Crosslinked, stable and reversible hydrogels obtained by additive manufacturing.

Abstract

The present invention relates to stereoformed hydrogels, in particular doubly cross-linked hydrogels, of a predetermined, reversible, and controllable shape, a method for preparing them, and their applications. (no figure)

Description

Title of the invention: Crosslinked, stable and reversible hydrogels obtained by additive manufacturing.

[0001] The present invention relates to stereoformed hydrogels, in particular doubly crosslinked, of predetermined reversible and controllable shape, a process for preparing them and their applications. STATE OF THE ART

[0002] A hydrogel is generally made up of a three-dimensional network of insoluble and hydrophilic polymers capable of absorbing a large quantity of water. Said network can be chemically crosslinked with covalent bonds or physically by non-covalent interactions of the ionic, hydrogen, hydrophobic type, or even by a combination of the two.

[0003] Polysaccharide polymers consist of a set of monosaccharide units linked together by glycosidic bonds. They are used for their biocompatibility and high water absorption capacity. Polymers from animal sources are typically hyaluronic acid, chondroitin sulfate and chitosan. Polymers from plant sources are typically alginate, cellulose, dextran, pectin and agarose.

[0004] Hydrogels are increasingly used in tissue engineering due to their similarity to the cellular microenvironment in vivo. Hydrogels can homogeneously encapsulate cells and provide cells with a 3D microenvironment similar to the native extracellular matrix.

[0005] Hydrogels are also widely used in controlled release applications. For example, intracellular delivery vehicles composed of methacrylated alginate (Alg-MA) have been developed for the internalization and release of doxorubicin hydrochloride (DOX).

[0006] We also know the document (4D Biofabrication Using Shape-Morphing Hydrogels, Alina Kirillova et al., 2017) which describes the preparation of a hydrogel in the form of a film obtained by dynamically reconfigurable 3D bioprinting.

[0007] This document particularly describes photo-crosslinked two-dimensional (2D) rectangular methacrylated alginate hydrogels that instantly fold into tubes after immersion in water or PB S. This bidirectional shape morphing behavior likely resides in the crosslinking gradient introduced when the films are crosslinked with light.

[0008] However, bioprinting techniques using hydrogels as inks offer resolutions that are generally less than a millimeter but above all do not do not allow to obtain stereoformed hydrogels, with a predetermined, reversible and controllable shape.

[0009] The inventors of the present application, by using another printing technique, based on stereolithography (for example, two-photon lithography) were able to obtain higher degrees of resolution but above all were able to more finely control the crosslinking gradients inside the hydrogel in order to generate isotropic and / or anisotropic three-dimensional and bidirectional transformations.

[0010] Surprisingly, the inventors of the present application were able to obtain more complex structures than with bioprinting and were able to achieve controllable adjustment of the degree of deformation of the stereoformed hydrogel, thanks to a stereolithography manufacturing technology, which makes it possible to produce a variety of custom-shaped hydrogels that can adopt a different pre-programmed shape after ionic stimulation. Equally unexpectedly, the stereoformed hydrogels exhibit a reversible shape after several swelling / contraction cycles. Summary of the invention

[0011] A first aspect of the present invention overcomes the above-mentioned limitations by providing hydrogels whose deformation obtained by ionic stimulation is reversible, controllable and essentially bidirectional.

[0012] The present invention describes a stereoformed hydrogel, in particular doubly crosslinked, of reversible and controllable predetermined shape, comprising at least one photocrosslinkable ionic natural or synthetic polymer prepared by stereolithography. After photocrosslinking, the hydrogels are ionically crosslinked by one or more bivalent or trivalent cations which modifies the shape of the hydrogel by contraction of the polymer chains and expulsion of water. The initial shape of the hydrogel is then recovered by swelling, after desorption of the cation. The cycle between the different shapes by contraction and swelling is reproducible. The degree of deformation of the hydrogel can be controlled by the degree of photocrosslinking of the polymer chains.

[0013] The present invention also relates to compositions comprising the stereoformed, doubly crosslinked hydrogel of reversible and controllable predetermined shape and other elements such as cells or active agents.

[0014] A second aspect of the present invention relates to the preparation of the stereoformed hydrogel, in particular doubly crosslinked, of predetermined reversible and controllable shape.

[0015] A third aspect of the present invention relates to applications of the stereoformed, doubly crosslinked hydrogel of reversible and controllable predetermined shape. DETAILED DESCRIPTION OF THE INVENTION

[0016] According to a first aspect of the invention, the present application relates to a stereoformed, doubly crosslinked hydrogel of predetermined, reversible and controllable shape.

[0017] Dual ionic and stereolithographic crosslinking of ionic polymers, in particular functionalized alginate macromers, has been used to prepare hydrogels with preprogrammed swelling and contraction behaviors leading to a variety of shape, or volume or density changes. The hydrogels according to the present invention are reversibly deformable by inducing and reversing the physical crosslinks of the already photocrosslinked polymer network.

[0018] The use of stereolithography allows control of the density and gradient of photolinks within a hydrogel, which can then lead to many anisotropic shapes and properties. Anisotropic behavior of a hydrogel can be achieved by alternating high and low density photocrosslinking areas. This can produce high swelling and lower mechanical properties in areas of low photocrosslinked density and low swelling and higher mechanical properties in areas of high photocrosslinking density when physically crosslinked by ion stimulation.

[0019] Alternatively, objects can be processed using stereolithography UV crosslinking on already physically crosslinked ionic polymer and in which the reversible and controlled shape change is achieved by removing the physical crosslinks after stereolithography. This will give different swelling profiles and mechanical properties since the stereolithographic ultraviolet crosslinking will occur on physical gels. The change in shape and density of the object is achieved depending on the pattern of the photochemical crosslinking. The subsequent reversal of the physical crosslinks creates local swelling at microscale.

[0020] Through a combination of stereolithographic photolinking and physical crosslink induction or inversion that attenuates or amplifies the repulsive electrostatic interaction groups on the photocrosslinked polymer chain, a variety of shapes and mechanical properties can be achieved in the device. This is only achievable through spatially controlled photocrosslinking with a specific pattern to induce a shape change upon physical crosslink inversion. Thus, the shape, gel density, and mechanical properties can be controlled through swelling and contraction through a combination of repulsive electrostatic interactions (from reversed physical crosslinking) and microscopically controlled photocrosslinking.

[0021] Physical crosslinking may be caused by the action of one or more divalent or trivalent cations when the ionic polymers of the hydrogel are negatively charged. tively.

[0022] Thus, according to a first aspect of the invention, the present application relates to a stereoformed hydrogel, of reversible and controllable predetermined shape, comprising at least a first component, a second component and optionally a third component.

[0023] in which:

[0024] the first component is a natural or synthetic ionic polymer, covalently crosslinked by a crosslinking agent,

[0025] the second component is an aqueous medium or water, and

[0026] the third component is one or more divalent or trivalent cations,

[0027] said hydrogel has

[0028] either

[0029] a state of swollen form, density or volume Eg having a degree (rate) of swelling DGg, in which said hydrogel is devoid of divalent or trivalent cations,

[0030] either

[0031] a state of contracted form, density or volume Ec having a degree (rate) of swelling DGc, wherein said hydrogel comprises one or more types of divalent or trivalent cations, and

[0032] wherein the degrees of swelling DGg of the hydrogel, obtained before and after a contracted state Ec, or the degrees of swelling DGC of the hydrogel, obtained before and after a swollen state Eg, respectively have substantially identical values, and / or

[0033] in which the contracted Ec and inflated Eg volume states exhibit three-directional volume changes.

[0034] According to the present invention, the transition between an inflated state Eg and a contracted state Ec is accompanied by a modification of shape or volume in a three-directional manner.

[0035] According to the present invention, the expression "the hydrogel is free of bivalent or trivalent cations" means that the hydrogel, after desorption of the cations by a PB S solution or any other acidic or basic solution, no longer contains any cations or only traces, at least 10 times, preferably 50 times less than in the contracted state of the gel.

[0036] According to the present invention, the term "stereoformed hydrogel" means a hydrogel manufactured by three-dimensional photopolymerization, for example by stereolithography.

[0037] According to the present invention, the term "hydrogel of predetermined shape" means a hydrogel whose shape is modeled by gold-aided design software. diner (CAD). The three-dimensional shape of the hydrogel is not particularly limited and can correspond, for example, to a sphere, a cube, a rod, a disc, a parallelepiped. All these shapes can be regular or irregular.

[0038] According to a variant of the present invention, the hydrogel is porous, microporous and / or macroporous.

[0039] According to the present invention, the microporosity corresponding to the meshes of the hydrogel network can vary from 100 to 800 A.

[0040] According to the present invention, the macroporosity corresponds to the desired design of the object established by design software, and can therefore vary from 10 μm to a millimeter.

[0041] These values ​​are determined using techniques or equations known to those skilled in the art.

[0042] According to the present invention, the term "hydrogel of reversible shape" means a hydrogel having a "swollen" state and a "contracted" state, said hydrogel being capable of returning to a swollen state after a contracted state, or being capable of returning to a contracted state after a swollen state.

[0043] For example, a hydrogel which comprises a chemically crosslinked ionic polymer, in the presence of an aqueous solution exhibits a so-called swollen state. After addition of ions with a charge opposite to the charges of the chemically crosslinked ionic polymer, physical crosslinking takes place and the hydrogel adopts a so-called contracted state. After desorption of the divalent or trivalent ions, in the presence of an aqueous solvent containing an ion chelator or by ion exchange or any other technique, the hydrogel returns to its swollen state, substantially identical to that obtained before physical crosslinking.

[0044] According to the present invention, the term "controllable shape hydrogel" means a hydrogel whose shape is obtained according to the principle of 4D printing which makes it possible to manufacture a 3D printed object, for example a hydrogel whose geometry and / or properties are modified with the impulse of an external energy such as temperature, light, ions or one of other environmental stimuli.

[0045] According to the present invention, the expression "substantially identical values" means values ​​exhibiting a variation of less than 5%, preferably less than 4%, 3%, 2% or 1%.

[0046] For example, according to a variant of the present invention, the stereoformed hydrogel, of reversible and controllable predetermined shape, is subjected to 1 to at least 10 cycles of adsorption / desorption of divalent or trivalent cations. The successive swollen and contracted states of said hydrogel obtained after 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 cycles of adsorption / desorption of cations have values ​​of degrees of swelling DGg of the hydrogel, obtained before and after a contracted state Ec, or the degrees of swelling DGC of the hydrogel, obtained before and after a swollen state Eg, which are substantially identical.

[0047] According to the present invention, "tri-directional modification of shape or volume" means a deformation of the hydrogel in three directions, front-back, right-left and up-down.

[0048] Typically, hydrophilic polymers create hydrogen bonds with water which modify the network and cause swelling of the hydrogel. This deformation can be modulated by the swelling kinetics or the total absorption capacity. Conversely, dehydration will generate a contraction of the hydrogel.

[0049] In some embodiments, the polyionic hydrogel is a polyanionic hydrogel.

[0050] Examples of polyanionic hydrogels include hydrogels comprising negatively charged carbohydrates such as xanthan, hyaluronic acid or alginate.

[0051] According to the present invention, the natural or synthetic ionic polymer is chosen from ionically charged polysaccharides such as: hyaluronic acid, chondroitin sulfate, chitosan, alginate, pectin, carrageenan, cellulose, and hemicellulose, preferably alginate.

[0052] According to the present invention, the molar mass of the polymer is between 5,000 and 2,000,000 g / mol.

[0053] According to the present invention, the polymer is alginate, in which the molar mass is from 20,000 to 80,000 g / mol.

[0054] According to the present invention, the crosslinking agent is chosen from compounds comprising acrylate / methacrylate, epoxide, thiol functions, or compounds such as fumarate or bismaleimide, itaconic acid, coumarin.

[0055] According to the present invention, the polymer is at least di-functionalized by acrylate, epoxide, thiol, fumarate or bismaleimide, or acrylate / methacrylic functions.

[0056] According to the present invention, the crosslinked polymer is hyaluronic acid methacrylate, chondroitin sulfate methacrylate, cellulose acrylate, dextran methacrylate, heparin methacrylate, chitosan methacrylate, alginate methacrylate or a combination thereof.

[0057] According to the present invention, the degree of crosslinking is from 80% to 120% per monomer unit of the polymer.

[0058] According to the present invention, the gel rate is from 80% to 95%.

[0059] According to the present invention, the term "gel rate" means the ratio of the mass of the dry hydrogel after washing by the mass of the dry hydrogel post-crosslinking.

[0060] The hydrogel can also be characterized by its mechanical properties, for example the viscoelastic modulus by rheology, or compression modulus measured by a traction / compression device.

[0061] According to the present invention, the crosslinking of the polymer is carried out either homogeneously, without a crosslinking gradient, allowing isotropic three-dimensional three-directional deformation, or heterogeneously, with a crosslinking gradient, allowing an anisotropic three-dimensional three-directional deformation.

[0062] The hydrogel according to the invention makes it possible to achieve controllable three-dimensional three-directional deformation.

[0063] The hydrogel according to the invention is not a single-layer or multi-layer film with only bidirectional deformation, that is to say, for example, by simple folding of a film to form a hollow tube.

[0064] The hydrogel according to the invention comprises a plurality of layers, each layer comprising a network of crosslinked polymers with either a plurality of zones (regions) having different degrees of crosslinking, or a plurality of layers, each layer comprising a zone of crosslinked polymer having a single degree of crosslinking.

[0065] In some embodiments, the shape-transforming hydrogel is ionically crosslinkable and shape transformation is actuated by increasing or decreasing the concentration of ionic crosslinking agent in the shape-transforming hydrogel.

[0066] In some embodiments, the hydrogel comprises a plurality of hydrogel-forming polymer layers wherein at least two of the layers may have different thickness and / or crosslink density. Extension or swelling of a hydrogel

[0067] When a hydrogel in the prepared state is immersed in aqueous solution, the polymer / aqueous solvent interactions are responsible for its more or less significant swelling. Determining the volume variation of the sample makes it possible to characterize on the one hand the macromolecular architecture of the network, fixed during the synthesis, which depends on the initial concentrations of polymer and crosslinking agent.

[0068] The amount of water absorbed by the hydrogel during the immersion time depends on the relaxation rate of the polymer chains relative to the diffusion rate of the solvent towards the center of the hydrogel. Measurement of the swelling rate of hydrogels

[0069] The main characteristic of hydrogels is their ability to swell, i.e. to absorb a greater or lesser quantity of water. At equilibrium, the degree (rate) of swelling DG (in %) is defined as follows:

[0070] DG = mass of swollen hydrogel - mass of dry polymer hydrogel / mass of dry hydrogel x 100 or DG = mass of swollen hydrogel - mass of contracted hydrogel / mass of the contracted hydrogel x 100.

[0071] The degree (rate) of swelling depends essentially on the crosslinking density (chemical or physical), the molar mass of the polymer, the nature of the polymer / polymer interactions, the polymer / solvent interactions, or the drying technique.

[0072] In simplified terms, the swelling process is due to an osmotic pressure gradient. The solvent thus diffuses towards the interior of the network and the latter begins to swell.

[0073] The degree (rate) of swelling is inversely proportional to the crosslinking density and therefore to the elastic modulus. Therefore, hydrogels with high elasticity generally have low swelling rates and vice versa.

[0074] According to the present invention, the mass of the hydrogel can be measured after drying, under vacuum, by lyophilization, or using a supercritical solvent, for example CO 2*

[0075] The mass of the hydrogel is measured at room temperature on a precision analytical balance.

[0076] In general, the stable or equilibrium state of swelling of a hydrogel according to the invention is obtained after immersion in an aqueous solution, at a temperature of 25°C after a few minutes or 1 hour, 6 hours or 12 hours at most. The stable or equilibrium state of swelling of a hydrogel can be verified by carrying out swelling kinetics (When a gel is immersed in an aqueous solution, the water molecules begin by swelling the surface of the hydrogel and then diffuse towards the center of the gel, thus creating a concentration gradient; the equilibrium state is reached when the hydrogel no longer absorbs water and the volume or mass of the swollen hydrogel no longer varies).

[0077] The swelling of the hydrogel according to the invention in an aqueous or humid medium is particularly rapid, for example the degree of swelling within ten minutes following its immersion in an aqueous medium having an osmotic pressure identical to that of biological fluids is between 150% and 600% in a 0.15 M PBS buffer.

[0078] According to a variant of the present invention, the degree (rate) of swelling of an alginate hydrogel (55,000 g / mol) with a methacrylation rate of 120% and a concentration of 6 to 12wt / v% is between 500 and 4000% (depending on the drying technique).

[0079] According to the present invention, it is possible to characterize a hydrogel by an expansion coefficient. This parameter corresponds to a dimensional measurement. For example

[0080] The volume expansion coefficient axy corresponds to the inflated volume / contracted volume or design x 100.

[0081] The volumedesign corresponds to the volume of the hydrogel as defined by the software of design. Contraction of a hydrogel

[0082] According to another aspect of the present invention, the stereoformed hydrogel, of reversible and controllable predetermined shape, and chemically crosslinked is brought into contact with a solution comprising one or more divalent or trivalent cations for a time sufficient to reduce the shape, volume or density of the hydrogel. The hydrogel of the invention passes from a swollen state to a contracted state.

[0083] A contracted hydrogel is a hydrogel that has a reduced shape, density, or volume compared to that of the swollen hydrogel. Preferably, the contracted hydrogel retains the same geometry it had before shrinking, but with reduced dimensions. The contraction of a hydrogel may vary depending on various factors, including, but not limited to, the chemical nature of the polymer (e.g., charge density), its molecular weight, or the electrostatic interactions between the anionic polymer and the divalent or trivalent cations.

[0084] The stable or equilibrium state of contraction of a hydrogel can be obtained after immersing the stereoformed hydrogel, of reversible and controllable predetermined shape, and chemically crosslinked in an aqueous solution with divalent or trivalent cations, at a temperature of 25°C after 1 hour, 6 hours or 12 hours at the most.

[0085] The contraction of the hydrogel according to the invention in an aqueous medium in the presence of divalent or trivalent cations is between 40% and 60%.

[0086] According to a variant of the present invention, the degree (rate) of contraction of an alginate hydrogel (55,000 g / mol) with a methacrylation rate of 120% and a concentration of 6 to 12wt / v% is between 45 and 55%.

[0087] According to the present invention, the one or more divalent cations is (are) chosen from Ca2+, Mg2+, Ba2+, Cu2+, Be2+, ​​Sr2+, Ra2+, Co2+, Ni2+, Zn2+, Cr2+, Cd2+, Mn2+, preferably Ca2+, and the one or more trivalent cations is (are) chosen from Fe3+, Al3+.

[0088] According to the present invention, the physical crosslinking can be carried out by a mixture of several divalent and / or trivalent cations.

[0089] In the present invention, by controlling the concentration and crosslinking time of the divalent or trivalent cation solution in the physical crosslinking process, the contraction degree of the hydrogel can be adjusted, and the concentration of divalent or trivalent cation is 20 to 200 ppm.

[0090] The content of the bivalent or trivalent cation in the hydrogel is from 0.05 g / g of dry polymer to 2 g / g of dry polymer.

[0091] The content of the bivalent or trivalent cation in the hydrogel is determined by isothermal complexometric titration.

[0092] This technique makes it possible to determine the thermodynamics of the interactions between two molecules. The enthalpy linked to the association of cations with the anionic polymer is measured using a microcalorimeter.

[0093] Alternatively, the content of the bivalent or trivalent cation in the hydrogel is determined by inductively coupled plasma mass spectrometry (ICP-MS), or compleximetric assay, or conductimetric assay, or atomic spectroscopy.

[0094] According to another aspect of the present invention, the stereoformed hydrogel, of reversible and controllable predetermined shape, is prepared by stereolithography. Mechanical properties

[0095] The mechanical properties of a hydrogel can vary and be modulated according to the desired application of the material.

[0096] The elastic modulus of a hydrogel is proportional to the crosslinking density and also depends on the nature and energy of the crosslinking bonds of the 3D network.

[0097] The crosslinking agent therefore plays a role in the mechanical behavior of the hydrogel, which becomes more rigid when larger quantities are used following the increase in crosslinking density. Gelling conditions can also affect the mechanical properties of hydrogels. Water is the major constituent of the 3D structure of a hydrogel (up to 90%), which limits their mechanical strength. Preparation methods

[0098] A second aspect of the invention relates to a method for preparing a stereoformed hydrogel, of reversible and controllable predetermined shape, by stereolithography.

[0099] Stereolithography is an additive manufacturing method that typically involves a multi-step process. For example, the first step involves designing and entering a precise mathematical geometric description of the desired structure shape into a computer-aided design (CAD) program and saving the description in STL (Standard Transform Language) file format. In a second step, the STL file is imported into the specific software of the stereolithography device that cuts the design of the object (the hydrogel) layer by layer. By calculating the build parameters, the software of the stereolithography device controls the fabrication of the object (the hydrogel) by a light source (e.g., an ultraviolet laser) sent onto a mirror that will reflect the spatially controlled radiation onto the area to be illuminated.The laser then selectively hardens the liquid photopolymer to form a first layer of the object (the hydrogel). Once the first layer is formed, the stereolithography device moves to the desired thickness to produce the next layer.

[0100] The hydrogel according to the invention is prepared from a functional ionic polymer tionalized by a crosslinking agent by spatially controlled photopolymerization by stereolithography. The chemically crosslinked hydrogel can be ionically crosslinked beforehand.

[0101] Control of the polymerization zones obtained by stereolithography on a physically crosslinked hydrogel makes it possible to obtain varied shapes with variable swelling.

[0102] The geometry and density of polymerization are determined by the distribution and density of the chemical crosslinking nodes.

[0103] The spatial control of the photo-crosslinking gradient obtained during the process of manufacturing the hydrogel by stereolithography makes it possible to anticipate zones with variable photocrosslinking on the same hydrogel and to anticipate morphological changes of a hydrogel.

[0104] Stereolithography allows an object to be formed layer by layer (or voxel by voxel in 2 photons) by chemical crosslinking. The degree of crosslinking can vary from layer to layer (or from voxel to voxel) so as to produce variable crosslinking gradients which allows spatial control of crosslinking in x, y and z, unlike other manufacturing processes. Thus these areas with variable crosslinking will have variable swelling or shrinking behavior inducing variable morphological changes anticipated by the degree of crosslinking obtained by stereolithography.

[0105] The present invention relates to a method for manufacturing a stereoformed hydrogel of reversible and controllable predetermined shape, in particular a doubly crosslinked stereoformed hydrogel of reversible and controllable predetermined shape

[0106] comprising

[0107] a step of chemical crosslinking (photocrosslinking) of an ionic polymer carried out by an additive manufacturing technique by stereolithography, said step consists of solidifying / crosslinking / gelling an ionic polymer, functionalized with a crosslinking agent in the presence of a photoinitiator using a UV laser beam to form layer by layer a chemically crosslinked hydrogel,

[0108] a step of physical crosslinking of an ionic polymer with one or more divalent or trivalent cations

[0109] in which the two steps of chemical and physical crosslinking of an ionic polymer are carried out without order of priority.

[0110] According to the method of the present invention, the ionic polymer, functionalized with a crosslinking agent, is chosen from ionically charged polysaccharides such as: hyaluronic acid, chondroitin sulfate, chitosan, alginate, pectin, carrageenan, cellulose, and hemicellulose, preferably alginate.

[0111] According to the present invention, the molar mass of the polymer is between 5,000 and 2 000 000 g / mol.

[0112] According to the present invention, the polymer is alginate, in which the molar mass is from 20,000 to 80,000 g / mol.

[0113] According to one embodiment of the present invention, the stereoformed hydrogel of reversible and controllable predetermined shape is prepared by a stereolithography technique selected from UV laser scanning stereolithography, volumetric stereolithography, continuous stereolithography, or 2-photon printing, preferably UV laser scanning stereolithography.

[0114] In stereolithography, controlling the thickness of a layer (solidified area) is essential in order to obtain the optimal manufacturing resolution. This parameter is controllable depending on the power of the light, the wavelength used and the irradiation time.

[0115] In practice, a “working curve” is applied to determine the crosslinking thickness.

[0116] This parameter is determined by the following equation: Cd = Dp In (E / Ec)

[0117] with “Cd” the crosslinked thickness (in pm), “Dp” the penetration of the irradiation (in pm) and “E” the energy supplied (in mJ / cm2) (“Ec” minimum solidification energy).

[0118] Different components constitute the formulation to control photopolymerization such as: (a) a photoinitiator, which absorbs light and generates the active species; (b) photosensitive monomers / oligomers containing at least two reactive functions in order to allow photocrosslinking, (c) a reactive or non-reactive diluent which has the role of adjusting the viscosity of the mixture, which is classically recognized to be optimal for values ​​of the order of 1-10 Pa.s.

[0119] The viscosity measurement is determined by rheology.

[0120] The photocrosslinking involved in stereolithography is based on the use of a liquid resin which solidifies by radical or cationic polymerization following exposure to a light source in the presence of a photoinitiator.

[0121] According to the present invention, the wavelengths vary from 365 to 405 nm for the stereolithography process and from 520 to 550 nm for the 2-photon polymerization process.

[0122] According to the present invention, the photoinitiator is chosen from type I photoinitiators (Irgacure 819, Irgacure 1173, Irgacure 2959, TPO Irgacure acyl phosphine oxide derivatives and in particular mono-acylphosphine oxide), type II photoinitiators (Camphorquinone, isopropylthioxanthone, Benzophenone, Ethyldimethylaminobenzoate, 4-(Dimethylamino)benzonitrile, Phenyltetrazole thiol, Eosin, Riboflavin).

[0123] According to one embodiment, the present invention relates to a method of manufacturing a stereoformed, preferably doubly crosslinked, hydrogel of pre-formed shape. determined reversible and controllable, in which no (isotropic) or at least one area having a gradient of crosslinking (anisotropic) of polymer is achieved by controlling the following parameters:

[0124] the irradiation (crosslinking) time,

[0125] the penetration depth (Dp),

[0126] the curing depth (layer thickness),

[0127] critical energy (Ec).

[0128] According to the present invention, the average thickness of a layer of the hydrogel is from 20 to 500 qm for stereolithography, and voxels from 100 x 100 nm to 400 x 400 nm for the 2-photon polymerization process.

[0129] According to a particular embodiment, the present invention relates to a hydrogel formed from a plurality of layers, each layer having an average thickness of from 20 to 500 qm, for example from 50 to 100 qm.

[0130] According to a particular embodiment, the present invention relates to a method for manufacturing a stereoformed hydrogel, preferably doubly crosslinked, of predetermined reversible and controllable shape, in which the chemical crosslinking of a layer of 100 pm requires an irradiation time (365 nm) between 7 and 15 s for a power of 10 mW / cm2.

[0131] According to one embodiment, the present invention relates to a three-dimensional, controllable shaping method, allowing a reversible three-directional transformation of a hydrogel according to the invention, comprising the following steps:

[0132] either

[0133] a) immersing a hydrogel comprising a natural or synthetic ionic polymer, covalently crosslinked by a crosslinking agent, in an aqueous medium or water, to obtain a swollen hydrogel having a state of form or volume Eg having a degree of swelling DGg,

[0134] b) immersing said swollen hydrogel in a solution comprising one or more types of di- or trivalent cations allowing reversible physical crosslinking, after adsorption of said cations, to obtain a contracted, doubly crosslinked hydrogel having a contacted form or volume state Ec having a degree of swelling DGC, and

[0135] c) immersing said contracted hydrogel in a phosphate buffer saline solution (PBS) or an EDTA solution (1% v / v) to obtain a swollen hydrogel after desorption of said cations, substantially recovering its state of form or volume Eg having a degree (rate) of swelling DGg,

[0136] d) subjecting said hydrogel to a succession of n cycles of desorption and adsorption of said cations by the hydrogel,

[0137] said desorptions and adsorptions of said cations being capable of bringing the hydrogel to a swollen state and to return the hydrogel to a contracted state, respectively,

[0138] either

[0139] a) immersing a stereolithographically formed, doubly crosslinked hydrogel of predetermined shape comprising a natural or synthetic ionic polymer, covalently crosslinked by a crosslinking agent, in an aqueous medium or water in the presence of one or more di or trivalent cations allowing reversible physical crosslinking

[0140] to obtain a contracted hydrogel having a state of form or volume Ec having a degree of swelling DGC,

[0141] b) immersing said contracted hydrogel in a phosphate buffer saline solution (PBS) or an EDTA solution (1% v / v) to obtain a swollen hydrogel at equilibrium,

[0142] c) immersing said swollen hydrogel in a solution comprising one or more divalent or trivalent cations allowing reversible physical crosslinking, to obtain a contracted, doubly crosslinked hydrogel substantially recovering its state of form or volume of Ec having a degree of swelling DGC,

[0143] d) subjecting said hydrogel to a succession of n cycles of desorption and adsorption of said cations by the hydrogel,

[0144] said desorptions and adsorptions of said cations being capable of bringing the hydrogel to a swollen state and of bringing the hydrogel back to a contracted state, respectively,

[0145] wherein the degrees of swelling DGg of the hydrogel, obtained before and after a state of contracted shape or volume Ec, have substantially identical values ​​or the degrees of swelling DGC of the hydrogel, obtained before and after a state of inflated shape or volume Eg, have substantially identical values, and / or

[0146] in which the contracted Ec and inflated Eg volume states exhibit three-directional volume changes.

[0147] According to the present invention, the transition between an inflated state Eg and a contracted state Ec is accompanied by a modification of shape or volume in a three-directional manner.

[0148] According to the present invention, the term "the number n of cycles is from 1 to at least 10" means the number n is equal to 1, 2, 3, 4, 5, 6, 7, 9, 10 or more, preferably from 1 to 10, from 10 to 20 or more.

[0149] According to the present invention, the stereoformed hydrogels, preferably doubly crosslinked, of predetermined reversible and controllable shape have the following advantages:

[0150] spatial control of the density, gradient or distribution of chemical crosslinking nodes

[0151] shaping or shaping control by stereolithography

[0152] reversibility or stimulation of the reversibility of physical crosslinking

[0153] swelling or shrinkage due to reversibility of physical crosslinking

[0154] In some embodiments, the stereoformed, particularly doubly crosslinked, reversible and controllable predetermined shape hydrogel may further optionally comprise other components, which are not particularly limited and may be appropriately selected to suit a particular application.

[0155] The invention relates to compositions comprising mammalian cells or medicaments.

[0156] According to a third aspect of the present invention, stereoformed hydrogels, in particular doubly crosslinked, of predetermined reversible and controllable shape are used in medical devices intended to interact with biological tissues. Said hydrogels can also be used for biomedical applications ranging from the formulation of active ingredients, targeted vectorization of drugs to tissue engineering.

[0157] According to other embodiments, the hydrogels of the present invention can be used in osteoconduction by trapping or releasing Ca2+ ions. List of figures [Fig 1]

[0158] [Fig.l] represents a swelling / contraction cycle of a macroporous cubic hydrogel, after immersion in a solution of PBS and CaCl2. [Fig 2]

[0159] [Fig.2] represents a cylindrical gel having crosslinking gradients allowing alternating degrees of swelling after immersion in a PBS solution. [Fig 3]

[0160] [Fig.3] represents the variations in the degree of swelling after several cycles of immersion in a solution of PBS and CaCl2. [Fig 4]

[0161] [Fig.4] represents the variations in the expansion coefficient of 3D printed cylinders at 8 and 10 w / v% methacrylated alginate at different UV penetration depths for a 100 qm layer printing process. Experimental part

[0162] The following examples illustrate the invention. Preparation of a hydrogel

[0163] Low viscosity sodium alginate (0.0136 Pa.s for 1 w / v%) was obtained from Sigma-Aldrich. Methacrylic anhydride (MA), calcium chloride (CaCl2), Tetrasodium ethylenediaminetetraacetic acid dihydrate (EDTA 4Na+, 2H2O), Orange-G, and phosphate-buffered saline (PBS) were purchased from Sigma-Aldrich. Triethylamine (TEA) was purchased from Fisher Chemicals. Milli-Q water (conductivity = 18.2 mQ.cm at 23°C) was used in this study. Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) was synthesized as described by Fairbanks, B. et al. (Biomaterials 2009, 30 (35), 6702-6707). All chemicals were used without further purification unless otherwise stated. Chemical modification of sodium alginate

[0164] Methacrylated alginate was obtained by reacting low viscosity Alg-Na+ with methacrylic anhydride. A 4% w / v aqueous Alg-Na+ was prepared by dissolving 12 g of sodium alginate in 300 mL of milli-q water in a 1 L round-bottom flask under mechanical stirring (600 rpm). In order to obtain a homogeneous mixture, the solution was allowed to stir for 4 hours. TEA was then added at 40 molar equivalents relative to methacrylic anhydride and the solution was allowed to stir for 30 minutes to ensure complete homogenization. Finally, methacrylic anhydride was added at 20 molar equivalents relative to the sugar unit (guluronic or mannuronic). The reaction was maintained at 24°C for 24 hours. Functionalized Alg-Na+ was obtained by precipitating the mixture in cold acetone followed by drying at 24°C under reduced pressure (103 mbar). 1H NMR characterization

[0165] In order to quantify the degree of modification (DM), 1H NMR was used. 20 mg of Alg-MA were dissolved in 1 mL of D2O and left stirring for 1 hour to obtain a homogeneous mixture. 1H NMR spectra were recorded on a Bruker Avance I 400 MHz (64 scans, 5 s relaxation time). The degree of modification, defined as the number of methacrylates per sugar unit (guluronic or mannuronic), was calculated as the ratio between the protons of the methacrylate (1.95 ppm, 5.8 ppm and 6.2 ppm) and the proton HGi of the alginate using the following equation: DM(%)=G% x 1H vinyl / 1h g

[0166] where IH represents the intensity of a vinyl proton of the grafted methacrylate at 6.2 ppm, represents the intensity of the anomeric protons of the guluronic or mannuronic moieties and G% represents the fraction of guluronic units in the polysaccharide. The G% is calculated by comparing the anomeric proton G at 5.8 ppm with the anomeric proton M at 4.47 ppm7wc1 Resin formulation and 3D printing

[0167] All structures were constructed by stereolithography using a digital light processing (DLP) printer (Max X27, Asiga Australia). The resins were prepared by dissolving appropriate amounts of Alg-MA to obtain 6, 8, and 10% w / v aqueous solutions. LAP was added at 3.75 w / w% relative to alginate and Orange G at 0.2 w / v% as a photoabsorber. Orange G was added to control the penetration depth of UV light (365-405 nm) for high-resolution printing. The UV intensity was set at 10 mW / cm2 and the layer thickness at 100 qm for all samples. Isotropic printing

[0168] Cylinder diameter 8mm and 4mm thickness were printed. Irradiation for each formulation is summarized in Table 1.

[0169] [Tables 1] Penetration depth 100 uni 300 uni 400 uni 8% w / v 6.2 20 35 10% w / v 4 12 20 Anisotropic printing

[0170] In order to achieve anisotropic swelling behavior, a crosslinking gradient is implemented. To maintain proper resolution, the gradient starts with the crosslinking time and decreases to a thickness of 100 qm.

[0171] Photopolymerizable alginate resin at a concentration of 8% w / v is used in stereolithography to manufacture three-dimensional objects such as porous cubes ([Fig.l]) or hollow cylinders ([Fig.2]). For each structure, the irradiation times are homogeneous and determined by the "working curve" to give objects that will generate isotropic transformations. Crosslinking gradients obtained by evolving irradiation times (for example, a 6mm zone composed of several 100 micron layers, each layer being formed after an irradiation time of 8s, a 7mm zone composed of several 100 micron layers, each layer being formed after an irradiation time of 12s, and a 7mm zone composed of several 100 micron layers, each layer being formed after an irradiation time of 20s) were established in order to generate transformation anisotropy.A predetermined area is crosslinked over an irradiation time corresponding to the desired degree of swelling: for example progressive swelling ( [Fig.l], Figure 2a) or alternating swelling (Figure 2b). Swelling kinetics.

[0172] To monitor the swelling kinetics of hydrogels in water, a washed dry hydrogel disc was placed in 40 ml of Milli-Q water. The hydrogel was then weighted at different time intervals after removing excess water with a wet KimTech paper. Between measurements, the gel was allowed to swell in a room at 24 ± 1°C. The swelling degrees (SDG) were measured.

[0173] The results obtained are presented in [Fig.3]. Cyclical swelling

[0174] Equilibrium cyclic swelling degrees were measured from the washed dry hydrogels. The cycles were between PBS and 5% wt / v CaCl2 to mimic physiological conditions. For each measurement, the hydrogel was allowed to swell for 24 h in a 24 ± 1°C room. The swelling degree was calculated. Values ​​are represented as the average of triplicate sets. Compression tests

[0175] Compression tests of the swollen hydrogels were performed on an Instron 3366L5885 mechanical tester equipped with a 100N load cell. Cylindrical samples of 10 x 4 mm were prepared in a Teflon mold as previously described, washed, dried and then allowed to swell in water. Hydrogels were sampled at different points of the cyclic swelling (the hydrogel was removed either from the swelling medium (PBS) or from the shrinking medium (CaCl2) and the measurement of the swelling rate was made at several times t and then reintroduced into the medium until the next point t+1) of the cycle and the compression tests were performed after measuring the dimensions using a caliper. The compression speed was set at 1 mm.s-1 with a sampling time of 200 ms. Each value is represented by the mean ± SD (n = 3) Swelling behavior

[0176] In order to study the influence of the degree of crosslinking on the hydrogel behaviors, gravimetric swelling tests were performed on 8x4mm printed cylinders. The printing layer thickness and UV intensity were set at 100 qm and 10 mW / cm2 for all samples respectively. However, different irradiation times were chosen when the penetration depth was 100, 300 and 400 qm. When the penetration depth is greater than the layer thickness, excessive crosslinking may occur. To evaluate the shape transformation during double crosslinking, the expansion coefficient was measured for all samples.

[0177] Swelling / contraction cycle of photocrosslinked alginate hydrogel in the presence of Ca2+

[0178] The absorption / desorption cycles were performed in water using 3 hydrogel discs fabricated by stereolithography. The absorption cycles are achieved in PBS solution while desorption and contraction are achieved once a Ca2+ solution is applied. PBS acts as a decomplexer due to the presence of Na+ ions. Specifically, each hydrogel was immersed in 10 mL of PB S solution at room temperature for 24 h to reach the equilibrium swelling state and then weighed.

[0179] The measurements were made on hydrogels dried under vacuum (10-3 bar, room temperature, for 24 h). The masses are measured at room temperature on a precision analytical balance.

[0180] Then, the samples were alternately immersed for 24 h in 10 ml of CaCl2 solution and 24 h in 10 ml of PBS solution. Between each immersion, disc-shaped hydrogels were weighed and the measurements of the expansion coefficient or swelling degree are reported on graphs (Figures 3 and 4). The weight variation of the photocrosslinked alginate hydrogels during the swelling cycles decreases considerably after immersion on the Ca2+ solution, with a mass loss of about 80%. However, soaking in the PBS solution allows the hydrogel to absorb water again close to its initial state. The results between the samples show good reproducibility of the experiment and also exhibit significant stability after several cycles, which means that the Ca2+ crosslinking does not degrade the hydrogel structure.It is also interesting to note that the swelling properties of these hydrogels are directly related to the proportion of methacrylated functions. The higher this proportion, the more crosslinked the gel will be after UV irradiation and therefore the less its network will be able to expand when water is absorbed. In short, the absorption capacity of a hydrogel is inversely proportional to its methacrylation rate. The stress applied to an ionically crosslinked alginate gel relaxes when the crosslinks dissociate and water is expelled from the gel, resulting in plastic deformation. Conversely, water migration in covalently crosslinked gels leads to stress relaxation, and the inability to dissociate and reform the bonds generates significant elastic deformation. In comparison, photocrosslinking allows the creation of alginate hydrogels independent of calcium levels.Photocrosslinking by stereolithography is also a convenient way to control the timing and kinetics of gelation. Based on calcium ion crosslinking, the addition of photocrosslinking can form hydrogels with more compact macromolecular chains and avoids the destruction of ionic crosslinked microfibers in physiological solution.

[0181] These physical properties could be adjusted by varying the extent of methacrylation of the alginate. Furthermore, within the scope of this invention it is shown that ionic crosslinking with Ca2+ can contract the hydrogel network to remove water. Thus, it is possible to have complete control over the absorption and desorption of this system, which can then be valuable for the molecule delivery system controlled. Alginate hydrogels are stable after multiple cation absorption / desorption cycles, allowing them to be used for various long-term applications. The materials developed in this study can be processed through additive manufacturing using 3D stereolithography. This allows precise control of the shape and morphology required for different drug delivery and medical device applications. In addition, the 3D printed object can recover a memorized shape through ion stimulation for what is now considered 4D printing.

Claims

Claims

1. A stereoformed hydrogel, of reversible and controllable predetermined shape, comprising at least a first component, a second component and optionally a third component wherein: the first component is an ionic natural or synthetic polymer, covalently crosslinked by a crosslinking agent, the second component is an aqueous medium or water, and the third component is one or more divalent or trivalent cations, said hydrogel has either a swollen state of shape, density or volume Eg having a degree of swelling DGg, wherein said hydrogel is devoid of divalent or trivalent cations, or a contracted state of shape, density or volume Ec having a degree of swelling DGC, wherein said hydrogel comprises one or more types of divalent or trivalent cations, and wherein the degrees of swelling DGg of the hydrogel, obtained before and after a contracted state Ec,or the degrees of swelling DGC of the hydrogel, obtained before and after a swollen state Eg, have substantially identical values ​​and / or in which the contracted volume states Ec and swollen Eg have three-directional volume changes.,

2. A stereoformed hydrogel of reversible and controllable predetermined shape according to claim 1, wherein the polymer is selected from ionically charged polysaccharides such as: hyaluronic acid, chondroitin sulfate, chitosan, alginate, pectin, carrageenan, cellulose, and hemicellulose.

3. A stereoformed hydrogel of reversible and controllable predetermined shape according to claim 2, wherein the molar mass of the polymer is from 5,000 to 2,000,000 g / mol.

4. Stereoformed hydrogel of reversible and controllable predetermined shape according to any one of claims 1 to 3, in which the polymer is alginate, preferably with a molar mass of 20,000 to 80,000 g / mol.

5. Stereoformed hydrogel of reversible and controllable predetermined shape according to claim 1, in which the crosslinking agent is chosen from compounds comprising acrylate / methacrylate, epoxide, thiol functions, or compounds such as fumarate or bismaleimide, itaconic acid, coumarin.

6. A reversible and controllable predetermined shape stereoformed hydrogel according to claim 1, wherein the degree of functionalization is from 80% to 120% per monomer unit of the polymer.

7. Stereoformed hydrogel of reversible and controllable predetermined shape according to claim 1, wherein the one or more divalent cations is (are) selected from Ca2+, Mg2+, Ba2+, Cu2+, Be2+, ​​Sr2+, Ra2+, Co2+, Ni 2+, Zn2+, Cr2+, Cd2+, Mn2+ preferably Ca2+, and the one or more trivalent cations is (are) selected from Fe3+, Al3+.

8. A stereoformed hydrogel of reversible and controllable predetermined shape according to claim 1, wherein the hydrogel is formed of a plurality of layers, each layer having an average thickness of 20 to 500 qm, for example 50 to 100 qm.

9. Stereoformed hydrogel of reversible and controllable predetermined shape according to claim 1, obtained or obtainable by stereolithography.

10. A method for three-dimensional, bidirectional, controllable, reversible deformation of a hydrogel according to one of claims 1 to 9 comprising the following steps: either a) immersing a hydrogel comprising a natural or synthetic ionic polymer, covalently crosslinked by a crosslinking agent, in an aqueous medium or water, to obtain a swollen hydrogel having a state of shape or volume Eg having a degree of swelling DGg, b) immersing said swollen hydrogel in a solution comprising one or more types of di- or trivalent cations allowing reversible physical crosslinking, after adsorption of said cations, to obtain a contracted, doubly crosslinked hydrogel having a state of shape or volume contacted Ec having a degree of swelling DGC, and c) immersing said contracted hydrogel,in a phosphate buffer saline (PBS) solution or an EDTA solution (1% v / v) to obtain a swollen hydrogel after desorption of said cations, substantially recovering its state of form or volume Eg having a degree (rate), of DGg swelling, d) subjecting said hydrogel to a succession of n cycles of desorption and adsorption of said cations by the hydrogel, said desorption and adsorption of said cations being capable of bringing the hydrogel to a swollen state and of returning the hydrogel to a contracted state, respectively either a) immersing a stereolithographically formed, doubly crosslinked hydrogel of predetermined shape comprising a natural or synthetic ionic polymer, covalently crosslinked by a crosslinking agent, in an aqueous medium or water in the presence of one or more di or trivalent cations allowing reversible physical crosslinking to obtain a contracted hydrogel having a state of shape or volume Ec having a degree of swelling DGC, b) immersing said contracted hydrogel in a phosphate buffer saline solution (PBS) or an EDTA solution (1% v / v) to obtain at equilibrium a swollen hydrogel, c) immersing said swollen hydrogel in a solution comprising one or more divalent or trivalent cations allowing reversible physical crosslinking, to obtain a contracted, doubly crosslinked hydrogel substantially recovering its state of form or volume of Ec presenting a degree of swelling DGC, d) subjecting said hydrogel to a succession of n cycles of desorption and adsorption of said cations by the hydrogel, said desorptions and adsorptions of said cations being capable of bringing the hydrogel to a swollen state and of returning the hydrogel to a contracted state, respectively, wherein the degrees of swelling DGg of the hydrogel, obtained before and after a state of contracted shape or volume Ec, have substantially identical values ​​or the degrees of swelling DGC of the hydrogel, obtained before and after a state of swollen shape or volume Eg, have substantially identical values, and / or wherein the states of contracted volume Ec and swollen volume Eg have three-directional volume changes.

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