METHOD FOR THE PRODUCTION OF BIOCOMPATIBLE AND BIODEGRADABLE POROUS THREE-DIMENSIONAL POLYMER MATRICES

Description

[0001] The present invention relates to a method for preparing a biocompatible and biodegradable three-dimensional porous polymer matrix, usable in particular as a support and for cell culture or in regenerative medicine, especially for cardiac cell therapy.

[0002] Cell therapy is a promising therapeutic strategy for ischemic heart disease. Indeed, these conditions are already treated in patients with autologous mesenchymal stem cell (MSC) transplantation, directly injected into the affected organ. However, the low cell survival rate (<15%) in the three days following injection limits the beneficial effects. Yet, the functional recovery of the affected organ is directly correlated with the number of surviving MSCs; recent studies have shown that the regeneration of the infarcted organ is primarily due to their paracrine effects. In this context, maintaining the viability and functionality of MSCs appears to be a major challenge for improving the benefit-risk ratio of cell therapy for ischemic heart disease.

[0003] The development of porous biomaterials, capable of generating a biomimetic environment favorable to the maintenance of MSCs, while allowing administration in the form of "patch" on the surface of the injured organ, represents an advance in this field, and is the subject of intensive research.

[0004] Such an approach, eliminating the need for direct cell injection into the heart, would avoid: 1) cell mortality, 2) side effects associated with intraparenchymal injection (particularly bleeding), and 3) the risk of mesenchymal stem cells transforming into an undesired phenotype (osteoblast, chondroblast, adipocyte, or worse, cancer cells). To achieve this, it is necessary to develop biomimetic, three-dimensional (3D) porous materials that localize cells to the damaged site, protect them during implantation, and maintain them in an undifferentiated state to prolong their paracrine effects. To this end, these three-dimensional porous materials must meet precise specifications regarding their porosity, mechanical strength, and elasticity.

[0005] Polymer matrices are widely used in tissue engineering. Generally, two main types of matrices are distinguished: those using synthetic polymers and those based on natural polymers, the latter being preferred due to their better biocompatibility and the possibility of synthesizing matrices without organic solvents or toxic reaction intermediates, at temperatures and pH levels compatible with cell survival.

[0006] Over the past 20 years, alginate has proven to be the polymer of choice in tissue engineering due to its excellent biocompatibility in vivoThis is attributed to its polysaccharide nature and the fact that its network structure is similar to that of the extracellular matrix (ECM) of living tissues. For example, Andersen T. et al. (BioMacromolecules, 2012, 13, 3703-3710) describe alginate-based macroporous sponges with open and interconnected pores and their potential applications for cell encapsulation, drug delivery, and wound healing. These macroporous sponges are prepared by gelling a solution of alginate, calcium carbonate, and a plasticizer in the presence of a divalent cation, after the solution has been agitated at high speed to incorporate air bubbles. The resulting gel is then dehydrated by air drying at 80°C to produce a highly porous structure called a xerogel. These alginate xerogels have limited commercial interest for all applications requiring implantation.Indeed, these materials exhibit a porosity that is favorable to cell culture but, conversely, gives them low mechanical resistance. Furthermore, it is difficult to maintain their initial 3D structure (responsible for their biocompatibility, biomimicry, and seeding capacity) after this type of drying.

[0007] International application WO 2007 / 103208 describes a process for preparing biodegradable, open-porosity sponges based on a polysaccharide such as chitosan or hyaluronic acid. The process involves forming a moist foam from an aqueous dispersion comprising such a polysaccharide, a foaming agent, and optionally one or more gel-forming ions, a plasticizer, a crosslinking agent, and a pH modifier. The resulting foam is then molded and air-dried. Like the previous alginate sponges, the polysaccharide sponges thus obtained (xerogels) exhibit an open pore network but lack sufficient mechanical properties for easy implantation.

[0008] US patent 6,425,918 further describes a process for preparing matrices based on polysaccharides such as alginates, carrageenans, gellan gum, xanthan gum, chitosan, etc., consisting, in a first step, of gelling a solution of a polysaccharide in the presence of a crosslinking agent, then in a second step of freezing the resulting gel, before drying it by lyophilization in a third step. The matrices obtained according to this process (cryogels) exhibit improved mechanical strength compared to the alginate xerogels described in Andersen T. et al. (cited above). However, it is difficult with this process to modulate the porosity generated by the freezing and drying method, and to adapt it to the intended applications; the possibilities of controlling the dimensions of the pores obtained are limited.

[0009] A matrix composed of a combination of three polymers—alginate, chitosan, and gelatin—has also been proposed, notably in the article by Chhavi Sharma et al., *Journal of Applied Polymer Science*, 2012, Vol. 127(4), pages 3228-3241. This matrix is ​​prepared from a foam obtained by agitating an aqueous solution of alginate (2%) and gelatin (5%) without using a surfactant, but in the presence of NaHCO3 to generate gas. This foam is then mixed with a glutaraldehyde solution. The crosslinked alginate / gelatin foam is then added as extruded beads in an acidic chitosan solution containing CaCl2 (the crosslinking agent for alginate). The beads are then subjected to a vacuum to modify their porosity. The use of glutaraldehyde allows for the cross-linking of gelatin or chitosan via covalent bonds. However, glutaraldehyde is toxic to cells.The subsequent addition of chitosan leads to the formation of a shell around the microspheres and its uneven distribution within the final material structure. Finally, the simple use of sodium bicarbonate results in the formation of bubbles and thus a non-interconnected porosity within the beads. The vacuum drying step at the end of the process causes the pores to interconnect. via the rupture of the thinnest walls of the porous structure but leads to a population of pores with random dimensions and which are not necessarily suitable for cell culture applications.

[0010] There is therefore a need for biocompatible and biodegradable polymer matrices that are highly porous while exhibiting good mechanical properties.

[0011] The inventors therefore set themselves the goal of developing a preparation process enabling access to three-dimensional, biocompatible and biodegradable polymer matrices which combine good mechanical resistance properties with a network of open and interconnected pores meeting the criteria of seeding capacity, biocompatibility and biomimicry in order to allow their use as a cell support and / or for cell culture, as well as in tissue therapy, particularly in cardiac tissue therapy.

[0012] The present invention therefore relates primarily to a method for preparing a biocompatible and biodegradable polymer matrix comprising a network of open and interconnected pores, said method comprising at least the following steps: 1) the preparation of an aqueous solution comprising at least one biocompatible anionic polysaccharide and at least one biocompatible cationic polymer, 2) the mechanical agitation of said solution obtained above in the previous step in the presence of a foaming agent or a gas under pressure, to form a foam, 3) the freezing of the foam obtained above in the previous step, to obtain a frozen foam, 4) the gelation of the frozen foam obtained above in the previous step, by adding to said foam at least one gelling agent in solution in a solvent, to obtain a gelled foam, 5) the dehydration of the gelled foam obtained above in the previous step, to obtain a dehydrated gelled foam, then 6) the drying of the dehydrated gelled foam obtained above in the previous step, by treatment with supercritical CO2, to obtain said polymer matrix.

[0013] Thanks to the process according to the invention, it is now possible to prepare biocompatible and biodegradable polymer matrices comprising a network of open and interconnected pores allowing their use for cell culture and having in addition sufficient mechanical resistance and elasticity to allow their use in cell therapy, in particular in cardiac cell therapy.

[0014] According to the invention, the term "biocompatible" used in this description to qualify said polymer matrix or substance, such as for example an anionic polysaccharide or a cationic polymer, means that said material or substance does not interfere with or degrade the biological environment in which it is used.

[0015] For the purposes of the present invention, the expression "open and interconnected pores" means that the polymer matrix has an open porosity, that is to say a porosity which is accessible from outside the matrix, and that said pores communicate with each other to form a three-dimensional network.

[0016] The biocompatible anionic polysaccharide(s) usable according to the invention preferably have an average molecular mass (Mw A) greater than or equal to 75,000 Daltons, preferably ranging from about 75,000 to 250,000 Daltons, and even more preferably from about 140,000 to 240,000 Daltons.

[0017] The biocompatible anionic polysaccharide(s) usable according to the invention are preferably chosen from alginates and modified alginates, pectins, cellulosic derivatives, polysaccharide gums such as agar-agar, xanthan gum and gellan gum, carrageenans, modified dextran and hyaluronic acid.

[0018] According to a preferred embodiment of the invention, the biocompatible anionic polysaccharide(s) are selected from alginates and modified alginates.

[0019] Among the alginates, particular preference is given to alginates comprising 32 to 61% mannuronic acid (M) units, 39 to 68% guluronic acid (G) units relative to the total number of units constituting said alginates, and in which the M / G ratio varies from 0.47 to 1.56.

[0020] Among such alginates, we can notably mention sodium alginates containing at least 60% by number of G units such as the products sold under the trade names Pronova UP MVG ®< , Pronova UP LVG ®< , Alginate SLG20 ®< and Alginate SLG100 ®< by the company Novamatrix, alginates containing at least 50% by number of M units such as the products sold under the trade names Pronova UP MVM ®< , Pronova UP LVM ®< , Alginate SLM20 ®< and Alginate SLM100 ®< by the company Novamatrix, or even sodium alginates sold under the trade names Alginate Medium Viscosity and Alginate High Viscosity by the company Sigma-Aldrich.

[0021] According to a particularly preferred embodiment of the invention, the biocompatible anionic polysaccharide is a sodium alginate having a viscosity greater than or equal to 2000 mPa-s and an average molecular mass between 80,000 and 120,000 Da inclusive.

[0022] For the purposes of this invention, "modified alginate" means an alginate whose basic structure is functionalized by one or more groups, in particular by one or more peptides such as, for example, tripeptides composed of L-arginine, glycine, and L-aspartic acid (RGD peptides), which are peptides involved in cell adhesion. For example, such modified alginates are sold under the trade names Novatach®<G RGD, Novatach®<M RGD, Novatach®<G VAPG, and Novatach®<M REGV by the company NovaMatrix.

[0023] According to a preferred embodiment of the invention, the amount of anionic polysaccharides present in the aqueous solution of step 1) varies from about 0.5 to 8% by mass, and even more preferably from about 1 to 3% by mass relative to the total mass of said aqueous solution.

[0024] The biocompatible cationic polymer(s) usable according to the present invention preferably have an average molecular mass (Mw C) greater than or equal to 100,000 Daltons, more preferably ranging from about 150,000 to 600,000 Daltons, and even more preferably from about 190,000 to 310,000 Daltons.

[0025] The biocompatible cationic polymer(s) usable according to the present invention are preferably chosen from cationic polysaccharides, in particular from the group including chitosan, particular saline forms of chitosan and chitosan derivatives; and polymers having basic reactive groups such as amine or imine groups, among which may be mentioned polyethyleneimines, poly(L-lysines), poly(vinylamines), poly(amino acids) and poly(alkylamines).

[0026] The biocompatible cationic polymer(s) are preferably chosen from chitosan, specific salt forms of chitosan, and chitosan derivatives. In particular, one can mention chitosans sold under the trade names Chitosan Low Molecular Weight, Chitosan Medium Molecular Weight, Chitosan High Molecular Weight, Chitosan High Purity Mw 60,000-120,000, Chitosan High Purity Mw 110,000-150,000, and Chitosan High Purity Mw 140,000-220,000 by Sigma-Aldrich, or Protasan UP CL 113, 114, 213, and 214 by NovaMatrix. Among these chitosans, particular preference is given to chitosans with a viscosity of approximately 200 to 800 mPa-s, an average molecular mass between 190,000 and 310,000 Da inclusive and a deacetylation rate greater than 80%.

[0027] The specific salt forms of chitosan (which can also be called chitosan salts) can notably be chosen from chitosan chloride, lactate, acetate and glutamate.

[0028] For the purposes of this invention, a "chitosan derivative" means a chitosan whose basic structure is functionalized by one or more functional groups, in particular, by one or more groups selected from among carboxymethyl, hydroxybutyl, or glyceryl phosphate-hydroxyethylcellulose groups. Examples of such chitosan derivatives include products sold under the trade names Chitoscience® or Chitoceuticals Carboxymethylchitosan® by HMC+.

[0029] According to a preferred embodiment of the invention, the quantity of cationic polymers present in the aqueous solution of step 1) varies from about 0.5 to 15% by mass, and even more preferably from about 1 to 2.25% by mass relative to the total mass of said aqueous solution.

[0030] According to a preferred embodiment of the invention, the mass ratio of anionic polysaccharides (M PA) / cationic polymers (M PC) present in the aqueous solution of step 1) varies from 20 / 80 to 80 / 20, and even more preferably from 40 / 60 to about 60 / 40.

[0031] The total concentration of polymers present in the aqueous solution of step 1), that is to say the sum of the quantities of anionic polysaccharides and cationic polymers, varies preferably from 1 to 10% by mass about, and even more preferably from 1.5 to 3.75% by mass about relative to the mass of the aqueous solution.

[0032] In addition to the anionic polysaccharide(s) and the cationic polymer(s), the aqueous solution prepared in step 1) preferably includes at least one hydrophilic surfactant whose presence helps to stabilize the foam formed in step 2).

[0033] For the purposes of the present invention, hydrophilic surfactant means a surfactant having an HLB value (“ Hydrophilic-Lipophilic Balance " : hydrophilic / hydrophobic balance) greater than or equal to 8, and preferably greater than or equal to 12. Their presence in the aqueous solution prepared in step 1) helps to stabilize the foam formed during step 2).

[0034] The hydrophilic surfactant(s) are preferably chosen from among the non-ionic surfactants.

[0035] Among such non-ionic surfactants, mention may be made in particular of ethoxylated fatty acid and sorbitan esters (polysorbates) such as the products sold under the trade names Eumulgin ®< SML20, SMS20 by BASF, Montanox ®< 20 PPI, Montanox ®< 20 API and Montanox ®< 80 PPI by Seppic, Alkest ®< TW20, TW60, TW80, TW80 K, TW327 by Univar, Carnacel ®< TW20 and TW80 by Quimica Delta, and Tween ®< 20, 40, 60, 65, 80 and 85 by Sigma-Aldrich; nonionic polyoxyethylene-polyoxypropylene block copolymers (also known as poloxamers) such as products sold under the trade names Pluronic ®<, in particular Pluronic ®< F68, Pluronic ®< F108 and Pluronic ®< F127, by Sigma-Aldrich, or Synperonic ®< or Kolliphor ®< also by Sigma-Aldrich; cellulose derivatives such as hydroxypropylmethylcellulose; glucosides and alkanolamides.

[0036] The hydrophilic surfactant(s) can also be chosen from anionic surfactants, and in particular from sodium dodecyl sulfate such as the products sold under the trade names Triton ®<, in particular Triton ®< X-405 by the company Sigma-Aldrich, and cetyl trimethylammonium bromide (known by the acronym CTAB).

[0037] More rarely, the hydrophilic surfactant(s) may be chosen from certain cationic polymers such as, for example, polyquaterniums, such as Polyquaternium-10 in particular.

[0038] Finally, proteins such as albumin and gelatin can also act as hydrophilic surfactants.

[0039] According to a preferred embodiment of the invention, the hydrophilic surfactant is a nonionic surfactant, and even more preferably a polysorbate or a poloxamer.

[0040] The hydrophilic surfactant(s) preferably represent 0.01 to 5% by mass, and even more preferably about 1% by mass, relative to the total mass of the aqueous solution from step 1).

[0041] Although not mandatory, the aqueous solution prepared in step 1) may also contain one or more plasticizing agents.

[0042] In this case, the plasticizing agent(s) are preferably chosen from glycerol, sorbitol and their mixture.

[0043] When used, the ratio of the plasticizing agent(s) to the polymers in aqueous solution prepared in step 1) can vary from 10:1 to 2:1, preferably from 8:1 to 3:1, and even more precisely from 6:1 to 4:1.

[0044] The mechanical agitation carried out during step 2) is preferably performed at a rotation speed greater than or equal to 1500 revolutions per minute (rpm), and even more preferably at a rotation speed ranging from approximately 1500 to 2100 rpm.

[0045] The duration of mechanical agitation generally varies from approximately 5 to 120 minutes. Typically, it is about 30 minutes.

[0046] Mechanical agitation is preferably carried out using a conventional paddle-type device.

[0047] According to a first embodiment of step 2), the foam is formed in the presence of a foaming agent which is then added to the solution prepared in step 1) just before carrying out step 2).

[0048] In this case, the foaming agent (also called "porogenous agent" or "gas-generating agent") is preferably chosen from sodium bicarbonate and sodium carbonate, and the aqueous solution from step 1) is then brought to an acidic pH, preferably varying from 4.0 to 6.5 by adding at least one acidifying agent.

[0049] According to this first embodiment of step 2), the foaming agent then preferably represents approximately 0.5 to 5% by mass, and even more preferably approximately 1 to 2% by mass, relative to the total mass of the aqueous solution prepared in step 1).

[0050] The addition of an acidifying agent allows for the release of gas from the foaming agent present in the aqueous solution.

[0051] The acidifying agent can, for example, be chosen from acetic acid, adipic acid, tartaric acid, glucono-δ-lactone and hydrogen peroxide.

[0052] In this case, the acidifying agent (other than hydrogen peroxide) preferably represents approximately 0.05 to 15% by mass relative to the total mass of the aqueous solution. If the acidifying agent is hydrogen peroxide, then its concentration can be up to approximately 40% by mass relative to the total mass of the aqueous solution.

[0053] According to a second embodiment of step 2), the foam is formed by introducing a gas under pressure into the aqueous solution prepared in step 1).

[0054] In this case, the gas is chosen, for example, from air, argon, and carbon dioxide.

[0055] According to this second embodiment, the pressure at which the said gas is introduced can vary from approximately 1 to 100 bars.

[0056] The duration of introduction of said gas under pressure into the aqueous solution can vary from approximately 20 minutes to 2 hours.

[0057] The freezing in step 3) is preferably carried out by bringing the foam obtained in step 2) to a temperature of -10°C or less, and even more preferably to a temperature of -18 to -20°C.

[0058] According to a particular and preferred embodiment, step 3) of freezing is carried out by maintaining the mousse obtained in step 2) at a temperature of about -18°C for 8 to 24 hours.

[0059] According to another particular embodiment, step 3) of freezing is carried out by immersing the foam obtained in step 2) in a bath of liquid nitrogen at a temperature of -180°C. In this case, freezing is rapid, on the order of a few seconds.

[0060] During the freezing stage, the mousse obtained in step 2) can be introduced into a mold so as to give a particular shape to said mousse, after freezing.

[0061] According to a particular embodiment of the process according to the invention, step 3) of freezing can be followed by a step 3bis) of freeze-drying the frozen foam obtained at the end of step 3).

[0062] In this case, step 3bis) of freeze-drying can be carried out under vacuum at a temperature of approximately -40 to -50°C and at a pressure of approximately 10 to 100 µm of mercury.

[0063] The gelling agent used in step 4) of gelling the frozen foam is preferably a solution of at least one salt of a divalent or trivalent cation in a solvent. Such cations are preferably chosen from inorganic cations such as copper, calcium, aluminum, magnesium, strontium, barium, zinc, chromium, as well as from organic cations such as alkylammonium salts, polyethyleneimine, poly(vinylamine), poly(amino acids) and poly(alkylamines).

[0064] The solvent for the gelling agent solution is preferably water, buffered or unbuffered.

[0065] According to a preferred embodiment of the invention, the gelling agent is an aqueous solution of calcium chloride, strontium chloride, calcium gluconate, calcium carbonate, or strontium carbonate.

[0066] The concentration of the gelling agent solution can vary from approximately 0.005 M to 1 M, and preferably from approximately 0.05 to 1 M.

[0067] At the end of step 4), the foam is preferably washed several times to remove the surfactant, for example with a pH neutral solution such as a buffer solution, for example a buffer based on 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES buffer).

[0068] Step 5), the dehydration of the gelled foam obtained in step 4), is preferably carried out progressively by immersing the gelled foam in successive baths of absolute ethanol of increasing concentrations. For example, the dehydration of the gelled foam can be achieved by immersing it in three baths of 20% absolute ethanol, then three baths of 40% absolute ethanol, then three baths of 80% absolute ethanol, and finally, three baths of 100% absolute ethanol, each immersion lasting approximately 10 to 15 minutes.

[0069] Step 6) of drying with supercritical CO2 of the dehydrated foam obtained at the end of step 5) is preferably carried out at a temperature of approximately 35 to 50°C, and at a pressure of approximately 45 to 95 bars.

[0070] According to a particular and preferred embodiment, the supercritical CO2 drying step of the dehydrated foam is carried out at a temperature of about 44°C, under about 85 bars of pressure, for about 25 minutes.

[0071] When step 6) of dehydration is complete, the polymer matrix is ​​ready for use or storage for later use. Before use, the matrix is ​​preferably sterilized using methods well known to those skilled in the art, such as exposure to UV radiation, γ-irradiation, the use of pulsed or non-pulsed electron beams, ethylene oxide, autoclaving, or contact with alcohol or a gas of the formula NOx, plasma gas sterilization (Sterrad®), provided that none of these methods affects the properties of the final matrix or the characteristics of its components.

[0072] The biocompatible and biodegradable polymer matrix obtained by implementing the process as defined according to the first object of the invention is new in itself and as such constitutes the second object of the invention.

[0073] A biocompatible and biodegradable polymer matrix is ​​obtained by implementing the process according to the present invention, said matrix being characterized in that it is in the form of an alveolar material consisting of a porous polymer matrix resulting from the gelation of a foam of at least one biocompatible anionic polysaccharide and at least one biocompatible cationic polymer, and in that said matrix: comprises open and interconnected pores having an average dimension d A ranging from 0.2 µm to 400 µm; has a pore volume ranging from 60 to 98% of the total matrix volume; has a modulus of elasticity at 50% strain (E 50%) ranging from 1 to 100 kPa.

[0074] The modulus of elasticity at 50% deformation, E 50%, is determined using a texture analyzer sold under the trade name TA-XT2 Texture Analyser by Stable Micro Systems. This analyzer uses a cylindrical piston made of Plexiglas® (poly(methyl methacrylate)) with a diameter of 20 mm and a compression speed of 2 mm / s to measure the force required to compress a polymer matrix sample to 50% of its initial height. The modulus of elasticity at 50% deformation is then calculated using the following formula: E 50 % = F 50 % / S 0 , 5 × 1000 where E 50% and F 50% are respectively the modulus of elasticity (in kPa) and the force (in N) required to obtain 50% deformation, and S is the surface area of ​​the polymer matrix sample (in mm²) in contact with the piston. Such a method was, for example, described by Shapiro L et al. (Biomaterials 1997, 18, 583-590).

[0075] According to a preferred embodiment of this material, the modulus of elasticity in compression varies from 30 to 45 kPa.

[0076] Furthermore, said polymer matrix in the rehydrated state preferably exhibits a tensile Young's modulus ranging from 0.3 to 20 kPa, preferably from 0.5 to 15 kPa, and even more preferably from 1 to 10 kPa. Tensile strength measurements of the polymer matrices according to the invention were carried out after rehydration of dumbbell-shaped samples in a medium " Minimum Essential Medium » (MEM) complete alpha (MEM α complete) until complete swelling. The measurements were carried out using a TAXT2 texture analyzer equipped with jaws, according to the protocol described in the article by Andersen et al., Biomacromolecules, 2012 (ASTMD638-10 (Type I) standard).

[0077] Finally, said polymer matrix preferably has storage moduli (G') and loss moduli (G") ranging from 100 to 25,000 Pa.

[0078] G* is a rheological quantity called the complex modulus. When a material is subjected to a dynamic shear test, a phase shift is observed between the applied stress and the material's deformation. This reflects viscoelastic energy conservation phenomena (expressed via the modulus G', called the conservation modulus) and energy dissipation (expressed via the modulus G" called loss modulus). The moduli G' and G" are measured during a dynamic (oscillatory) rheological test according to the method described by Kong HJ et al. (Polymer, 2002, 43, 6239-6246).

[0079] Also according to a preferred embodiment of this material, the moduli (G') and (G") vary from 12,000 to 25,000 Pa for G' and from 1,000 to 3,000 kPa for G".

[0080] As previously mentioned, the very nature of the material (high porosity biocompatible polymer matrix) makes its use for cell culture particularly advantageous.

[0081] Therefore, the biocompatible and biodegradable polymer matrix obtained by the process as defined in the object of the invention can be used as a support for animal or human cells and / or for the culture of animal or human cells in vitro, in particular undifferentiated mammalian cells such as, for example, mesenchymal stem cells.

[0082] Indeed, studies conducted by the inventors have shown that such a matrix constitutes a porous environment conducive to the viability and maintenance of stem cell functionality. Furthermore, it is possible to vary the porosity and mechanical properties of the matrices of the invention within the ranges indicated above, depending on the choice of polymers used in their preparation and the type of cells to be supported or cultured.

[0083] Thus, a porous polymer matrix containing animal or human cells, in particular undifferentiated mammalian cells, can be obtained, said support being characterized in that the porous polymer matrix is ​​a matrix such as is obtained according to the process as defined according to the object of the invention, and in that said cells are predominantly present in the pores of said matrix.

[0084] The excellent biocompatibility and mechanical properties of such a cell scaffold make its use in tissue therapy, and in particular in cardiac tissue therapy, especially advantageous.

[0085] Thus, cell support can be used in regenerative medicine, particularly in cell therapy, especially in cardiac cell therapy.

[0086] Such a support allows, after implantation, particularly at the site of a lesion in the heart muscle, the localization of cells at the implantation site, while protecting them during implantation and maintaining them in an undifferentiated state so as to prolong their paracrine effects.

[0087] The present invention is illustrated by the following embodiments, to which it is not, however, limited. EXAMPLES

[0088] The raw materials used in the following examples are listed below: Sodium alginate from brown algae, with an average molecular weight between 80,000 and 120,000 Da, and a viscosity ≥ 2000 mPa-s (at 2% wt. in water, at 25°C), sold under the trade name Alginic acid sodium salt from brown algae - Medium viscosity by Sigma-Aldrich; 80% deacetylated chitosan, with an average molecular weight between 190,000 and 300,000 Da, and a viscosity between 200 and 800 mPa-s (at 1% wt. in 1% acetic acid, at 25°C), sold under the trade name Chitosan medium molecular weight by Aldrich; NaCl and calcium carbonate (BDH Prolabo), pure acetic acid (Fisher Chemical), absolute ethanol, HEPES buffer (Sigma-Aldrich) Physico-chemical characterizations Scanning electron microscopy (SEM) :

[0089] The polymer matrices prepared in the following examples were silver-plated by argon sputtering using an apparatus sold under the trade name Sputter Coater S 150B ® by Edwards, and then observed using a Jeol JSM-6400 scanning electron microscope at a voltage of 10 kV. For each sample, 10 pore diameter measurements were taken on the surface and in cross-section, and the average was calculated. Environmental scanning electron microscopy:

[0090] Cubic samples, approximately 5 x 5 x 2.5 mm in size, were cut from the polymer matrices prepared in the following examples and placed in a dry state in the chamber of an ESEM Quanta® 250 FEG microscope from FEI. The samples were progressively hydrated by controlling the humidity level in the chamber, which was gradually increased from 85% to 99% by regulating the water vapor pressure. The accelerating voltage was set to 15 kV and the temperature to 2°C. Mechanical resistance (Uniaxial compression test):

[0091] Uniaxial compression tests were performed on samples of the polymer matrices prepared in the following examples, after hydration for 24 hours in a cell culture medium. Each sample was subjected to three uniaxial compression tests, and measurements were taken in triplicate. The measurements were performed using a texture analyzer sold under the trade name TA-XT2 Texture Analyser by Stable Micro Systems, with a cylindrical aluminum piston with a diameter of 20 mm and a compression speed of 2 mm / s to measure the force required to compress the samples to 50% of their initial height. The modulus of elasticity at 50% deformation was then calculated using the following formula: E 50 % = F 50 % / S 0 , 5 × 1000 in which E 50% and F 50% are respectively the modulus of elasticity (in kPa) and the force (in N) required to obtain 50% deformation and S is the surface area of ​​the sample (in mm²) in contact with the piston. Mechanical resistance (Uniaxial tensile tests):

[0092] In the following examples, tensile tests were performed on samples of dumbbell-shaped polymer matrices (according to ASTM D638-10 (Type I)) after rehydration for 24 hours in a cell culture medium. The Young's modulus of the hydrated samples was determined in uniaxial tension using a texture analyzer sold under the trade name TA-XT2 Texture Analyzer by Stable Micro Systems, at a constant speed of 0.5 mm / s to measure the force required to break. The stress curve (equal to the force in Newtons per unit area in mm²) versus the strain curve (in %) is plotted, and the Young's modulus is then calculated as the slope at the origin in the linear portion of this curve. Measurements are taken on 3 to 5 samples for each alginate / chitosan ratio. Such a method is described, for example, in the article by Andersen et al., 2012, Biomacromolecules. Stem cell cultures:

[0093] Rat bone marrow mesenchymal stem cell (mSMC) cultures were performed as follows: A cell culture medium “ Minimum Essential Medium » (MEM) complete alpha (MEM α complete) was prepared by mixing 450 mL of MEM α medium, GlutaMAX ®< sold under reference 32561 by Gibco, 5 mL of a Penicillin / Streptomycin mixture (10000 U / mL) sold under reference 15140 by Gibco and 50 mL of Fetal Bovine Serum sold under reference A15-043 by PAA.

[0094] MSCs were thawed in 15 mL of pre-warmed, complete MEM α culture medium at 37°C. After centrifugation for 5 min at 1200 rpm, the supernatant was aspirated, and the cell pellet was resuspended in 25 mL of complete MEM α culture medium. The cells were then seeded into a culture flask at a density of 10,000 cells / cm². The complete MEM α culture medium was changed every 2–3 days. The cells were passed to confluence and re-seeded at a density of 10,000 cells / cm². Biocompatibility assessment in vitro:

[0095] MSCs were washed twice with 1x PBS buffer, then detached with trypsin and counted. They were then centrifuged for 5 min at 1200 rpm, and the cell pellet was resuspended in complete MEM α culture medium. 15 µL of cell suspension containing 100,000 MSCs were loaded onto the dry polymer matrix samples in a 48-well plate, centrifuged for 1 min at 400 g and 25°C to achieve homogeneous seeding, and then hydrated in complete MEM α culture medium. The plates were maintained under culture conditions at 37°C in a 5% CO₂ atmosphere. Evaluation of cell viability within polymer matrices:

[0096] Physiological saline: 0.9% NaCl solution by mass in deionized water, Fluorescent markers: viability and cytotoxicity test kit sold under the name using calcein AM and ethidium-III, sold under the name " ViabilitylCytotoxicity Assay Kit for Live & Dead cells » ,reference FP-BF4710 by the company Interchim Fluo Probes ®< , France.

[0097] A cell labeling solution was prepared just before use by diluting the markers 1 / 10 in a 1 / 1 (v / v) physiological saline / MEM α culture medium mixture to obtain a marker solution containing 2 µM ethidium-III and 1 µM calcein AM. The labeling solution was kept in the dark until use.

[0098] MSCs were washed once with a 1:1 (v / v) mixture of physiological saline and MEM α culture medium, then incubated with the labeling solution for 30 min at 37°C in the dark. After incubation, the cells were washed with physiological saline and stored in physiological saline at 37°C until observation.

[0099] The labeling was observed using a Zeiss 780 confocal microscope: excitation wavelength of calcein AM: 495 nm; emission wavelength of calcein AM: 515 nm; excitation wavelength of Ethidium-III: 495 nm; emission wavelength of Ethidium-III: 635 nm. Successive depth images of the matrices were acquired, and then a 3D reconstruction was obtained using the software associated with the microscope. Biocompatibility assessment in vivo :

[0100] Biocompatibility in vivoThe use of polymer matrices was evaluated in three female Lewis rats with an average weight of 200 g. For anesthesia, the animal was placed in a gas induction chamber and received a gas mixture of O₂ + 4% isoflurane. After complete loss of peripheral reflexes, the animal was placed on its back. Gas anesthesia was maintained with 2% or 3% isoflurane. A subcutaneous injection of buprenophrine (100 µg / kg) was administered. The abdomen was extensively shaved, followed by disinfection with 70% alcohol. After verifying the depth of anesthesia and the complete loss of peripheral reflexes, a skin incision was made to expose the pectoral muscles. A flexible retractor was inserted. The polymer matrix was then placed between two muscle layers. The muscle pocket was then closed with a single suture using 7 / 0 Prolene monofilament. The retractor was removed and peritoneal lavage was performed.The skin was closed using 5 / 0 Ethilon suture. The entire procedure was performed by a surgeon under an operating microscope (Zeiss OPM1 FC). Evaluation of angiogenic effects in vivo :

[0101] Angiogenic effects in vivo Polymer matrices were evaluated in rats after intramuscular implantation. The implantation of the matrices was performed according to the same protocol used above for biocompatibility assessment. in vivo. The formation of capillaries and more mature vessels (arterioles) was studied after 28 days of implantation, using immunofluorescence with antibodies directed against Von Willebrand factor (VWF) to detect endothelial cells and against alpha actin of smooth muscle (α-SMA) to detect arteriolar muscles according to the following protocols: Histology - Immunostaining :

[0102] Twenty-eight days after implantation, the matrices were harvested, rinsed with physiological saline, and immediately fixed with 4% paraformaldehyde (in 1X PBS, pH 7.4) for 48 hours, then transferred to 70% ethanol. After paraffin embedding, histological sections 4 to 6 µm thick were prepared using a microtome. Hematoxylin and eosin histological staining and immunofluorescence staining with anti-α-SMA (smooth muscle actin) and anti-VWF (Von Willebrand Factor) were performed. For immunofluorescence staining, the slide sections were first deparaffinized in xylene (three 5-minute baths), then rehydrated in successive ethanol baths (5 minutes each), and finally in water (5 minutes). Antigenic site unmasking was performed in Tris (10mM) - EDTA (1mM) buffer containing 0.05% of Tween20 at 121° for 3 min.The samples were then permeabilized with Triton (0.5%), and any unreacted aldehyde groups of the fixative were neutralized in a 0.1 M glycine solution (two 10-minute baths). Non-specific antigenic sites were saturated with a PBS buffer containing 2% goat serum, 1% bovine serum albumin, and 0.2% Triton (30 minutes). The slides were then stained with an anti-α-SMA antibody (monoclonal mouse anti-alpha-SMA, A2547, Sigma, 1 / 1000 dilution) and an anti-VWF antibody (polyclonal rabbit anti-human VWF, A0082, Dako, 1 / 200 dilution) diluted in the saturation buffer. After three washes (PBS-Tween20 0.2%, 10 min each), secondary antibodies were added: Alexa Fluor ®< 568 goat anti-mouse (A11019, Life Technologies) and AlexaFluor ®< 488 goat anti-rabbit (A11008, Life Technologies) for 30 min in the dark.After three washes, the nuclei were labeled with DAPI (D9542, Sigma, 0.05 µg / ml dilution in PBS, 10 min). Finally, the slides were washed and mounted with a coverslip using a fluorescence mounting solution (F4680, Sigma). All steps were performed at room temperature. Observation of immunostaining by confocal microscopy

[0103] Immunostaining was observed using a Zeiss LSM 780 confocal microscope (Carl Zeiss Microscopy) at 63x magnification. For each animal, the number of α-SMA-positive vessels with a closed lumen and a diameter greater than or equal to 5 µm was counted in the implant area on at least 5 non-overlapping photographs. Knowing the total area of ​​the optical field (in mm²), the arteriolar density was calculated as equal to the number of vessels / mm². Statistics on immunolabeling

[0104] For comparing the number of vessels per unit area between implanted groups (L+ or reference matrices, acellular or containing MSCs), the test t A two-sided Student's t-test was used. Statistical analysis was performed using software sold under the commercial name GraphPadPrism version 4 (PrismGraphPad, San Diego, CA). The Gaussian distribution of the data was tested with a normality test, and the results are expressed as mean ± standard error of the mean. A test was considered significant if the p-value was less than 0.05. EXAMPLE 1 Preparation of porous polymer matrices according to the present invention and of comparative porous polymer matrices not forming part of the invention - Characterizations

[0105] In this example, porous polymer matrices according to the invention were prepared based on alginate as an anionic polysaccharide and chitosan as a cationic biocompatible polymer, using different alginate / chitosan mass ratios according to Table 1 below: TABLE 1 Matrix M100 / 0 (*) Matrix M60 / 40 50 / 50 Matrix Matrix 40 / 60 Matrix 0 / 100 (< * )< Alginate / chitosan mass ratio 100 / 0 60 / 40 5050 40 / 60 0 / 100 (*) : Comparative matrices not forming part of the invention 1) Preparation of polymer matrices

[0106] The following compositions have been prepared: Alginate solvent (for 200 g): 1.8 g NaCl, make up to 200 g with deionized water; Chitosan solvent (for 100 g): 0.9 g NaCl + 1.5 g pure acetic acid (i.e., 0.25 M) and make up to 100 g with deionized water; Buffer I (for 1000 g): 9.0026 g NaCl + 3.2540 g HEPES buffer, make up to 1000 g with Milli-Q® water and adjust the pH to 7.4 with 1 M or 2 M hydrochloric acid (HCl). Gelling buffer II (per 500 mL): 5 g calcium chloride (0.1 M) + 50 g pure acetic acid, then make up to 500 g with Milli-Q® water (Merck) and homogenize. Tested pore-forming agent: Sodium bicarbonate: NaHCO3 (Sigma-Aldrich) - introduced in step 1. Tested surfactant: Polysorbate 20, sold under the trade name Montanox® 20 DF (Seppic), introduced in step 1.

[0107] Solutions A, B, C, and D, whose specifications are given in Table 2 below, were then prepared: TABLE 2 Solutions A B C D Alginate solvent (g) - - - 200 Alginate powder (g) - - - 6 Chitosan solvent (g) 100 100 100 - Chitosan powder (g) 2 3 4,5 -

[0108] Solutions A, B, C and D were stirred between 1600 and 1800 rpm for 60 min.

[0109] The foams with the composition indicated in Table 3 below (mass percentages) were then prepared according to the protocol described above (steps 1 to 6 of the process according to the invention): TABLE 3 alginate / chitosan mass ratio Foam 100 / 0 (< * )< 60 / 40 Foam 50 / 50 Foam 40 / 60 Foam Foam 0 / 100 (< * )< Solution A (g) - 50 - - - Solution B (g) - - 50 - 50 Solution C (g) - - - 50 - Solution D (g) 50 50 50 50 - Alginate solvent 50 - - - - Chitosan solvent - - - - 50 % Final Alginate 1,5 1,5 1,5 1,5 0 % Final Chitosan 0 1 1,5 2,25 1,5 % final foaming agent 0,9 0,9 0,9 0,9 0,9 % Montanox® < 20 final 1 1 1 1 1 (*) : Comparative matrices not forming part of the invention

[0110] The different ingredients composing the mousses were mixed and the resulting compositions were stirred at 1800 rpm for 30 minutes.

[0111] Each of the foams thus obtained was then poured into a 48-well plate at a rate of 500 µL per well and then immediately frozen at -20°C.

[0112] After freezing, a portion of the foams (referred to as "L<") was freeze-dried at a temperature of -50°C and a pressure between 10 and 100 µm of mercury under vacuum (according to step 3bis of the process according to the invention). Another portion of the frozen foams (referred to as "L<") did not undergo this freeze-drying step.

[0113] The frozen and lyophilized foams L+< and the frozen foams L-< were then gelled by adding 500 µL of gelling buffer II to each of the wells, it being understood that to achieve the gelling of the 0 / 100 Foam, 500 µl of a 1M NaOH solution was used instead of the gelling buffer II since it is known that the sodium hydroxide used at this concentration causes chitosan to gel.

[0114] After one hour, the plates were rinsed several times using swab I in order to completely remove the surfactant (3 washes).

[0115] The gelled foams were then dehydrated by immersing the plates in successive baths of increasing concentration in absolute ethanol: 20%, 40% and 80% with 3 successive immersions of 10 min in each of the baths, the last dehydration having been carried out in a bath of absolute ethanol at 100% with 3 successive immersions of 15 minutes.

[0116] The dehydrated, gelled foams were then dried with supercritical CO2. To do this, the foams were removed from the 48-well plates and placed in sample holders, which were then introduced into the chamber of a Quorum Technologies E3000 Series Critical Point Dryer. Supercritical CO2 drying was carried out at a temperature of 44°C and a pressure of 85 bar for 25 minutes. The chamber was depressurized at a rate of 2 bar / min until atmospheric pressure was reached. The chamber was then opened, and the expected matrices were recovered. 2) Characterization results

[0117] The macroscopic appearance of the matrices thus obtained is shown by the figure 1The attached image shows the microscopic appearance of the internal structure of dry or hydrated L+ matrices. Images of dry matrices were obtained using scanning electron microscopy (SEM). Images of hydrated matrices were obtained using environmental scanning electron microscopy (ESM) by progressively increasing the humidity level from 85% to 99%. The scale bar corresponds to 100 µm.

[0118] In this figure, the figure 1a corresponds to the SEM images of the dry matrices. The images of the hydrated matrices were obtained by environmental SEM by progressively increasing the humidity level from 85% ( figure 1b ) at 99% ( figure 1d ), there figure 1c corresponding to an intermediate state of hydration. On the figure 1 , the scale bar corresponds to 100 µm.

[0119] These photos show in all the matrices presented the presence of interconnected pores which unfold under the effect of rehydration; microscopic observation makes it possible to visualize the porosity open to the outside of the matrices obtained.

[0120] The quantitative evaluation of the porosity of the matrices thus obtained is given by the figure 2 attached: quantification of the pore size of dry matrices L+ (corresponding to the photos on the figure 1a The values ​​shown are in the form mean ± standard deviation of the mean. A p-value < 0.05 is considered significant. * p < 0.05; ** p < 0.01; *** p < 0.001.

[0121] The surface porosity (pore diameter measured by SEM at the surface) is reported on the figure 2a (pore diameter in µm depending on the prepared matrices). The cross-sectional porosity (transverse pore diameter measured by SEM) is plotted on the figure 2b(transverse diameter of pores in µm as a function of prepared matrices).

[0122] The results show a similarity in porosity between alginate matrices and alginate / chitosan matrices, which are generally higher than that of chitosan-only matrices, although they all fall within the porosity range recognized as favorable to cell survival and proliferation. Alginate and alginate / chitosan matrices therefore appear better suited for 3D seeding.

[0123] The results for the compressive strength of the matrices are given in the figure 3 attached, representing the compressive mechanical properties of L+ matrices hydrated in cell culture medium subjected to three successive compression cycles, as well as the figure 4attached, which compares the results obtained with matrix M40 / 60 L +< that underwent the intermediate step 3bis of lyophilization to those obtained with matrix M40 / 60 L -< that did not undergo said step. Regarding the results presented to the figure 3 , statistical comparisons are made versus M100 / 0.* p<0.05 ; *** p<0.001.

[0124] On the figure 3 The differential elastic modulus (in kPa) is a function of the nature of the matrices prepared; the white bars correspond to the first compression cycle, the grey bars to the second compression cycle, and the black bars to the third compression cycle.

[0125] On the figure 4 The force (in Newtons, N) is a function of the deformation (in %), the solid line curves correspond to the first compression cycle, the dashed line curves to the second compression cycle and the dotted line curves to the third compression cycle.

[0126] The results of the figure 3 demonstrate the synergy resulting from the interaction between alginate and chitosan, leading to matrices with optimized mechanical properties compared to matrices based on alginate alone (M100 / 0) or chitosan alone (M0 / 100). The supercritical CO2 drying method according to step 6 of the process according to the invention enabled the production of aerogels that preserved the porosity of the foam generated in step 2) and during gelation in step 4), as well as the mechanical properties associated with the formation of polyelectrolyte complexes with opposite charges. The matrix according to the invention, in which the alginate / chitosan ratio is 50 / 50 (matrix M50 / 50), exhibits the best mechanical strength properties. Conversely, matrices not conforming to the invention (M0 / 100 and M100 / 0 matrices) exhibit weaker mechanical properties which may be a hindrance to their use for implantation.

[0127] The results presented on the figure 4 show that step 3bis of lyophilisation, although optional, improves the mechanical properties of matrices according to the invention, the compressive strength being greater when the matrix has undergone said step.

[0128] The results of the viability tests of MSCr cells in the M0 / 100, M40 / 60 and M100 / 0 matrices after 7 days of culture are reported in Table 4 below: TABLE 4 Alginate / chitosan ratio L-matrices -< L+< matrices M100 / 0 (< * )< + + M40 / 60 ++ ++ M0 / 100 (< * )< ++ + (*) : Comparative matrices not forming part of the invention

[0129] In Table 4, the "+" signs indicate the presence of live cells (cells appearing green under confocal microscopy because they are labeled with calcein AM). The number of "+" signs represents the number of detectable live cells. All matrices contain live cells after 7 days of culture, demonstrating the biocompatibility of the matrices according to the invention.

[0130] Finally, the biocompatibility assessments after implantation in rats (evaluation 1 week after intramuscular implantation in the pectoral region) show that: The matrices obtained by the process according to the invention are well suited to surgical manipulation and implantation without being damaged; the implantation of the matrices does not cause the animals any discomfort in moving, feeding, etc.; the implantation does not cause a massive inflammatory reaction or any other physiological reaction that could endanger the health of the implanted animals; one week after their implantation, the matrices are kept in place (at the site of implantation) and retain their integrity (no matrix debris). EXAMPLE 2 Study of the rehydration behavior of two matrices according to the present invention.

[0131] In this example, we compared the rehydration behavior of the M40 / 60 matrix according to the invention and having undergone an intermediate step 3bis of lyophilization, as prepared according to the process described above in example 1 (Matrix M40 / 60 L +< ) to that of the M40 / 60 matrix according to the invention but not having undergone this intermediate step 3bis of lyophilization, as also prepared according to the process described above in example 1 (Matrix M40 / 60 L -< ).

[0132] To do this, samples of identical diameter from each of these two matrices were immersed in water for 5 minutes. Photographs of each matrix taken before and after immersion are provided at the figure 5 attached.

[0133] It can be observed that the M40 / 60 L matrix, having undergone the intermediate freeze-drying step 3bis of the process according to the invention, swells more rapidly in water and reaches its final swelling level (maximum hydration state) in less than 5 minutes, whereas the M40 / 60 L matrix, having not undergone said step, swells more slowly and does not reach its maximum hydration level within this time. Therefore, these results show that, although optional, this intermediate freeze-drying step improves the rehydration properties of the matrices according to the invention. EXAMPLE 3 Evaluation of the angiogenic effects of a matrix conforming to the invention compared to a matrix not conforming to the present invention.

[0134] In this example, the angiogenic effects of the M40 / 60 L +< matrix according to the invention and as prepared above in Example 1 were evaluated in comparison with those of a matrix not according to the invention, obtained according to a preparation process identical in all respects to that of the M40 / 60L +< matrix except that the last two steps of dehydration and drying with supercritical CO2 were replaced by a new freezing step and then a new freeze-drying step, said steps being carried out under the same conditions as the freezing of the foam and freeze-drying of the frozen foam steps described above in Example 1. Said matrix, doubly freeze-dried, is designated M40 / 60 REF.

[0135] Each of these two matrices was tested after implantation in rats (matrix M40 / 60 L +< conforming to the invention and matrix M40 / 60 REF not conforming to the invention), as well as after prior seeding with 500,000 rCSMs (matrix M40 / 60 L +< -CSM conforming to the invention and matrix M40 / 60 REF-CSM not conforming to the invention). Each matrix was tested on two rats.

[0136] The assessments were carried out after 28 days of implantation.

[0137] There figure 6 The attached figure gives the number of α-SMA positive vessels per mm2 after 28 days of implantation for each of the matrices tested.

[0138] Fluorescence microscopy observations (not shown) demonstrated that the implanted M40 / 60 L+< matrix is ​​richly vascularized and that vessels are distributed throughout the granular tissue that forms following its biodegradation. The vessels present in the matrices are functional (as they contain red blood cells). In the presence of rSMSCs seeded in the matrix, the vascularization of the latter is greater (237 ± 44 vessels per mm² in the M40 / 60 L+< group). versus 391±127 vessels per mm² in the 40 / 60 L group (CSM). In the case of the M40 / 60 REF matrix not conforming to the invention, the same trend is observed in the presence of CSM (166±11 vessels per mm²). versus 199±21 vessels per mm 2< ). However, it is observed that the vascularization of the M40 / 60 REF matrix is ​​significantly less abundant than that of the M40 / 60 L +< matrix dried with supercritical CO 2 according to the present invention.

[0139] These tests demonstrate that the choice of the final drying step with supercritical CO2 is not a simple alternative to a freeze-drying step, but that on the contrary this drying method influences the properties of the resulting matrix, in particular its angiogenic properties after implantation.

Claims

1. Process for the preparation of a biocompatible and biodegradable polymer matrix comprising a network of open and interconnected pores, said process comprising at least the following stages: 1) the preparation of an aqueous solution comprising at least one biocompatible anionic polysaccharide and at least one biocompatible cationic polymer, 2) the mechanical stirring of said solution obtained above in the preceding stage in the presence of a foaming agent or of a pressurized gas, in order to form a foam, 3) the freezing of the foam obtained above in the preceding stage, in order to obtain a frozen foam, 4) the gelling of the frozen foam obtained above in the preceding stage, by addition to said foam of at least one gelling agent in solution in a solvent, in order to obtain a gelled foam, the frozen foam obtained above in the preceding stage being optionally lyophilized, 5) the dehydration of the gelled foam obtained above in the preceding stage, in order to obtain a dehydrated gelled foam, then 6) the drying of the dehydrated gelled foam obtained above in the preceding stage, by treatment with supercritical CO2, in order to obtain said polymer matrix.

2. Process according to Claim 1, characterized in that the biocompatible anionic polysaccharide(s) is (are) chosen from alginates and modified alginates, pectins, cellulose derivatives, polysaccharide gums, such as agar, xanthan gum and gellan gum, carrageenans, modified dextrans and hyaluronic acid, preferably from alginates and modified alginates.

3. Process according to Claim 1 or 2, characterized in that the amount of anionic polysaccharides present within the aqueous solution of stage 1) varies from 0.5% to 8% by weight, with respect to the total weight of said aqueous solution.

4. Process according to any one of the preceding claims, characterized in that the biocompatible cationic polymers are chosen from cationic polysaccharides, in particular in the group comprising chitosan, particular salt forms of chitosan, and chitosan derivatives; and polymers having basic reactive groups, such as amine or imine groups, among which may be mentioned polyethyleneimines, poly(L-lysines), poly (vinylamines), poly(amino acids) and poly(alkylamines), preferably from chitosan, specific salt forms of chitosan, and chitosan derivatives.

5. Process according to any one of the preceding claims, characterized in that the amount of cationic polymers present within the aqueous solution of stage 1) varies from 0.5% to 15% by weight, with respect to the total weight of said aqueous solution.

6. Process according to any one of the preceding claims, characterized in that the ratio by weight anionic polysaccharides (WAP) / cationic polymers (WCP) which are present in the aqueous solution of stage 1) varies from 20 / 80 to 80 / 20.

7. Process according to any one of the preceding claims, characterized in that the aqueous solution prepared during stage 1) additionally comprises at least one hydrophilic surfactant.

8. Process according to Claim 7, characterized in that the hydrophilic surfactant(s) represent(s) from 0.01% to 5% by weight, with respect to the total weight of the aqueous solution of stage 1).

9. Process according to any one of the preceding claims, characterized in that the foam is formed in the presence of a foaming agent which is then added to the solution prepared in stage 1) just before carrying out stage 2).

10. Process according to any one of Claims 1 to 8, characterized in that, in stage 2), the foam is formed by introduction of a pressurized gas into the aqueous solution prepared in stage 1).

11. Process according to any one of the preceding claims, characterized in that the freezing stage 3) is followed by a stage 3a) of lyophilization of the foam obtained on conclusion of stage 3).

12. Process according to any one of the preceding claims, characterized in that the gelling agent used during stage 4) of gelling the frozen foam is a solution of at least one salt of a divalent or trivalent cation in a solvent.

13. Process according to any one of the preceding claims, characterized in that stage 6) of drying, with supercritical CO2, the dehydrated foam obtained on conclusion of stage 5) is carried out at a temperature ranging from 35°C to 50°C and at a pressure ranging from 45 to 95 bar.