco-supplementation systems of the invention require fewer applications and the effect is manifested for an extended period, of at least two weeks. This prolonged mechanical visco-supplementation of the diffusing agent which is located, fixed, protected then released by the hydrogel is made possible thanks to the viscoelastic matrix structure of the hydrogel. The effects of viscoelastic hydrogel, whose delayed and prolonged release of the diffusing agent, persist significantly. Under these conditions, the hydrogel gradually diffuses after a few weeks after use and this for several weeks, as if it resulted in continuous use in wounds or the joint, during such a lapse of time. This results, for example, in a significant simplification of support for joints by providing lasting visco-supplementation and significantly reducing the number of applications or implantations.

According to a fourth object, the invention relates to an external or internal visco-supplementation kit comprising a copolymer of acrylamide and of chitosan crosslinked with N, N'-methylenebisacrylamide, and a diffusing agent in solid phase or in suspension, said copolymer and said diffusing agent being premixed in the form of a hydrogel or mixed extemporaneously to form a hydrogel.

This kit comprises a copolymer formed at least from acrylamide and chitosan and crosslinked with N, N’-methylenebisacrylamide, and a diffusing agent in solid phase or in suspension. The copolymer and the diffusing agent are either premixed in the form of a hydrogel, or mixed extemporaneously to form a hydrogel.

EXAMPLES

EXAMPLE 1: PREPARATION OF THE COPOLYMER CONSTITUTING

HYDROGEL ACCORDING TO THE INVENTION

a) Products involved in the mixture

- 40% acrylamide solution in water, Sigma Aldrich ™ (Grade molecular biology / embryo transfer / pharma)

- Solution N, N'-methylene bis acrylamide 2% in water, Sigma Aldrich ™ (Grade molecular biology / embryo transfer / pharma) - 1% Chitosan solution in 0.1 M acetic acid, Sigma Aldrich ™ (350 kDa> 75% deacetylation, molecular biology grade / embryo transfer / pharma)

- Potassium Persulfate (KPS) (> 99%) powder, Sigma Aldrich ™ (Grade molecular biology / embryo transfer / pharma)

- Water for injections (ppi water)

- 1 L glass flask

- Magnetic stirrer (Fischer Bioblock Scientic ™ brand), rotation 500 rpm

- Temperature of 40 ° C (regulation by Heidolph ™ EKT 3001 thermocouple) c) General protocol

1. Step 1

Pour the Chitosan solution into the flask preheated to 40 ° C, keep this temperature and keep stirring at 500 rpm

2. Step 2

Mix the acrylamide solution with the N, N’-methylenebisacrylamide solution. Pour the mixture into the flask 1 minute after the chitosan

3. Step 3

Dissolve the APS (ammonium persulfate) or KPS in part of the additional water, stirring until completely dissolved.

Gradually pour this solution, 1 minute after pouring the solution (acrylamide and N, N ’, methylene bis acrylamide)

4. Step 4

Pour the ppi water up to the desired total volume

5. Step 5

Keep the magnetic stirring and the temperature for a period of 45 min for a copolymerization of Chitosan on the polyacrylamide and for crosslinking with N, N ’methylene bis acrylamide

6. Step 6

Let stand 24 hours to allow the gel to fully form

7. Step 7

Carry out several (3 to 5) rinses in ppi water for 96 hours then drain the gel and store it at room temperature

d) Different formulations tested

EXAMPLE 2. CHARACTERIZATION OF THE COPOLYMER CONSTITUTING

HYDROGEL ACCORDING TO THE INVENTION

a) Characterization by infrared with Fourier transform (FTIR) of the copolymer present in the hydrogel

FTIR measurements, on dry matter, were carried out to compare the spectra of the chitosan-polyacrylamide-MBA copolymer present in the copolymer hydrogel of the invention at 3.75%, without diffusing agent (V14), at 3 , 75% with diffusing agent (V20) versus “pure” polyacrylamide to more than 3.5% without chitosan (V 5) and “pure” chitosan (Chitosan). The dry matter weighings are contained in Figure 2A.

The spectra are shown in Figures 2B and 2C A more precise analysis of the spectra focused on the differences between the hydrogel without diffusing agent (V14) versus pure polyacrylamide hydrogel (V 5) and chitosan. The specific links are shown in Figure 2D.

The experimental conclusions confirm the literature published on grafting of the copolymer type.

1. The peaks of V14 at 1450 cm ^-1 and 3182 cm ^-1 are the unique characteristics of a copolymerization between chitosan and polyacrylamide.

2. The peak at 3350 cm ^-1 (NH) observed on the chitosan and less on the copolymer is characteristic of the consumption of this bond for grafting according to the bibliography. 3. The peaks at 1375 cm ^-1 (CH) and 1 1 13 cm ^-1 (C-0) observed on the chitosan but which disappear on the copolymer and the pure polyacrylamide gel: the literature attributes this to the chitosan grafting with the polyacrylamide.

Six specific and distinctive peaks of the chitosan-polyacrylamide-MBA copolymer are identified in the FTIR measurements, which confirms the copolymerization.

This FTIR analysis verifies that the chitosan and the polyacrylamide are covalently linked giving rise to a single copolymer.

b) Dissolution test of the copolymer in 1% acetic acid

Dissolution test of chitosan in acetic acid diluted to 1/100:

We weigh 49.5 of water, 0.5 g of pure acetic acid, 0.5 g of chitosan.

Mix in a beaker and leave to act, as shown in FIG. 3A. After 30 min, with mechanical stirring by hand, the chitosan is dissolved. Dissolution test of the chitosan-polyacrylamide-MBA copolymer in acetic acid diluted to 1/100:

We weigh 49.5 of water, 0.5 g of pure acetic acid, 20 g of chitosan-polyacrylamide-MBA.

Mix in a beaker and leave to act, as shown in Figure 3A. After 60 min, with mechanical stirring by hand, a filter is used, as illustrated in FIG. 3B.

A gel is collected which is weighed and the weight of 20.00 g is found.

It can be concluded that the copolymer is well crosslinked since it is insoluble in acetic acid.

c) Characterization of the dynamic viscoelasticity of the copolymer

The dynamic viscoelasticity of the copolymer obtained after washes is measured and compared to the dynamic viscoelasticity of the copolymer obtained before washes, to that of the copolymer obtained after addition of acetic acid and to that of the copolymer obtained after shearing.

As recalled by the conclusions of the tests in FIG. 4 which illustrates the loss tangents (G ”/ G ') of the chitosan-polyacrylamide-MBA copolymer before washing (top left), after washing (top right), after 1 h in acetic acid (bottom left) and after shearing with a syringe (bottom right), this measure of viscoelasticity reveals significant values of the elastic modulus characteristic of crosslinking. Thus, it proves the existence of a three-dimensional network in the case of the hydrogel of chitosan-grafted-polyacrylamide copolymer, as in the case of the hydrogel of pure polyacrylamide. In parallel with the modulus values of shear obtained, the loss tangent values show a marked presence of elasticity vis-à-vis the viscosity, characteristic of a crosslinked system.

In addition, this study also shows that the crosslinking of the copolymerized system is effective, because the gel formed is not dissolved in acetic acid, unlike the linear molecules of chitosan.

This state of crosslinking remains during the various operations (washing, shearing by the entry and exit of the syringe).

EXAMPLE 3: IMPROVED BIOCOMPATIBLITY OF HYDROGEL ACCORDING TO

THE INVENTION RELATING TO A HYDROGEL INCLUDING ONLY

POLYACRYLAMIDE

The hydrogel according to the invention has improved biocompatibility compared to a hydrogel containing only polyacrylamide. Indeed, the hydrogel according to the invention comprises chitosan copolymerized with polyacrylamide, chitosan comprises polyglucosamine sequences having a biocompatibility, which shows in vitro results of a better biocompatibility, with in particular an absence of cytotoxicity.

An XTT cytotoxicity test is performed by Eurofins Medical Device ™, in accordance with ISO 10993-5: 2009, under conditions of good laboratory practice (GLP). The test relates to the hydrogel described by the invention, namely a 5% polyacrylamide-chitosan NVC-0 hydrogel copolymer and a 4% polyacrylamide hydrogel containing silver ions (Bioform / Noltrex ™ brand). An extraction was carried out with stirring for 24 h in a cell culture medium, the extracts being incubated for 24 h to 48 h with L929 cells. The value of the drop in the mitochondrial dehydrogenase level of the culture media, compared to the reference control measure, served as a measure of the cytotoxicity, this at 4 different concentrations of hydrogel in the medium (100%, 66, 7%, 44.4%, 29.6%). Mitochondrial dehydrogenase activity greater than 70% indicates biocompatibility of the product according to the standard.

Results:

For a concentration of 100% (ie no dilution of the hydrogels in the medium), the mitochondrial dehydrogenase of the NVC-0 polyacrylamide-chitosan hydrogel is 101% (SD 0.07) and that of the hydrogel of 4% polyacrylamide is 95% (SD 0.05). As shown in Figures 13A and 13B, the NVC-0 hydrogel according to the invention has a total absence of cytotoxicity (no reduction in mitochondrial dehydrogenase, at all dilutions), unlike the other hydrogel. The hydrogel of the invention has, on this XTT test, an improved biocompatibility of + 6% compared to the hydrogel containing only polyacrylamide.

EXAMPLE 4: IMPROVED E3IODEGRADAE3ILITE OF HYDROGEL ACCORDING TO

THE INVENTION RELATING TO A HYDROGEL INCLUDING ONLY

POLYACRYLAMIDE

The hydrogel of the invention has the advantage of biodegradability of the chitosan which it contains, in addition to glucosamine and N-acetylglucosamine, substances naturally present in the human body. Glucosamine is known for its support in particular of the osteo-articular system. The biodegradability of chitosan can vary from a few tens of days to a few months depending on the enzyme media and the characteristics of the polyglucosamine. The formula shown in Figure 14 describes chitosan, thus indicating the presence of glucosamine as a constituent unit of the polymer.

Fourier Transformed Infrared Spectroscopy (IR-TF) between 4000 and 400 cm ^-1 is carried out in order to determine the chemical nature of the constitutive bonds of the 5% hydrogel NVC-0 copolymer and a hydrogel containing only polyacrylamide . A sample of each sample was placed in an oven at 30 ° C for 40 hours in order to carry out the analyzes by IR-TF spectroscopy in ATR (Total Attenuated Reflection) mode on dry extract. The spectrum is recorded between 4000 cm ^-1 and 400 cm ^-1 . ATR is a non-destructive technique that adapts to materials with high absorbency.

Results:

A characteristic peak appears around 1072 cm ^-1 (identified on the spectra) which is present for the sample of the copolymer hydrogel NVC-0 as shown in FIG. 15. It is a band d characteristic absorption observed only thereon. This band around 1072 cm ^-1 can be characteristic of the vibration modes of deformation type of the COC groups (of chitosan). The IR-TF analysis indicates the presence of the chitosan bonds in the hydrogel proposed by the invention. This hydrogel brings a biodegradability, in particular in glucosamine naturally present in the human body, compared to a hydrogel only of polyacrylamide. EXAMPLE 5 CAPACITY FOR CARRYING OUT HYDROGEL ACCORDING TO THE INVENTION

The copolymer hydrogel according to the invention has a carrying capacity, in particular of submicron capsules. The microstructure of the three-dimensional network of this hydrogel has alveoli the size of which is compatible with the particles carried away, which polyacrylamide gels produced according to the prior art do not offer.

The Joint Center for Applied Microscopy (CCMA) of the University of Nice carried out cryoimaging by scanning electron microscope (SEM) of the NVC-0 hydrogel according to the invention, containing microcapsules of organic silicon. Samples of NVC-0 hydrogel and NVC-0-C hydrogel containing microcapsules were successively immersed in liquid nitrogen, sublimated and then fractionated to pass to SEM. The analysis was carried out 7 weeks in post production of the batches.

Results:

The polyacrylamide-chitosan copolymer hydrogel has a carrying capacity which is illustrated by SEM. The results of this imaging also show biocompatible protection of the hydrogel against microcapsules, which are not deteriorated for 7 weeks in the hydrogel. As shown in FIG. 15A (Cryoimaging by SEM (x10,000) of the copolymer hydrogel NVC-0), a dense and relatively homogeneous crosslinked network is observed, with cells of size 0.1 -0.5 microns on the copolymer hydrogel samples. As shown in FIG. 15B (Cryoimaging by SEM (x23,000) of the copolymer hydrogel NVC-0-C (7 weeks post production)), a microcapsule with a size of 0.6 microns is observed by SEM on the hydrogel containing microcapsules, 7 weeks after production and integration of organic silicon microcapsules of 0.6-0.8 microns.

EXAMPLE 6: HYDROGEL SUPPLEMENTATION CAPACITY ACCORDING TO

THE INVENTION

The copolymer hydrogel according to the invention offers a capacity for supplementation, either by the degradation of chitosan to glucosamine (Example 4), or by the water contained in the hydrogel (Example 8), or by the degradation of microcapsules, such that contains for example orthosilicic acid. The organic silicon during its degradation makes it possible to supplement in silicon, substance naturally present in the body but not renewed with age. It appears that the hydrogel according to the invention proposes a release capacity for on-board microcapsules, with delayed release over time. Thanks to the biodegradability of the hydrogel containing chitosan (Example 4), the on-board microcapsules (Example 5) could be released as a function of time. This characteristic provides supplementation over time and locally of substances carried by the hydrogel according to the invention.

Summary of the experience:

A first preliminary study was carried out to assess the release kinetics of microcapsules carrying orthosilicic acid in a synthetic medium of synovial fluid type. The particles were incubated in the artificial fluid containing 3 g / L of hyaluronic acid at a concentration of 0.6 mg / ml in particles. A dialysis / filtration system made it possible to separate the dissolved fractions from the particles (below). The dissolved orthosilicic acid was then assayed by ICP-AES.

Results:

The analysis has shown that a significant amount of orthosilicic acid is detectable in the "dissolved" compartment. After 15 days of incubation, particles are still visible in the "particles" compartment and have been titrated by ICP. These preliminary data suggest slow degradation of the particles to supplement orthosilicic acid.

EXAMPLE 7: MANUFACTURE OF HYDROGEL - DIALYSIS

The hydrogel according to the invention is manufactured by implementing an efficient dialysis step, making it possible to remove the residual monomers from the synthesis, under very low quantification limits (less than 4 ppm), as mentioned in the table in figure 16.

The copolymer in the form of a hydrogel is extruded several times through extrusion screens between 50 and 500 microns depending on the hydrogels. This extrusion allows an optimal dialysis which begins with the choice of dialysis bags with pores adapted according to the consistency of the hydrogel, ideally from 6 to 50 kD MWCO (Molecular weight cut-off in kilo Daltons). The extruded hydrogel is bagged and then placed in water according to different durations, ideally from 2 to 7 days, depending on the quality of the synthesis and the viscosity of the chitosan retained.

The GCMS or UPLC / UV method measures residual monomers such as acrylamide or methyl-bis-acrylamide.

Results:

The 5% polyacrylamide-chitosan copolymer hydrogel was tested and the concentration measurements of any residual monomers indicate values below the quantification limits of 4 ppm by the various methods.

EXAMPLE 7 "HYDROPHILIC SWELLING" OF HYDROGEL ACCORDING TO

THE INVENTION

As illustrated in FIG. 10, the PAAG-CH copolymer hydrogel according to the invention has a capacity 3 times greater in the water transport (hydrophilic swelling) compared to a hydrogel of the same polyacrylamide concentration only. It therefore requires less substance with a copolymer hydrogel to provide as much hydration as with a polyacrylamide gel at equal initial concentration.

Two hydrogels are synthesized, one 5% polyacrylamide copolymer containing chitosan (NVC-0) and the other containing only polyacrylamide (PAAG). Following their extrusion and then dialysis, weighings are carried out every 12 hours to measure the relative difference in weight gain in water. Studies have also been conducted by varying the rates of crosslinking or percentage of polyacrylamide. The presence of the copolymerized chitosan in the hydrogel brings a significant increase in the relative difference in water intake.

Results:

From 3 days of dialysis, the 5% polyacrylamide hydrogel reaches a swelling plateau of 25% maximum of its weight. The 5% polyacrylamide-chitosan copolymer exceeds the water carrying capacity of a polyacrylamide hydrogel upon dialysis and continues to swell beyond 75% of its weight in water after 5 days, for example.

EXAMPLE 8: COPOLYMERIZATION OF CHITOSAN ON THE

POLYACRYLAMIDE IN A HYDROGEL ACCORDING TO THE INVENTION

As shown in FIG. 17, according to the invention, the copolymerization of chitosan on the polyacrylamide takes place by the creation of covalent bonds between the macromolecular chain of chitosan via free radicals formed by the action of a peroxide and the polyacrylamide chain in formation from acrylamide monomers.

The copolymer is formed creating a macromolecular chain which is then organized in the form of a three-dimensional network thanks to the crosslinking agent (methyl bisacrylamamide).

Thus the polyacrylamide / chitosan copolymer is a specific polymer which is different from polyacrylamide. The hydrogel obtained by the crosslinking of these copolymer and crosslinking chains forms a different network of acrylamide which is verified in particular by the much higher swelling rate in the case of the copolymer than in the case of acrylamide (Example 7 ).

Summary of the experience:

A polyacrylamide-chitosan copolymer hydrogel and a polyacrylamide hydrogel are tested by the NMR method. The samples are evaporated, the dry matter hydrated with D20 water and then evaporated again. The dry materials were dissolved in a preparation D20 / Ac0D 1/1. The 1 H NMR spectrum was recorded on a Bruker ™ 400 MHz device at 50 ° C.

Results:

The NMR of the copolymer (Figure 18A) has several distinctive points, in particular specific to chitosan at 3.2, 3.6 and 4.9 ppm (Figure 18B) and in significant proportion compared to the NMR of the polyacrylamide hydrogel alone (Figure 18C).

The NMR of the copolymer hydrogel described in the patent confirms the different nature of the polymer compared to a polyacrylamide hydrogel or a simple mixture with polyacrylamide.

Claims

1. Biocompatible hydrogel comprising, on the one hand, between 0.3% and 30% by weight of dry matter of a copolymer formed at least of acrylamide, chitosan and N, N'-methylenebisacrylamide and, on the other hand, a diffusing agent.

2. Hydrogel according to claim 1, characterized in that the copolymer formed at least of acrylamide, of chitosan and of N, N’-methylenebisacrylamide has the formula:

3. Hydrogel according to one of claims 1 or 2, characterized in that the diffusing agent is chosen from inert ingredients, which have biomechanical properties or active agents, preferably substances of plant origin such as genepi extracts , substances of marine origin such as extracts of New Zealand green mussels (Perna canaliculus), orthosilicic acid, organic silicon, silanol, vitamins such as vitamins A, D3, E or C , metals such as gold or silver, pain relievers such as lidocaine, xylazine, detomidine, nonsteroidal anti-inflammatory drugs, such as flunixin, ketoprofen, aspirin, corticosteroids such as prednisolone, triamcinolone, l hyaluronic acid, glycosaminoglycans, chondroitin sulfate, methylsulfonylmethane, bromelain, arnica, collagen, antioxidants, fatty acids.

4. Hydrogel according to any one of claims 1, 2 or 3, characterized in that the diffusing agent is included in a cargo ship chosen from microcapsules, microparticles and polymeric vehicles.

5. Hydrogel according to any one of the preceding claims, characterized in that the diffusing agent forms microparticles of size between 200 nm and 20,000 nm.

6. Hydrogel according to any one of the preceding claims, characterized in that it is substantially free of pyrogen.

7. Hydrogel according to any one of the preceding claims, characterized in that the mass ratio between N, N'-methylenebisacrylamide and acrylamide is between 1/50 and 1/1000, preferably between 1/100 and 1/500.

8. Hydrogel according to any one of the preceding claims, comprising the following ratio of constituents in% by weight of the total weight of the hydrogel: polyacrylamide: between 0.3% and 20%

chitosan: between 0.0375% and 10%

N, N’-methylenebisacrylamide: between 0.004% and 0.4%

diffusing agent: between 0.001% and 30%

H2O: supplement up to 100%.

9. A method of manufacturing a hydrogel according to one of the preceding claims, comprising the following steps: copolymerization of acrylamide and chitosan, in the presence of N, N'-methylenebisacrylamide and a radical initiator, in an aqueous medium , to obtain a copolymer; washing the copolymer with water to obtain a washed copolymer; and adding the diffusing agent to obtain the hydrogel.

10. Method according to claim 9, characterized in that the step of adding the diffusing agent is prior to the step of copolymerization of acrylamide and chitosan in the presence of N, N'-methylenebisacrylamide and l radical initiator, so that the three-dimensional network formed by the copolymer is built around the diffusing agent.

1 1. Method according to one of claims 9 or 10, comprising the following steps of: copolymerization of acrylamide and chitosan at a temperature between 20 ° C and 60 ° C, preferably between 40 ° C and 60 ° C, in the presence of N, N'-methylenebisacrylamide introduced with a mass ratio relative to the acrylamide of between 1/50 and 1/1000, preferably between 1/100 and 1/500, and of a radical initiator with a mass ratio compared to the acrylamide between 1/100 and 1/10 chosen from potassium persulfate or ammonium persulfate, preferably potassium persulfate, optionally in combination with tetramethylethylenediamine with a mass ratio compared to acrylamide between 1/2000 and 1/20, in an aqueous medium to obtain a copolymer; washing the copolymer with water to obtain a washed copolymer; and addition of the diffusing agent between 0.001% and 30% by weight of the total weight of the hydrogel, the diffusing agent being chosen from inert ingredients, which have biomechanical properties or active agents, these agents preferably being substances of vegetable origin such as extracts of genepi, substances of marine origin such as extracts of New Zealand green mussels (Perna canaliculus), ortho-silicic acid, organic silicon, silanol, vitamins such as vitamins A, D3, E or C, metals such as gold or silver, pain relievers such as lidocaine, xylazine, detomidine, nonsteroidal anti-inflammatory drugs, such as flunixin, ketoprofen, aspirin, corticosteroids such as prednisolone, triamcinolone, hyaluronic acid, glycosaminoglycans, chondroitin sulfate, methylsulfonylmethane, bromelain, arnica, collagen, antioxidants, fatty acids, possibly com taken from a cargo ship chosen from microcapsules, microparticles or polymeric vehicles, preferably biodegradable microcapsules, to obtain the hydrogel.

12. Method according to one of claims 9 to 1 1, characterized in that the washing step of the copolymer is carried out by dialysis, by means of dialysis membranes.

13. Use of a hydrogel according to one of claims 1 to 8, in a mechanical visco-supplementation system, for external or internal use.

14. Use of a hydrogel according to claim 13, in a mechanical visco-supplementation system, for internal use.

15 Use of a hydrogel according to claim 14, for the support of soft tissues of mammals, see bones and cartilage.

16. Use according to claim 13, characterized in that the visco-supplementation system is a moisturizer.

17. Use according to any one of claims 10 to 16, characterized in that the diffusing agent begins to diffuse between the 2nd and the 30th day after administration, preferably between the 10th and the 20th day, in particular at from the 15th day.

18. Use according to any one of claims 10 to 17, characterized in that the diffusing agent is released over a period of between 2 weeks and 12 months, preferably between 1 month and 6 months.

19. External or internal mechanical visco-supplementation kit comprising a copolymer of acrylamide and of chitosan crosslinked with N, N'-methylenebisacrylamide, and a diffusing agent in solid phase or in suspension, said copolymer and said diffusing agent being previously mixed in the form of a hydrogel during manufacture or mixed extemporaneously to form a hydrogel.