How to cure alginate / gelatin hydrogels

Abstract

The present invention relates to a method for hardening a hydrogel comprising alginate and gelatin, comprising a hardening step by crosslinking said hydrogel with a suitable solution, in order to give said hydrogel particularly advantageous mechanical properties. The use of this crosslinking solution for hardening the structure of a hydrogel comprising alginate and gelatin, and the hydrogel obtained by this method are also another aspect of the present invention. The present invention also relates to a method for hardening a hydrogel comprising alginate and gelatin, comprising preparing the hydrogel, shaping it, and then contacting it with a suitable hardening agent. The hydrogel obtained by this method is also another aspect of the present invention. In all these aspects of the present invention, the hydrogel may further comprise fibrinogen and / or viable cells.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates generally to the field of materials manufacturing, and in particular to biomaterials or biocompatible materials.

[0002] The present invention relates to a method for hardening a hydrogel comprising alginate and gelatin, comprising a hardening step by crosslinking the hydrogel with a suitable solution, in order to give it particularly advantageous mechanical properties. The use of this crosslinking solution for hardening the structure of a hydrogel comprising alginate and gelatin also constitutes another aspect of the present invention. The hydrogel obtained by this method is also another aspect of the present invention.

[0003] The present invention also relates to a method for hardening a hydrogel comprising alginate and gelatin, comprising preparing the hydrogel, shaping it, and then contacting the hydrogel with at least one divalent cation, preferably calcium, and transglutaminase. The hydrogel obtained by this method is also another aspect of the present invention.

[0004] In all embodiments of the present invention, the hydrogel may further comprise fibrinogen and / or viable cells. [Background technology]

[0005] 2. Background of the Invention Structures based on hydrogels, including alginate and gelatin, are known from the prior art, but these components often have limited elasticity (in particular a low Young's modulus), which means they lack sufficient mechanical strength, making the structures obtained difficult to handle.

[0006] International patent application published under number W02017115056 describes the production of a hydrogel-based body substitute comprising alginate, gelatin and fibrinogen, which is then treated with a cross-linking solution containing calcium and thrombin.

[0007] Nevertheless, the mechanical properties of the structures obtained in the prior art are still insufficient to make them suitable for being manipulated. In addition, in the case of structures intended to be implanted in the human or animal body and, if necessary, sutured, the methods of the prior art do not make it possible to obtain structures with suitable mechanical properties, the degradation of which, upon contact with living cells or tissues, is not very rapid.

[0008] Moreover, if the structure is likely to be cellularized during manufacture, it is necessary that it is capable of maintaining cell viability, and therefore the methods for preparing such structures must be compatible with the presence and maintenance of viable cells. Summary of the Invention [Problem to be solved by the invention]

[0009] overview One of the aims of the present invention is to overcome these drawbacks of the prior art and to make it possible to produce cellularized or non-cellularized hydrogels having particularly advantageous and innovative characteristics, in particular with regard to (i) the mechanical strength of the components, (ii) their stability over time, (iii) their flexibility, (iv) their tear and impact resistance, and (v) their colonization by cells.

[0010] To this end, according to a first embodiment of the invention, it is proposed to provide a method for hardening a hydrogel comprising alginate and gelatin, comprising preparing a hydrogel and then contacting it with a hardening solution comprising at least one divalent cation, preferentially calcium, a transglutaminase and optionally one or more other divalent cations.

[0011] Optionally, if the hydrogel contains fibrinogen in addition to alginate and gelatin, the hardening solution may also contain thrombin. Moreover, the hardening step is compatible with the fact that the hydrogel may contain viable cells.

[0012] According to another aspect of the invention, the use of the previously described solution for hardening hydrogel structures comprising alginate and gelatin is described, which solution has particular advantages when preparing hydrogels intended to be implanted in the human or animal body, i.e. internal implants.

[0013] This use may also be used to harden hydrogels further comprising fibrinogen. Moreover, hardening may also be performed on hydrogels further comprising viable cells.

[0014] In summary, the present invention relates to a method for hardening a hydrogel comprising alginate and gelatin, comprising preparing a hydrogel and contacting it with a hardening solution comprising at least one divalent cation, preferentially calcium, a transglutaminase and optionally one or more other divalent cations. [Means for solving the problem]

[0015] Preferably, contacting the hydrogel with the curing solution is carried out by immersion, preferably total immersion, of the hydrogel in the curing solution or by spraying the curing solution onto the hydrogel.

[0016] Preferably the divalent cation(s) is selected from the group including calcium, strontium and barium.

[0017] Preferably the hydrogel comprises 0.5-3% alginate and 1-17.5% gelatin.

[0018] Preferably, the hydrogel further comprises fibrinogen, in which case the setting solution further comprises thrombin.

[0019] Preferably the hydrogel contains up to 2% fibrinogen.

[0020] Preferably the hardening solution is: - 0.003 to 30% divalent cations; - 0.004-16% transglutaminase and optionally - 2-25U / ml thrombin Includes.

[0021] Preferably, the hydrogel further comprises viable cells that are incorporated during the hydrogel preparation prior to its hardening.

[0022] Preferably, the contact with the hardening solution comprises: - at temperatures between 15 and 40°C, and / or - For a period ranging from 30 minutes to 6 hours It will be executed.

[0023] Preferably, the hydrogel is shaped before its hardening, preferentially said shaping is carried out by extrusion of the material, even more preferentially said shaping is carried out by moulding or by additive manufacturing techniques.

[0024] According to another aspect, the present invention also relates to the use of a solution comprising a divalent cation, preferably calcium, transglutaminase and optionally one or more other divalent cations, for hardening a hydrogel comprising alginate and gelatin.

[0025] According to another aspect, the invention also relates to a hydrogel comprising hardened alginate and gelatin intended to be implanted in a human subject, the hydrogel being obtainable by the hardening method previously described.

[0026] According to a second embodiment of the invention, there is provided a method for hardening a hydrogel comprising alginate and gelatin, comprising the following steps: - preparation of a hydrogel, - shaping the prepared hydrogel; - contacting the hydrogel with at least one divalent cation, preferably calcium, and transglutaminase, and optionally one or more other divalent cations. It is also proposed to provide a method comprising, in sequence:

[0027] Preferably, preparation of the hydrogel does not require shaping of one or more components prior to preparation of the hydrogel.

[0028] Preferably, the preparation of the hydrogel does not require the addition of fibers.

[0029] Preferably the divalent cation(s) is selected from the group including calcium, strontium and barium.

[0030] According to another aspect, the invention relates to a hydrogel comprising hardened alginate and gelatin intended to be implanted in a human subject, the hydrogel being obtainable by the hardening method previously described.

[0031] definition For purposes of the present invention, the following terms are defined as follows: - "crosslinking agent" in the context of the present invention refers to an agent capable of crosslinking the components of the hydrogel, in particular alginate, gelatin and fibrinogen. - "Simultaneous" in the context of the present invention refers to events occurring at the same time. Thus, simultaneous contacting of a hydrogel with calcium and transglutaminase means that a hydrogel comprising alginate and gelatin is contacted with calcium and transglutaminase at the same time, thus allowing simultaneous cross-linking of alginate and gelatin. - "fiber" in the context of the present invention refers to any element of thread-like appearance, generally in the form of a bundle. Examples of fibers are given below: - "Shaping" in the context of the present invention consists in giving a particular shape, structure and / or configuration to a shaped element. If it is the shaping of a hydrogel, this consists, for example, in giving the hydrogel a particular shape, structure and / or configuration that is particularly suited to the final goal of the hardened hydrogel. If it is the shaping of one or more components of a hydrogel during its preparation, this consists, for example, in giving said components a particular structure during the hydrogel preparation, such as a structure in the form of fibers. - "Crosslinking" or "hardening" in the context of the present invention are equivalent terms and refer to the fact of hardening the hydrogel by crosslinking the alginate, gelatin and / or fibrinogen. - "Sequentially" in the context of the present invention refers to events that do not occur at the same time. Thus, sequential contact of a hydrogel with calcium and transglutaminase means that the hydrogel is contacted with calcium and then with transglutaminase, or with transglutaminase and then with calcium. - "hardening solution" or "crosslinking solution" in the context of the present invention are equivalent terms and refer to the solution used in the method according to the invention to harden the hydrogel by crosslinking.

[0032] Detailed Description The present invention provides a method for preparing hydrogels comprising cellularized or non-cellularized alginate and gelatin. By crosslinking the hydrogel components with at least one divalent cation, preferably calcium, and transglutaminase, the structure of the hydrogel is hardened, giving the hydrogel particularly advantageous mechanical properties, especially with regard to the mechanical strength of the components, their stability over time, and their remarkable tear and impact resistance.

[0033] According to a first embodiment, the present invention relates to a method for hardening a hydrogel comprising alginate and gelatin, comprising preparing a hydrogel and then contacting said hydrogel with a hardening solution comprising at least one divalent cation, preferentially calcium, a transglutaminase and optionally one or more other divalent cations.

[0034] In the context of the present invention, said hardening solution can be obtained by alternative but equivalent methods: it can be obtained by adding the various elements, i.e. at least one divalent cation, preferably calcium, and transglutaminase, and optionally one or more other divalent cations, in the same solution, or by mixing at least two solutions, i.e. a solution comprising at least one divalent cation, preferably calcium, and optionally one or more other divalent cations, and a solution comprising at least one transglutaminase.

[0035] Alginate is a linear polysaccharide extracted from marine algae, mainly from species of the Phaeophyceae family, Brown algae. This biocompatible polymer is composed of homopolymer blocks of the acid 1,4-β-D mannuronic acid (M) and its epimer acid, C-5α-L guluronic acid (G). The biopolymer consists of sequences of M and G blocks intercalated with sequences of MG blocks. Only the G units appear to participate in intermolecular cross-linking during polymerization. Sodium alginate is widely used as a hydrogel.

[0036] Gelatin is a collagen-derived polymer that contains bioactive sequences such as the RGD (arginine-glycine-aspartic acid) motif for cell adhesion. It is obtained by denaturing the natural triple helical structure of collagen through acid treatment (type A gelatin) or alkali (type B gelatin). The amino acid composition of gelatin is similar to that of collagen, but differs from that of collagen after denaturation (deamination of glutamine to glutamic acid during the process of making type B gelatin). The structure of gelatin changes during gelation.

[0037] The preparation of hydrogels (E.M. Ahmed; Journal of Advanced Research, 2015, 6, 105-121), as well as the polymerization and cross-linking of alginate and gelatin (Chen Q, Tian X, Fan J, Tong H, Ao Q, Wang X An Alginate / Gelatin Network for Three-Dimensional (3D) Cell Cultures and Organ Bioprinting. Molecules. 2020; 25(3): 756.) are well known in the art.

[0038] With respect to alginates and based on the above, alginates rich in M ​​units are more flexible since the chains have a more linear configuration, whereas gels containing more G units will be more rigid since they are more polymerized. In the context of the present invention, the alginates used have an M / G ratio, for example, between 1 and 2, in particular between 1 and 1.9, or between 1 and 1.5. In the context of the present invention, the alginates used have an M / G ratio of, for example, 1.9.

[0039] Preferably the gelatin contained in the hydrogel is of type A.

[0040] In the context of the present invention, the hydrogel is preferably prepared from an alginate solution to which a gelatine solution has been added, or vice versa, to obtain a hydrogel comprising 0.5-3% alginate and 1-17.5% gelatine, more preferably 1-2.5% alginate and 2-10% gelatine. Advantageously, the hydrogel comprises 2% alginate and 5% gelatine.

[0041] Unless otherwise indicated, percentages referred to herein are expressed by weight / volume of the total composition.

[0042] Preferably, when the hydrogel to be cured contains alginate and gelatin, these components are present in a weight ratio within the range of 1:0.3 to 1:35, respectively, most particularly in a weight ratio of 1:2.5.

[0043] The hardening solution preferably comprises: - 0.003-30% divalent cation(s); - 0.004 to 16% transglutaminase; Includes.

[0044] More preferably the hardening solution contains 1-6% divalent cation(s), advantageously 3% divalent cation(s).

[0045] More preferably the hardening solution contains 1-10% transglutaminase, advantageously 4% transglutaminase.

[0046] Transglutaminase enzymes (TAGs) are extracellular aminoacyltransferases. They are monomeric proteins that contain a single cysteine ​​catalytic residue (active site).

[0047] In the context of the present invention, the hardening solution preferably contains type 2 transglutaminase. This TAG is commercially produced as a recombinant microbial protein by fermentation of the microorganism Streptoverdicillium mobaraense. The hardening solution used in the context of the present invention may also contain multiple TAGs.

[0048] Any other divalent cation, preferably a non-toxic divalent cation, can be used in the context of the method according to the invention.

[0049] For example, the divalent cation(s) is selected from the group including or consisting of calcium, strontium, barium, zinc, copper, iron and nickel. Preferably, the divalent cation(s) is selected from the group including or consisting of calcium, strontium and barium. Preferably, the divalent cation is calcium, but may be strontium or barium.

[0050] It is also possible to envisage the use of multiple divalent cations in the mixture. Moreover, the divalent cation(s) are present as salts in the hardening solution. Any salt, preferably anhydrous salts, can be used.

[0051] Preferably the hardening solution contains only divalent cations, preferably potassium and transglutaminase. Advantageously, if one or more divalent cation(s) other than calcium are present, it will preferably be a single other divalent cation, in particular barium.

[0052] Preferably the hardening solution contains calcium chloride as the only divalent cation.

[0053] According to the present invention, simultaneous cross-linking of the hydrogel components, i.e., conversion from linear polymers to three-dimensional polymers, is carried out by contacting the hydrogel components with a hardening solution in which (i) divalent cations, preferably calcium or other divalent cation(s), enable cross-linking of the alginate, and (ii) transglutaminase induces enzymatic cross-linking of the gelatin.

[0054] Preferably, during the curing step according to the invention, the contacting of the hydrogel with the curing solution is carried out by immersion, during which the hydrogel is entirely immersed in the curing solution, which is also referred to herein as a consolidation bath. The contacting of the hydrogel with the curing solution can also be carried out by spraying, by imbibition, by means of a drip, trickle or similar system. The contacting preferably refers to the entire hydrogel. Preferably, the hydrogel is entirely immersed in the curing solution.

[0055] The hydrogels cured according to the present invention may further comprise fibrinogen in addition to alginate and gelatin.

[0056] The fibrinogen monomer is composed of three α, β and γ chains linked by a central E domain and two repeats of fibrinopeptides A and B (FpA, FpB) that link the α chains to the E domain. It has multiple cell adhesion motifs and can therefore increase cell development within the hydrogel.

[0057] Preferably, the hydrogels prepared for hardening by the method of the present invention are composed of alginate and gelatin, or alginate, gelatin and fibrinogen, without any other components capable of forming a gel.

[0058] Preferably, when the hydrogel comprises fibrinogen in addition to alginate and gelatin, it is prepared to contain up to 6% fibrinogen, particularly 0.0001% to 6% fibrinogen, especially 2% fibrinogen.

[0059] Also preferably, when the hydrogel contains alginate, gelatin and fibrinogen, these components are present in a weight ratio ranging from 1:0.3:0.00003 to 1:35:12, respectively, most particularly in a weight ratio of 1:1:2.5.

[0060] When the hydrogel contains fibrinogen in addition to alginate and gelatin, the hardening solution used in the method according to the invention further comprises thrombin, i.e. in addition to calcium and TAG, and optionally one or more other divalent cations, it comprises thrombin.

[0061] In the context of the present invention, said hardening solution further comprising thrombin can be obtained by alternative but equivalent methods.The hardening solution can be obtained by adding different elements, namely at least one divalent cation, preferentially calcium, transglutaminase, thrombin and optionally one or more other divalent cations, to the same solution, or by mixing at least three solutions, namely a solution comprising at least one divalent cation, preferably calcium and optionally one or more other divalent cations, a solution comprising at least transglutaminase and a solution comprising thrombin.It is also possible to incorporate thrombin into the solution comprising at least one cation or into the solution comprising transglutaminase. In this case, the hardening solution can be obtained by mixing at least two solutions: i) a solution comprising at least one divalent cation, preferably calcium and optionally one or more other divalent cations and thrombin, with a solution comprising transglutaminase; or ii) a solution comprising at least one divalent cation, preferably calcium and optionally one or more other divalent cations, with a solution comprising transglutaminase and thrombin.

[0062] In addition to those mentioned above for cross-linking of alginate and gelatin, thrombin in this case cross-links fibrinogen to fibrin, a natural biological polymer resulting from polymerization that mimics the final step of the coagulation cascade when thrombin acts on fibrinogen. Thrombin first cleaves fibrinopeptide A, leading to the formation of protofibrils. Cleavage of fibrinogen B leads to the release of the α-chain and then side chain polymerization of fibrinogen to form fibrin.

[0063] When the hydrogel cured by the method of the present invention contains fibrinogen, the curing solution used contains thrombin in addition to calcium and TAG, and optionally other divalent cations. In this case, and preferably, the curing solution contains: - 2-25 U / ml thrombin; - 0.003-30% divalent cation(s); - 0.004 to 16% transglutaminase; Includes.

[0064] As mentioned above, more preferably the hardening solution comprises 1-6% divalent cation(s), advantageously 3% divalent cation(s).

[0065] More preferably the hardening solution contains 1-10% transglutaminase, advantageously 4% transglutaminase.

[0066] More preferably, when the setting solution further comprises thrombin, the thrombin is preferably present at 2-10 U / ml.

[0067] More preferably, the hardening solution contains 4 U / ml thrombin, 3% calcium, and 4% transglutaminase.

[0068] In the context of the present invention, the alginates, gelatines and, optionally, fibrinogens used are selected from those having characteristics most similar to the following: - Alginates: viscosity of 130-300 mPa.s in a 2% solution; - Gelatin: Type A, porcine, Bloom value 280 (strength or resistance to depression); - Fibrinogen: Human, clottable protein level ≥ 91mg / mL; - Thrombin: human, activity ≥ 500 U / ml.

[0069] The hydrogel advantageously contains alginate, gelatin, and optionally fibrinogen as natural components of the hydrogel.

[0070] However, other naturally occurring components such as chitin, chitosan, cellulose, agarose, chondroitin sulfate, hyaluronic acid, glycogen, starch, pullulan, carrageenan, hepatin, collagen, albumin, fibrin, fibroin, dextran, xanthan, gellan, as well as any components extracted from the extracellular matrix such as collagen, laminin, Matrigel-type proteoglycan, GelMa-type methacrylate gelatin may or may not be present within the hydrogel.

[0071] In addition to the natural components and especially those listed above, the hydrogels of the present invention may or may not also contain synthetic components such as polyolefins (PE, PP, PTFE, PVC), silicones (PDMS), polyacrylates (PMMA, pHEMA), polyesters (PET, Dacron, PGA, PLLA, PLA, PDLA, PDO, PCL), polyethers (PEEK, PES), polyamides, polyurethanes, PEG, Pluronic F127.

[0072] Textile fibers of natural or synthetic origin may or may not be present in the hydrogel composition.

[0073] Examples of fibers of natural origin include, but are not limited to, cellulosic fibers.

[0074] Examples of synthetic fibers include, but are not limited to, polyester fibers, nylon fibers, polyethylene fibers, polypropylene fibers, and acrylic fibers.

[0075] The hydrogel may further comprise viable cells.

[0076] In the context of the present invention, said hydrogel may comprise alginate, gelatin, optionally fibrinogen, and optionally viable cells.In the context of the present invention, said hydrogel may consist of alginate, gelatin, optionally fibrinogen, and optionally viable cells.

[0077] These viable cells may be of any type, except human embryonic stem cells obtained by destruction of the embryo, and preferably several types of viable cells may coexist. The viable cells are preferably selected from cells of epithelial, connective, adipose, endothelial tissue, and in particular from fibroblasts, keratinocytes, stem cells of adipose tissue, adipocytes, melanocytes, endothelial cells, macrophages, leukocytes, etc. In this implementation of the method of the invention, the cells are therefore manipulated under conditions that can be determined by the skilled person in order to maintain viability and proliferation, and ideally differentiation.

[0078] These cells can be incorporated during the preparation of the hydrogel before its hardening, when alginate, gelatin and optionally fibrinogen solutions are mixed to prepare the hydrogel. In that case, it is called cellularized hydrogel. They can also be added to the hardening solution or after the hydrogel hardens. Preferably, the hydrogel hardened according to the present invention comprises viable cells, which are incorporated during the preparation of the hydrogel before its hardening. For example, viable cells can be suspended in a fibrinogen solution, to which alginate and gelatin are added in one or more steps, to obtain a hydrogel containing the amount of alginate / gelatin / fibrinogen mentioned above.

[0079] To illustrate different implementations of the present invention, the following sequence is preferably used to prepare a hydrogel that is cured by the method of the present invention: (i) adding alginate to a viable cell suspension followed by addition of gelatin; (ii) adding alginate to fibrinogen followed by gelatin; (iii) suspending viable cells in fibrinogen, followed by addition of alginate, followed by addition of gelatin. Advantageously, sequence (iii) is used to prepare the cellularized hydrogel according to the invention.

[0080] It is also possible that the hydrogel does not contain viable cells and is therefore acellular.

[0081] In these particular sequences, the priorities of the ranges of each of the components mentioned above also apply. Whether the components are alginate and gelatin, and optionally fibrinogen and / or viable cells, it is also possible to prepare the hydrogel to be cured in a single step, i.e. by mixing all the components at the same time. Preferably, the curing step, which consists in contacting the hydrogel with the curing solution, is carried out at a temperature in the range of 15-40°C, preferably 20-40°C, even more preferably 21-37°C.

[0082] Preferably, this curing step is carried out for a period ranging from 10 minutes to 6 hours, preferentially from 30 minutes to 6 hours, and ideally from 1 hour to 3 hours.

[0083] Thus, preferably the curing step is carried out at 37° C. for 1 hour and 30 minutes.

[0084] Preferably the hydrogel consists of alginate and gelatin or of alginate, gelatin and fibrinogen and the hardening step is carried out at 37° C. for 1 hour and 30 minutes.

[0085] Preferably, the hydrogel consists of alginate and gelatin or of alginate and gelatin and fibrinogen, and the hardening step is carried out by total immersion of the hydrogel in the hardening solution at 37° C. for 1 hour and 30 minutes.

[0086] In the context of the curing method of the present invention, the latter may include a step of shaping the hydrogel after preparation and prior to its curing.

[0087] Thus, the hydrogels described in detail above can be prepared and then shaped before hardening by various methods known to those skilled in the art that allow the configuration of volumes (in particular in 3D), in particular by adding or aggregating material by layer-by-layer or successive deposition.The hydrogels described above can therefore be obtained by additive manufacturing methods.

[0088] Among these methods, the inventors may particularly mention injection, extrusion, and especially molding, or additive manufacturing, especially 3D printing.Thus, the hydrogels described above may be obtained by extrusion of material, preferably by molding techniques, or by additive manufacturing, especially 3D printing.

[0089] In particular, hydrogels composed only of alginate and gelatin may have viscosities in the range of 50-6000 Pa.s when measured at temperatures of 5-45° C., so the skilled artisan will take care to select methods that allow for molding of highly viscous materials.

[0090] In the context of the present invention, the hydrogel cured by the curing method is preferably shaped, preferably by 3D printing techniques, prior to curing.

[0091] As previously indicated, the implementation of the method according to the invention makes it possible to endow the hydrogel, after hardening, with advantageous mechanical properties that are particularly suitable for providing a hydrogel intended to be implanted in the human or animal body, i.e. an internal implant.

[0092] To do this, using 3D printing to mold the hydrogel before its hardening offers the advantage of being able to prepare hydrogels of custom structure, whose dimensions and / or fill rate / porosity are defined in relation to the requirements of the body intended to receive the intracorporeal implant and the role / function it should play in this recipient organism. Indeed, the porosity of the implant is a crucial parameter that is adjusted according to the tissue or organ to be replaced and / or augmented. The porosity reflects the cavities present in the implant and can be adapted to provide the most material, thus conferring a specific mechanical strength as close as possible to the native tissue of the transplanted section. In particular, the porosity can be expressed in two different but correlated and therefore equivalent or alternative ways, namely the pore size expressed in pore micrometers and / or the fill rate of the hydrogel expressed as a percentage (volume of hydrogel / total volume of implant).

[0093] To harden a hydrogel containing viable cells with such an end goal (cellularized hydrogel, or cellularization after production of the hydrogel), it is further particularly advantageous in terms of safety that the viable cells are autologous cells, i.e., cells originating from the recipient organism.

[0094] Thus, in an exemplary embodiment, the hydrogel previously described makes it possible to obtain a three-dimensional body implant comprising one or more sections, each of which has a total porosity comprised between 100 μm and 10,000 μm, while having a mechanical strength between 1 kPa and 1000 kPa. The total porosity of a porous section corresponds to the average of the pore sizes measured in the porous section.

[0095] The pores of the porous section may have uniform pore sizes, i.e., may differ from each other by no more than 15%.

[0096] The pores of the porous section may be uniformly, i.e. evenly distributed.

[0097] The holes in the porous section may each extend along a central axis having a uniform orientation, i.e. orientations differing from each other by no more than 20°. The central axes of the holes in the porous section may be uniformly spaced, i.e. spacings that do not differ from each other by more than 15%.

[0098] The pores of the porous section may each have a uniform geometric structure, i.e. their contours may overlap by more than 50% with overlapping or parallel portions.

[0099] The holes in the porous section may be separated from one another by strands of material each having a uniform thickness, ie thicknesses that differ from one another by no more than 15%.

[0100] In particular, the implant may comprise at least two porous sections in which the pores have different pore sizes and / or shapes.

[0101] The porous sections may be arranged to form a gradient of pore size distributed throughout the implant, with the porous sections following one another along the direction of the gradient in an order selected from ascending and descending pore size.

[0102] In particular, implants a first porous section forming a base, representing between 5% and 40%, preferably between 20% and 40%, of the total volume of the implant and having a pore size comprised between 500 micrometers and 5000 micrometers, in particular between 250 micrometers and 800 micrometers; a second porous section forming a core, representing 20% ​​to 70%, preferably 30% to 50%, of the total volume of the implant and having a pore size comprised between 500 micrometers and 2500 micrometers, in particular between 100 micrometers and 250 micrometers; a third porous section forming an outer shell, representing between 5% and 40%, preferably between 10% and 40%, of the total volume of the implant and having a pore size comprised between 1000 micrometers and 10000 micrometers, in particular between 1000 micrometers and 2500 micrometers; may include.

[0103] The implant may include at least one non-porous section, the non-porous section having a fill factor of greater than 99%.

[0104] At least one non-porous section may include a perimeter surrounding the porous section.

[0105] The porous section(s) may cover a substantial part of the implant, ie at least 50%, preferably at least 75%, in particular at least 90%, for example at least 95%.

[0106] The implant may be made up of multiple layers, each having a mesh of multiple meshes, stacked on top of each other such that the meshes form pores.

[0107] The mesh in each layer may have a uniform mesh size, i.e. mesh sizes differing from each other by no more than 15%.

[0108] The mesh in each layer may be uniformly, i.e. evenly distributed.

[0109] The meshes of each layer may extend about a central axis having uniform orientations, ie orientations that do not differ from each other by more than 20°.

[0110] The central mesh axes of the meshes in each layer may be uniformly spaced, i.e., not differing by more than 15% relative to each other.

[0111] The meshes of each layer may have a uniform geometric structure, i.e. their contours may overlap by more than 50% with parts that overlap or are parallel.

[0112] The meshes of each layer may be separated from one another by strands of material each having a uniform thickness, ie thicknesses that differ from one another by no more than 15%.

[0113] The implant may have a volume in the range of 0.05 mL to 3 L, preferably 100 mL to 600 mL.

[0114] The implant may be a breast implant.

[0115] According to another aspect, the present invention relates to the use of a solution comprising a divalent cation, preferably calcium, transglutaminase and optionally one or more other divalent cations, for hardening a hydrogel comprising alginate and gelatin.

[0116] This hardening solution can also be used to harden hydrogels containing fibrinogen in addition to alginate and gelatin, such that the solution contains divalent cations, preferentially calcium and transglutaminase, as well as thrombin in addition to any other divalent cation(s).

[0117] All the preferences mentioned above regarding the hardening methods apply mutatis mutandis to the use of the hardening solutions according to the invention.

[0118] According to another aspect, the present invention relates to a hydrogel comprising hardened alginate and gelatin, preferably intended to be implanted in a human subject, the hydrogel being obtainable by the hardening method previously described.

[0119] According to a second embodiment, the present invention relates to a method for hardening a hydrogel comprising alginate and gelatin, comprising the following steps: - preparation of a hydrogel, - shaping the prepared hydrogel; - contacting the hydrogel with at least one divalent cation, preferably calcium, and transglutaminase, and optionally one or more other divalent cations. in order.

[0120] Hydrogels comprising alginate and gelatin can be prepared as previously described.

[0121] Preferably, the preparation of the hydrogel does not require shaping of one or more components prior to preparation of the hydrogel.Preferably, the preparation of the hydrogel does not require shaping of one or more components, such as alginate and / or gelatin, in the form of fibers prior to preparation.

[0122] Methods for forming components in the form of fibers are well known to those skilled in the art and include, for example, electrospinning, extrusion, fragmentation, freeze-drying followed by fragmentation.

[0123] Preferably, the preparation of the hydrogel does not require the addition of fibers. Preferably, the hydrogel is fiber-free.

[0124] Examples of fibers are mentioned above.

[0125] The hydrogel can be shaped as described above. Preferably, the shaping of the prepared hydrogel is carried out by extrusion of the material, preferably by molding or by additive manufacturing, in particular 3D printing.

[0126] Contacting the hydrogel with at least one divalent cation, preferably calcium, and transglutaminase, and optionally one or more other divalent cations, may be carried out simultaneously with a setting solution comprising at least calcium, transglutaminase, and optionally one or more other divalent cations.

[0127] Preferably the curing solution and contacting the hydrogel with said solution is as described above.

[0128] Contacting the hydrogel with at least one divalent cation, preferably calcium, and transglutaminase, and optionally one or more other divalent cations, may also be carried out sequentially, i.e., the crosslinker is not added at the same time during curing.

[0129] After preparation, the hydrogel can be contacted with the solutions described below in the following order: a solution comprising a divalent cation, preferably calcium, and optionally one or more other divalent cations, - A solution containing transglutaminase.

[0130] After preparation, the hydrogel can be contacted with the solutions described below in the following order: - a solution containing transglutaminase, A solution comprising a divalent cation, preferably calcium, and optionally one or more other divalent cations.

[0131] If the hydrogel contains fibrinogen in addition to alginate and gelatin, curing further includes cross-linking the fibrinogen with thrombin, which can be carried out sequentially with (e.g., before or after) or simultaneously with the cross-linking of the alginate and gelatin.

[0132] If the hydrogel contains fibrinogen in addition to alginate and gelatin, the hydrogel can be contacted after preparation with the solutions described below in the following order: a solution comprising a divalent cation, preferably calcium, and optionally one or more other divalent cations, - a solution containing transglutaminase, - A solution containing thrombin.

[0133] If the hydrogel contains fibrinogen in addition to alginate and gelatin, the hydrogel can be contacted after preparation with the solutions described below in the following order: a solution comprising a divalent cation, preferably calcium, and optionally one or more other divalent cations, - a solution containing thrombin, - A solution containing transglutaminase.

[0134] If the hydrogel contains fibrinogen in addition to alginate and gelatin, the hydrogel can be contacted after preparation with the solutions described below in the following order: - a solution containing transglutaminase, a solution comprising a divalent cation, preferably calcium, and optionally one or more other divalent cations, - A solution containing thrombin.

[0135] If the hydrogel contains fibrinogen in addition to alginate and gelatin, the hydrogel can be contacted after preparation with the solutions described below in the following order: - a solution containing transglutaminase, - a solution containing thrombin, A solution comprising a divalent cation, preferably calcium, and optionally one or more other divalent cations.

[0136] If the hydrogel contains fibrinogen in addition to alginate and gelatin, the hydrogel can be contacted after preparation with the solutions described below in the following order: - a solution containing transglutaminase, a solution comprising thrombin and a divalent cation, preferably calcium; A solution containing a divalent cation, preferably calcium.

[0137] If the hydrogel contains fibrinogen in addition to alginate and gelatin, the hydrogel can be contacted after preparation with the solutions described below in the following order: - a solution containing thrombin, - a solution containing transglutaminase, A solution containing a divalent cation, preferably calcium.

[0138] If the hydrogel contains fibrinogen in addition to alginate and gelatin, the hydrogel can be contacted after preparation with the solutions described below in the following order: - a solution containing thrombin, - a solution containing a divalent cation, preferably calcium, - A solution containing transglutaminase.

[0139] If the hydrogel contains fibrinogen in addition to alginate and gelatin, the hydrogel can be contacted after preparation with the solutions described below in the following order: - a solution containing thrombin, a solution comprising transglutaminase and a divalent cation, preferably calcium, A solution containing a divalent cation, preferably calcium.

[0140] During curing, contacting the hydrogel with the above-mentioned solution(s) can be carried out by immersion, where the hydrogel is immersed in its entirety in the aforementioned solution(s), or by spraying, by imbibition, by trickle, or by similar system.

[0141] According to another aspect, the present invention also relates to a hydrogel comprising hardened alginate and gelatin, preferably intended to be implanted in a human subject, the hydrogel being obtainable by the hardening method previously described.

[0142] Further characteristics, objects and advantages of the present invention will become apparent from the following description which is purely illustrative and non-limiting and which must be read in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0143] [Figure 1] 1 shows a comparison of Young's modulus (A) and viscosity (B) of AG and FAG hydrogels that make up an implant according to the invention. [Diagram 2] Figure 1 shows a comparison of Young's modulus E (Pa) of AG hydrogels in which gelatin was crosslinked in the presence and absence of transglutaminase and stored at 37°C for up to 7 days. [Diagram 3] Comparison of Young's modulus E0 (Pa) of AG hydrogel and commercial hydrogel crosslinked in the presence or absence of transglutaminase. *: Liquid compound at 37°C. +: Polymerization is visible at 37°C by DMA measurement, but gel stiffness is insufficient. [Figure 4] FIG. 1 represents the viability and cell growth measured by kinetics in FAG and AG hydrogels constituting an implant according to the invention and colonized in vitro with fibroblasts after production. [Diagram 5] FIG. 1 represents the viability and cell growth measured by kinetics in FAG and AG hydrogels constituting the implants according to the invention and populated in vitro with adipose tissue stem cells after production. [Figure 6] FIG. 1 shows the metabolic activity of an AG implant according to the invention at different culture times after in vitro colonization of purified adipose tissue fractions after production. [Figure 7] Figure 1 represents a histological analysis by hematoxylin-phloxine-saffron (HPS) staining of an AG implant according to the invention after 2 days (four left images) or 7 days (two right images) of in vitro incubation with purified adipose tissue fraction after manufacture (top: outer edge of the matrix; bottom: inner hole of the matrix; images taken under white light; magnification x100; scale 100 µm). [Figure 8]Figure 1 shows perilipin 1 immunostaining and Dapi staining of cell nuclei on an AG implant according to the invention after 2 days (upper image) or 7 days (lower image) of in vitro incubation with purified adipose tissue fraction after manufacture (fluorescence image; magnification 200x; scale 50 μm). [Figure 9] 1 depicts a comparison of Young's modulus of AG implants for various periods of crosslinking at 21° C. (B) and 37° C. (A). [Figure 10] Figure 1 shows a comparison of Young's modulus E0 (A–C) and viscosity (D–F) of AG and FAG implants after crosslinking with different concentrations of CaCl2, TAG, and thrombin. [Figure 11] Figure 1 shows a comparison of Young's modulus E0 (A–B) and viscosity (C–D) of AG and FAG implants after sequential or simultaneous crosslinking with CaCl2, TAG and thrombin. [Figure 12] 1A-B show a comparison of Young's modulus E0 (A) and viscosity (B) of AG and FAG implants after crosslinking with solutions containing calcium chloride or barium chloride. [Figure 13A] Illustrates the variation in dimensions (A1-A2) and pores (A3-A4) of AG and FAG implants according to the invention before and after crosslinking. [Figure 13B] The effect of sterilization on the dimensions (B1-B2) and Young's modulus (B3-B4) of these implants is shown. [Figure 14] 1 illustrates the reproducibility of the production of AG implants according to the invention with respect to dimensions (A), volume (B) and porosity (C). [Figure 15] 1 illustrates the reproducibility of shrinkage of an AG implant according to the invention after curing. [Figure 16] 1 illustrates the reproducibility of shrinkage of an AG implant according to the invention as a function of sterilization method. [Figure 17A] This illustrates the reproducibility of the extrusion diameter. [Figure 17B] 4 illustrates the pore length (B1-B2) of the AG implant according to the present invention. [Figure 18] 13 depicts images of pores of various sizes in an AG implant according to the present invention. [Figure 19] 1 depicts the surgical plan (left side) for in vivo subcutaneous implantation (right side) of AG and FAG implants according to the present invention. [Figure 20] 1 shows a histological analysis of sections of an AG implant according to the invention after 3 weeks of subcutaneous in vivo implantation in the back of a rat, stained with Masson's Trichrome (low, medium and high magnification images). [Figure 21A] Analysis of cell survival, as measured by lactate accumulation, over 28 days is shown. [Figure 21B] 13 shows analysis of calcein labeling in cellularized FAG hydrogels after growing cells for 28 days. [Figure 22] FIG. 13 depicts Young's modulus (E0) measurements as a function of initial cell concentration in cellularized FAG hydrogels. [Diagram 23] Figure 2 depicts the evolution of lactate concentrations measured in culture supernatants from cellularized and cured AG or FAG hydrogels over 21 days. [Figure 24] FIG. 13 depicts histological analysis after HPS staining of bioprinted full (left panel) or porous (right panel) FAG fibroblast / endothelial cell hydrogel constructs after 21 days of culture. [Diagram 25] 13A-13D depict histological analysis after HPS staining of bioprinted non-porous (left panel) or porous (right panel) AG fibroblast / endothelial cell hydrogel constructs after 21 days of culture. [Figure 26] CD31-DAB immunolabeling (black) in non-porous bioprinted FAG fibroblast / endothelial cell hydrogel constructs after 21 days of culture. [Figure 27] CD31-DAB immunolabeling (black) in porous bioprinted FAG fibroblast / endothelial cell hydrogel constructs after 21 days of culture. [Figure 28] 13 depicts histological analysis after HPS staining of skin reconstructed from 3D bioprinted FAG hydrogel cured with TAG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0144] Working Example The invention will be better understood on reading the following examples which illustrate the invention in a non-limiting manner.

[0145] Materials and Methods Protocol #1 Preparation of AG hydrogel: To prepare the AG hydrogel, 2 g of alginate (ultra-low viscosity, Alpha Aesar, France), 5 g of gelatin (Sigma-Aldrich, France) are dissolved in 100 mL of 0.1 M NaCl solution (Labelians, France) for 12 h at 37 °C.

[0146] Protocol #2 Preparation of FAG hydrogel: To prepare the FAG hydrogel, 2 g of alginate (ultra-low viscosity, Alpha Aesar, France), 5 g of gelatin (Sigma-Aldrich, France) and 2 g of fibrinogen (Sigma-Aldrich, France) are dissolved in 100 mL of 0.1 M NaCl solution (Labelling, France) for 12 h at 37 °C.

[0147] Protocol #3 Molding of AG or FAG hydrogels: Place 1.8 mL of hydrogels prepared according to protocol #1 or #2 into a well of a 6-well culture plate and incubate at 21 °C for 30 min.

[0148] Protocol #4 Crosslinking of AG hydrogels: Prepare a crosslinking solution by dissolving 4 g of transglutaminase (Ajinomoto, Japan), 3 g of CaCl2 (Sigma Aldrich, France) in 100 mL of 0.1 M NaCl solution (Labelling, France). The crosslinking solution is then placed in contact with the hydrogel for 1 h 30 min at 37 °C (unless otherwise stated).

[0149] Protocol #5 Cross-linking of FAG hydrogels: Prepare a cross-linking solution by dissolving 4 g of transglutaminase (Ajinomoto, Japan), 3 g of CaCl2 (Sigma Aldrich, France) and 400 units of thrombin (Sigma Aldrich, France) in 100 mL of 0.1 M NaCl solution. The cross-linking solution is then placed in contact with the hydrogel for 1 h 30 min at 37 °C (unless otherwise stated).

[0150] Protocol #6 Dynamic Mechanical Analysis (DMA) in Compression: The mechanical properties of FAG and AG hydrogels are measured in triplicate by rotational rheometer (DHR2, TA Instrument, France), Peltier plane (TA Instrument, France) and 8 mm notch geometry (TA Instrument, France). Three 8 mm diameter disks are cut from the hydrogels molded according to protocol #3. The disks are placed on the lower notch geometry for 60 s at 37 °C, after which a 10 μm oscillatory compression procedure is carried out at 0.1-10 Hz at 100 μm / s and 37 °C. The values ​​of Young's modulus E0 (Pa) and viscosity η0 (Pa.s) of the hydrogels are obtained from viscous hyperelastic solid modeling using the E' and E'' values ​​obtained during this test.

[0151] Protocol #7 Preparation of cellularized hydrogels: To prepare the cellularized FAG hydrogel, 0.12 g alginate (ultra-low viscosity, Alpha Aesar, France), 0.3 g gelatin (Sigma-Aldrich, France) are dissolved in 6 mL DMEM culture medium (Gibco Cell Culture, Invitrogen, France). The freshly trypsinized cells are suspended in 2 mL 8% fibrinogen solution (Sigma-Aldrich, France). This cell suspension is then added to the preceding solution to form the cellularized FAG. To prepare the cellularized AG hydrogel, 0.12 g alginate (ultra-low viscosity, Alpha Aesar, France), 0.3 g gelatin (Sigma-Aldrich, France) are dissolved in 6 mL DMEM culture medium (Gibco Cell Culture, Invitrogen, France). The freshly trypsinized cells are suspended in 2 mL 0.1 M NaCl solution (Fabelians, France). This cell suspension is then added to the previous solution to form cellularized FAG.

[0152] Protocol #8 3D printing of hydrogels: Hydrogels prepared according to protocols #1 and #2 are transferred into a 3 mL cartridge (Nordson EFD) equipped with a 410 μm diameter extrusion nozzle (Nordson EFD). The cartridge-nozzle assembly is then placed in a 3D printer (BioassemblyBot, Advanced Solution Lifescience, USA) and a constant pressure is applied to the cartridge while moving in all three directions in space. Printing parameters are a speed of 10 mm / s, a pressure of 25-35 PSI, and a temperature of 21 °C. Different filling rates are obtained by an internal slicer in the printer control software (Tsim, Advanced Solution Lifescience, USA).

[0153] Protocol #9 In vivo implantation in rats: In vivo implantation studies in rats were performed at BIOVIVO (technical platform for preclinical trials at the Institut Claude Bourgelat, Lyon, France). The experiments were performed in accordance with the European Directives 2010 / 63 / EU. Sixteen animals (Sprague-Dawley rats, 250-300 g) were anesthetized by inhalation (oxygen and 5% isoflurane). The dorsal implantation site was shaved, disinfected with povidone and sterile gauze, covered with a sterile drape, and the surgical area was outlined. General anesthesia was maintained with isoflurane (2%) and oxygen inhalation. Preoperative analgesia was performed by subcutaneous injection of 1 mg / kg meloxicam and morphine, respectively. The body temperature and pulse rate of the rats were monitored during the surgery. Two skin incisions of 2-3 cm were made in the dorsal region. The bioprosthesis was implanted in the dorsal subcutaneous region of each animal. In the control group, only incision and dissection were performed. Four surgical sites were performed in one animal per group, three bioprosthesis and one control section. The surgical sites were closed in multiple layers using subcutaneous and cutaneous sutures with absorbable sutures (PDS® Polydioxanone, 4 / 0 and Nylon 3 / 0, Ethicon J&J). Postoperatively, the animals were monitored for signs of morbidity and the surgical wounds were examined daily for skin healing and absence of infection. Explantation was performed 21 days after implantation.

[0154] Protocol #10 Histological analysis: Implants were fixed in 4% formalin solution (Alphapat, France) for 24 hours, then dehydrated in successive baths of absolute ethanol (vwr chemicals, France) and methylcyclohexane (vwr chemicals, France) using a STP120 dehydrator (Myr, Spain) and then embedded in kerosene (Sakura, Japan). 5 μm thick sections were made with a HM340e microtome (Microm, France). Hematoxylin-phloxine-saffron (HPS), Masson's trichrome and DAPI staining were performed.

[0155] Example 1 – Mechanical properties of alginate / gelatin (AG) and fibrinogen / alginate / gelatin (FAG) hydrogels AG and FAG hydrogels were prepared by protocols #1 and #2, molded according to protocol #3, then crosslinked using protocols #4 and #5, and their mechanical properties tested by DMA using protocol #6.

[0156] The results are shown in Figure 1 (A-B). The measured Young's modulus and viscosity values ​​are similar between the AG and FAG hydrogels after their crosslinking by the process of the present invention. The Young's modulus under the specific conditions of this test is around 68000 Pa.

[0157] Example 2 - Effect of cross-linking with transglutaminase on the mechanical properties of alginate / gelatin hydrogels (AG) Molding samples of AG were prepared by protocols #1 and #3 and cross-linked by a variation of protocol #4, where the cross-linking solution consisted of 30 mg / mL potassium chloride solution alone or 30 mg / mL calcium chloride and 40 mg / mL transglutaminase solution. Four gels of each condition were cast and tested by DMA on the same day and after 1, 4 and 7 days of storage at 37°C, respectively, to mimic physiological conditions.

[0158] The samples were then tested by DMA using protocol #6.

[0159] The results are shown in Figure 2. This study shows the beneficial effect of the use of transglutaminase in crosslinking on the mechanical properties of the hydrogel, which is even greater when the gel is transformed at 37°C, justifying the particular interest of crosslinking according to the invention for hydrogels intended to be implanted.

[0160] Example 3 - Effect of cross-linking with transglutaminase on the mechanical properties of commercial gelatin and / or collagen hydrogels Molded samples of AG were prepared according to protocols #1 and #3 and crosslinked according to protocol #4. Commercially available hydrogel samples listed in Table 1 below were prepared according to protocols provided by the supplier and molded according to protocol #3. [Table 1]

[0161] Hydrogels were crosslinked by a modification of protocol #4 using either a solution containing 30 mg / mL calcium only (no TAG) or a solution containing 30 mg / mL calcium and 40 mg / mL transglutaminase to observe the effect of TAG.

[0162] Uncrosslinked or crosslinked with TAG samples were then tested by DMA using protocol #6.

[0163] The results are grouped in Figure 3. Six of the seven commercial hydrogels tested were cross-linked with transglutaminase. The collagen-based hydrogels (Co4Cell, rat collagen) are not stiff enough to be analyzed by DMA, whereas the gelatin-based hydrogels (Gel4cell, Gel4cell-VEGF and GelMa) have significantly higher Young's modulus after cross-linking with transglutaminase (7.3, 9.9 and 50 kPa, respectively). This study shows the effect of cross-linking with transglutaminase on the stiffness of commercial hydrogels.

[0164] Example 4 - Effect of the amount of alginate and gelatin in fibrinogen / alginate / gelatin (FAG) hydrogels on the mechanical properties FAG hydrogels were prepared by a variation of protocol #2, molded by protocol #3, then crosslinked using protocol #5, and then mechanical properties were tested by DMA using protocol #6. In this variation, we tested the mechanical properties of FAG hydrogels by preparing them with 1 or 3 or 2 g alginate, 10 or 7.5 or 5 g gelatin, and 2 g fibrin, respectively.

[0165] The results are grouped below in Table 2. Young's modulus under the specific conditions of this test is in the range of 200-800 kPa. [Table 2]

[0166] Example 5 – Evaluation of fibroblast colonization of fibrinogen / alginate / gelatin (FAG) and alginate / gelatin (AG) hydrogels AG and FAG hydrogels were prepared by protocols #1 and #2. Square implants with size 1.5 cm and thickness 0.2 cm were then printed using protocol #8 and crosslinked using protocols #4 or #5. The printed implants were produced with a 410 μm inner diameter extrusion nozzle at 50% fill factor. A negative control (empty well) was also used.

[0167] Normal human fibroblasts from route 6 are thawed and expanded in a culture medium containing DMEM supplemented with 10% bovine serum and 1% antibiotics in a 175 cm2 culture flask. Each implant was seeded on the surface with a cell suspension of normal human fibroblasts at a concentration of 4 million fibroblasts / ml. 250 μl of this suspension, i.e. 1 million fibroblasts / implant, were dispensed dropwise onto each implant. After 1 hour of adhesion, the implants were immersed in culture medium. The implants were cultured at 37° C. and 5% CO2 in a culture medium composed of DMEM containing 10% bovine serum supplemented with vitamin C and EGF (epidermal growth factor). The implants were cultured for 21 days with the same medium replaced every 3 days.

[0168] The metabolic activity of fibroblasts in the implants was tested by colorimetry with Alamar Blue 3, 5, 8, 10, 14 and 21 days after seeding. The solution was made by 10-fold dilution of a solution of Alamar Blue (DAL1100, Invitrogen) in DMEM. After 19 hours of incubation at 37°C, 100 μl of supernatant was collected and their absorbance at 570 nm and 600 nm was measured by a spectrophotometer (NanoQuant® infinite M200PRO, TECAN).

[0169] Cell viability and growth was monitored over 21 days of culture using kinetics at six time points: days 3, 5, 8, 10, 14 and 21. The results are shown in FIG.

[0170] Results confirmed that all implants allowed fibroblast attachment and survival as early as day 3 of culture. Cell growth was observable in each porous implant over 21 days of culture for both types of hydrogels (FAG and AG) and for each total porosity used.

[0171] Example 6: Evaluation of Adipose Tissue Stem Cell (ASC) Engraftment of Fibrinogen / Alginate / Gelatin (FAG) and Alginate / Gelatin (AG) Hydrogels AG and FAG hydrogels were prepared by protocols #1 and #2. Then, square implants with size 1.5 cm and thickness 0.2 cm were printed using protocol #8 and crosslinked using protocols #4 or #5. The printed implants were produced with a 410 μm inner diameter extrusion nozzle at 50% and 75% filling rate. Sterilization was performed by irradiating the implants with a 30 kGy dose of gamma radiation by IONISOS (France).

[0172] Normal human adipocyte stem cells from routes 2-5 were thawed and expanded in a culture medium containing DMEM supplemented with 10% serum and 1% antibiotics in 175 cm2 culture flasks. Each implant was seeded on the surface with a cell suspension of ASCs at a concentration of 6, 12, or 24 million ASCs / ml. 250 μl of these suspensions, i.e. 1.5, 3, or 6 million ASCs / implant, were dispensed dropwise onto each implant. After 1 hour of adhesion, the implants were immersed in culture medium. The implants were cultured for 7 days in culture medium containing DMEM supplemented with 10% serum and 1% antibiotics, and then for 14 days in medium containing DMEM supplemented with 10% serum, insulin, rosiglitazone, and 1% antibiotics. The culture medium was changed every 3 days.

[0173] The metabolic activity of fibroblasts in the implants was tested by colorimetric analysis with Alamar Blue on days 3, 5, 7, 14 and 21 after seeding. The solution was made by diluting a solution of Alamar Blue (DAL1100, Invitrogen) in DMEM 10-fold. After 5 hours of incubation at 37°C, 100 μl of supernatant was collected and their absorbance at 570 nm and 600 nm was measured by a spectrophotometer (NanoQuant® infinite M200PRO, TECAN).

[0174] Cell viability and growth was monitored over 21 days of culture using kinetics at six time points: days 3, 5, 7, 14 and 21. The results are shown in FIG.

[0175] Results confirmed that all implants allowed the attachment and survival of adipocyte stem cells from day 3 of culture. Cell growth was observable in each porous implant over 21 days of culture for both types of hydrogels (FAG and AG) and each seeding density.

[0176] Example 7: Evaluation of retention of alginate / gelatin (AG) hydrogels in contact with purified adipose tissue fractions AG hydrogels were prepared by protocol #1. Then, cubic implants with size 1.5 and thickness 0.8 cm were printed using protocol #8 and crosslinked using protocol #4. The printed implants were produced with a 410 μm inner diameter extrusion nozzle at 50% fill factor.

[0177] The lipoaspirate was centrifuged at 1500 rpm for 2 minutes and then rinsed with 1×PBS. The lipoaspirate was centrifuged again at 1500 rpm for 30 seconds and then the 1×PBS was removed. The lipid aspirate was considered purified.

[0178] Each implant was then immersed in 6 mL of purified lipid aspirate, and the entire set was placed into a culture insert in a 6-well plate and incubated for 2 or 7 days at 37°C, 5% CO2 in medium containing DMEM supplemented with 10% serum and 1% antibiotics.

[0179] Following contact with lipid aspirates, implants were cultured in 6-well plates in culture medium containing DMEM supplemented with 10% serum, insulin, rosiglitazone, and 1% antibiotics with medium changes three times a week for up to 21 days.

[0180] The cell metabolic activity in the implants was tested by colorimetric analysis with Alamar Blue on days 2, 7 and 21 of culture after seeding. The solution was made by 10-fold dilution of a solution of Alamar Blue (DAL1100, Invitrogen) in DMEM. After 5 hours of incubation at 37°C, 100 μl of supernatant was collected and their absorbance at 570 nm and 600 nm was measured with a spectrophotometer (NanoQuant® infinite M200PRO, TECAN).

[0181] Cell viability and growth were monitored over a period of 21 days and the results are shown in Figure 6.

[0182] Significantly higher metabolic activity than the negative control was observed in implants contacted with purified lipoaspirate.

[0183] Histological analysis was performed to complete this study according to protocol #10, and the results are shown in FIG.

[0184] The images reveal the presence of aggregated polygonal uniform unilocular bulky adipocytes, morphological features characteristic of healthy adipocytes that can be found in adipose tissue.

[0185] Immunostaining for perilipin 1 was also performed. Samples were included in OCT (Cellpath, KMA-0100-00A) and then stored at -80°C. 16 μm thick sections were made from each sample by cryostat (Microm, HM520). Sections were then fixed in acetone / methanol (v / v) solution for 20 min and rinsed three times with 1×PBS. Incubation in 4% PBS-BSA solution for 1 h at room temperature was performed to saturate specific sites. Sections were then incubated overnight at room temperature with perilipin 1 specific primary antibody solution. The next day, sections were rinsed three times with 1×PBS and then incubated with Alexa fluor568 coupled secondary antibody solution for 45 min at room temperature. Sections were then rinsed three times with 1×PBS and mounted with Dapi fluoromount-G® mounting medium (SouthernBiotech) between slides and coverslips. The resulting images are grouped together in FIG.

[0186] The images show adipocytes with large spherical or polygonal vacuoles depending on the clustering of the cells. The adipocytes appear unilocular and their size is similarly physiological, being in the range of 50-200 µm.

[0187] Taken together, these results confirm the attachment, survival and regeneration of human adipose tissue in contact with the implant. The specific structure and composition of the implant therefore creates a favorable environment for the regeneration of healthy adipose tissue.

[0188] Example 8 - Effect of temperature and crosslinking time on the mechanical properties of alginate / gelatin hydrogels (AG) Molding samples of AG were prepared by Protocol #1 and Protocol #3 and crosslinked by a variation of Protocol #4, in which the crosslinking time and temperature were varied from 10 min to 14 h and from 37° C. to 21° C.

[0189] The samples were then tested by DMA using protocol #6.

[0190] The results are shown in Figure 9 (A-B). The crosslinking time as well as the crosslinking temperature have very little effect on the final mechanical properties (Young's modulus) of the hydrogel. However, it seems that the optimum solution can be found around 1 hour and 30 minutes, regardless of the temperature.

[0191] These Young's moduli are very stable over 7 days after cross-linking at 37° C. The cross-linking of gelatin was efficient since there was no loss of gelatin dissolved in the medium.

[0192] Example 9 - Effect of the component concentrations of the crosslinking solution on the mechanical properties of alginate / gelatin (AG) and fibrinogen / alginate / gelatin (FAG) hydrogels after crosslinking Molding samples of AG and FAG were prepared by protocols #1, #2, and #3 and cross-linked by modifications of protocols #4 and #5, in which the concentrations of the cross-linking solution components (transglutaminase, calcium chloride, and thrombin) were varied.

[0193] The samples were then tested by DMA using protocol #6.

[0194] The results are grouped in Figure 10 (A-F). No significant changes were observed in this range of reagent concentrations (all of the E0s were very similar).

[0195] Example 10 - Effect of sequential or simultaneous crosslinking of alginate / gelatin (AG) and fibrinogen / alginate / gelatin (FAG) hydrogels Molding samples of AG and FAG were prepared by protocols #1, #2 and #3 and crosslinked by a variant of protocols #4 and #5. In this variant, we investigated sequential crosslinking with FAG and AG, which involves crosslinking the hydrogel in multiple steps. Each step took 1 hour, and three rinses with 0.1M NaCl solution were performed between each step to remove residual crosslinker. The conditions tested for sequential crosslinking are listed in the table below (each step consisted in immersing the hydrogel in the listed solution for 1 hour). [Table 3]

[0196] The samples were then tested by DMA using protocol #6.

[0197] The results are shown in Figure 11 (A-D). The sequential crosslinking set (FAG and AG) produces hydrogels with lower Young's modulus than single step crosslinking.

[0198] It can be observed that if calcium is not added first, a very soft and fragile gel is obtained, but indeed TAG and thrombin are calcium-dependent, and therefore their activity is greatly reduced without the addition of CaCl2. Therefore, the gel is difficult to manipulate without calcium cross-linking. If thrombin is added first, the gel has very low mechanical strength and pores appear.

[0199] Example 11 - Effect of the nature of the divalent cation on the crosslinking of alginate / gelatin (AG) and fibrinogen / alginate / gelatin (FAG) hydrogels Molding samples of AG and FAG were prepared by protocols #1, #2 and #3 and crosslinked by a variant of protocols #4 and #5, in which we tested crosslinking in the presence of 30 mg / mL barium chloride.

[0200] The samples were then tested by DMA using protocol #6.

[0201] The results are shown in Figure 12 (A-B). Crosslinking in the presence of barium leads to gels with Young's modulus very similar to that obtained with CaCl2. However, barium increases the viscosity of the gel, so the formation of additional side chains can be presumed.

[0202] Example 12 - Maintenance of three-dimensional structure and mechanical properties of alginate / gelatin (AG) and fibrinogen / alginate / gelatin (FAG) hydrogel implants after sterilization AG and FAG hydrogels were prepared by protocols #1, #2 and #3, crosslinked using protocols #4 and #5, optionally observed, and then tested by DMA using protocol #6. The printed geometries were 2 cm diameter hemispheres, generated with variable packing fractions (30, 50 and 75%).

[0203] Sterilization was performed by IONISOS (France) by irradiating the implants with various doses of gamma radiation (30 kGy and 40 kGy).

[0204] The effect of the cross-linking step on the dimensions of alginate / gelatin and fibrinogen / alginate / gelatin hydrogel implants was studied, these dimensions being measured from macroscopic images.

[0205] The pore dimensions obtained as a function of the filling rate were also studied: these dimensions were measured from images made under a microscope (Olympus, magnification x4).

[0206] The results are shown in Figure 13 (A-B). The implants shrink by an average of 10% after the crosslinking step, but the pore size does not change significantly (Figure 13A (A1-A4)).

[0207] In relation to sterilization, the 40 kGy dose appears to result in greater shrinkage of the construct than the 30 kGy dose. For E0, sterilization does not result in any change in the mechanical properties of the material at both doses (FIG. 13B (B1-B4)).

[0208] Example 13: Production quality of large alginate / gelatin (AG) hydrogel implants: Reproducibility of implant cavity dimensions, hardening and post-sterilization dimensions by multiple methods AG hydrogels were prepared according to protocol #1. Hemispherical implants with a diameter of 6 cm and a thickness of 2 cm were then printed according to protocol #8, crosslinked using protocol #4, and then observed and measured as appropriate. The printed shapes were produced using an extrusion nozzle with an inner diameter of 410 or 840 μm with various fill rates (25-65%). Sterilization was performed by IONISOS (France) by irradiating the implants with two doses of beta radiation (30 kGy and 40 kGy) or a range dose of 30 kGy.

[0209] The effect of cross-linking and sterilization steps on the dimensions of large alginate / gelatin hydrogel implants was examined, these dimensions being measured from macroscopic images.

[0210] The resulting pore dimensions as a function of the filling rate were also studied: these dimensions were measured from images made under a microscope (Olympus, magnification x4).

[0211] The post-printing results are shown in Figure 14 (A-C). These results show high reproducibility of the dimensions of the 3D printed large implants, reflecting high production quality.

[0212] The results after curing of the implants are grouped in Figure 15. This graph shows the high reproducibility of the shrinkage of the large implants after the curing phase.

[0213] The results after sterilization of implants by three methods (beta radiation at doses of 40 and 30 kGy, and gamma radiation at 30 kGy) are grouped in Figure 16. These results show less shrinkage of large implants at 30 and 40 kGy of beta radiation.

[0214] Large implants were printed using two extrusion nozzles with inner diameters of 410 and 840 μm, with fill rates ranging from 25 to 65%. The reproducibility of the extrusion diameter and the resulting hole length were measured. The results are shown in Figure 17 (A-B).

[0215] Figure 17A shows the high reproducibility of the size of the extruded beads, and Figure 17B (B1-B2) shows the variation in pore length with the filling rate of the hydrogel.

[0216] Images of various pore sizes were taken and are listed in FIG.

[0217] These data demonstrate the wide range of pores that can be obtained in the implants, as well as their high reproducibility and production quality.

[0218] Example 14 – Testing the in vivo resistance of implants AG and FAG hydrogels were prepared by protocols #1, #2 and #8 and crosslinked via protocols #4 and #5. The printed geometries were 1 cm diameter hemispheres and were generated with various filling fractions (30, 50 and 75%).

[0219] The porous hemispheres were sterilized with a dose of 30 kGy and then implanted subcutaneously in rats according to protocol #9.

[0220] Details of the implantation groups are set forth in Table 3 below, which references the surgical implantation plan set forth in FIG. [Table 4]

[0221] Histological analysis was performed using protocol #10 and the results are summarized in Figure 20. Explants were used to verify the resistance of the implants to skin tension. Histological analysis was used to evaluate cell colonization, angiogenesis, extracellular matrix synthesis, and the presence of inflammatory areas.

[0222] Example 15 – Fibrinogen / Alginate / Gelatin (FAG) Viability and Cell Growth and Mechanical Properties Cellularized FAG hydrogels were prepared by protocol #7 in the presence of different concentrations of human dermal fibroblasts (0.5 million / 0.25 million / 0.125 million cells / mL hydrogel). 2 Slabs of 0.2 cm thickness and 100% loading were prepared using protocol #8 and then crosslinked using protocol #5.

[0223] The different slabs were then cultured for 28 days at 37°C and 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) / Glutamax medium (Gibco Cell Culture, Invitrogen, France) supplemented with 10% (v / v) calf serum (Gibco Cell Culture, Invitrogen, France), 0.5% (v / v) amphotericin B (Gibco Cell Culture, Invitrogen, France) and 1% penicillin / streptomycin.

[0224] To follow cell growth, administration of produced L-lactic acid was carried out every 2 days by a commercial kit (L-Lactic Acid Assay kit, Megazyme). Optical density measurements were carried out by an INFINITE plate reader (TECAN, France).

[0225] To observe the presence of viable cells within the hydrogel slabs along the culture, viability labeling was performed every 5 days in the presence of 1 mM calcein AM (Thermofisher, France).

[0226] The results are summarized in Figure 21A. The synthesis of lactate is an indicator of cell activity. A sharp increase in the amount of lactate was observed from D15 at all cell concentrations, indicating cell growth within the cured bioprinting hydrogels according to the present invention.

[0227] Calcein AM labeling images, labeled in FIG. 21B, show the presence of viable and growing cells from D11, and cell spreading from D17 at 0.25 M cells / mL, and from D22 at other cell concentrations.

[0228] Hydrogels of cellularized FAG prepared and cultured as described above were tested according to protocol #6.

[0229] The results are summarized in Figure 22. The Young's moduli of the cellularized hydrogels after 28 days in culture are independent of the cell concentration used in their generation. However, these Young's moduli are lower than those obtained from non-cellularized hydrogels, indicating remodeling of the hydrogel by cells.

[0230] Example 16 – Bioprinting of dermal equivalents in hardened alginate / gelatin (AG) and fibrinogen / alginate / gelatin (FAG) hydrogels Cellularized AG and FAG hydrogels were prepared by protocol #7 in the presence of a mixture of human dermal fibroblasts (250,000 cells / mL hydrogel) and human dermal microvascular endothelial cells (1 million cells / mL hydrogel). 2 Slabs of 0.2 cm thickness and 50 and 100% infill were prepared using protocol #8 and then cured using protocols #4 and #5.

[0231] The different constructs were then cultured for 21 days at 37°C and 5% CO2 in a culture medium suitable for culturing human dermis, consisting of DMEM supplemented with 10% calf serum, 1% antibiotics, vitamin C and EGF.

[0232] To follow cell growth, administration of the produced L-lactic acid was carried out every 2 days using a commercial kit (L-Lactic Acid Assay kit, Megazyme) according to the protocol recommended by the supplier. Optical density measurements were carried out by an INFINITE plate reader (TECAN, France).

[0233] The results are summarized in Figure 23. An increase in lactate production by cells is observed from D11 under all conditions. Lactate production is higher in AG-based hydrogels and porous constructs. These results indicate cell proliferation in hydrogels cured for 21 days.

[0234] Histological analysis was performed according to protocol #10. HPS staining was performed for all conditions. To assess the presence of endothelial cells within the hydrogels, CD31 immunohistochemical labeling with endothelial cell-specific DAB revelation was performed on 5 μm thick paraffin sections.

[0235] The results are summarized in Figures 24 to 27.

[0236] Figure 24: Images of non-porous FAG constructs show that numerous cells are present within the gel and appear to grow in the form of aggregates at adhesion sites on the gel. In the porous constructs, clusters of proliferation within the gel are also observed, but also a layer of proliferation on the pore surface. We can also observe the beginning of degradation of the hydrogel around the cells within the gel.

[0237] Figure 25: Images of non-porous AG constructs show that numerous cells are present within the gel. In porous constructs, clusters of proliferation are also observed within the gel, but also in the superficial growth layer and in areas of high cell density at the corners of the pores. Degradation of the hydrogel around the cells is also observed within the gel.

[0238] Figure 26: Images of non-porous FAG constructs reveal the presence of small clusters of endothelial cells located around and within the fibroblast clusters as a result within the gel.

[0239] Figure 27: Images of the porous FAG construct reveal the presence of cellular clusters of fibroblasts within the gel, along with clusters of endothelial cells located around and within the fibroblast clusters. A proliferative layer of fibroblasts is also observed at the surface.

[0240] Example 17 – Generation of skin equivalents by epidermalization of bioprinted products from cured fibrinogen / alginate / gelatin (FAG) hydrogels Cellularized FAG hydrogels were prepared by protocol #7 in the presence of a mixture of human dermal fibroblasts. Two separate conditions were tested: bilayer bioprinting at 1,000,000 fibroblasts / ml, and a hybrid bilayer with an acellular bottom layer and a cellularized top layer at 2,000,000 fibroblasts / ml. For the cellularized bilayer constructs, slabs with dimensions 2.2 cm x 2.2 cm x 0.2 cm (bilayer) and 100% infill were printed using protocol #8 and then cured using protocol #5. For the hybrid cellularized bilayer constructs, a first acellular layer with dimensions 2.2 cm x 2.2 cm x 0.1 cm and a second cellularized layer with dimensions 2.2 cm x 2.2 cm x 0.1 cm and 100% infill (2,000,000 fibroblasts / ml) were printed using protocol #8 and cured using protocol #5.

[0241] The various constructs were then cultured for 21 days at 37°C and 5% CO2 in a culture medium suitable for culturing human dermis composed of DMEM supplemented with 10% calf serum, 1% antibiotics, vitamin C and EGF. After 3 weeks of culture, a suspension of 4,000,000 c / ml of normal human keratinocytes was prepared in a suitable culture medium composed of DMEM / HAMF12 supplemented with 10% calf serum, 1% antibiotics, insulin, hydrocortisone, vitamin C and EGF. 250 μl of this suspension was added to give a total of 250,000 keratinocytes / cm. 2 After seeding, the various constructs were cultured for 21 days at 37° C. and 5% CO2 in a culture medium suitable for culturing human skin equivalents, composed of DMEM / HAMF12 supplemented with insulin, hydrocortisone, vitamin C and 1% antibiotics.

[0242] The collagen content of the bioprinted constructs was evaluated under various conditions. A quantitative analysis of the collagen present in the support was carried out using the Sircol test (Kit S1000, Biocolor). The control for this test was acellular FAG constructs produced according to the same methodology as the samples with bioprinted surfaces. The pellet resulting from collagen degradation was dissolved in 250 μl of basic reagent solution (kit). The absorbance was then measured at 555 nm ((NanoQuant® infinite m200PRO, TECAN) and the results were compared with those of a standard range, allowing the evaluation of the collagen concentration in the digestion supernatant. This concentration was then added to each sample mass to evaluate their collagen concentration.

[0243] The results are grouped in Table 4 below, where the nascent collagen concentration is shown for each type of construct. These results were obtained by subtracting the concentration / mg of the control sample from the concentration of the various constructs tested (μg collagen mass per mg wet weight of the sample at the end of the cell culture). [Table 5]

[0244] Since the value corresponding to the control sample is not zero, it can be inferred that the Sircol test identifies the gelatin in the FAG hydrogel as collagen. The constructs showed a higher collagen concentration than the control, confirming the presence of nascent collagen.

[0245] Histological analysis was performed at the end of the culture. Constructs were cut in half. The first half was fixed in 4% formalin for 24 h, then dehydrated in successive baths of absolute ethanol and methylcyclohexane in a STP120 dehydrator (Microm) and embedded in kerosene. 5 μm thick sections were cut using a HM340e microtome (Microm). Hematoxylin-phloxine-saffron (HPS) staining was then performed on these sections.

[0246] The results are shown in FIG. 28, and the images reveal that both conditions tested supported the establishment of a thick healthy dermis accompanied by extracellular matrix synthesis, and a differentiated, multilayered epidermis.

[0247] This study demonstrated the feasibility of producing reconstituted tissue from a FAG hydrogel support cured by TAG. Different conditions resulted in reconstituted skin with a thick dermal equivalent and properly differentiated epidermis. The two methods tested here produced mature dermal-epidermal assemblies that demonstrated collagen neogenesis.

Claims

1. A method for curing a hydrogel comprising alginate and gelatin, the method comprising preparing the hydrogel and then contacting the hydrogel with a curing solution comprising at least one divalent cation and transglutaminase.

2. The method according to claim 1, wherein the contacting of the hydrogel with the curing solution is carried out by immersing the hydrogel in the curing solution or by spraying the curing solution onto the hydrogel.

3. The method according to claim 1, wherein the divalent cation(s) is selected from the group consisting of calcium, strontium and barium.

4. The hydrogel is - comprising 0.5 to 3% alginate and 1 to 17.5% gelatin, or - further comprising fibrinogen, and the curing solution further comprises thrombin, or - comprising up to 2% fibrinogen, or - further comprising viable cells incorporated during the preparation of the hydrogel prior to its curing The method according to claim 1, characterized in that.

5. The curing solution is: - 0.003 to 30% divalent cation, and - 0.004 to 16% transglutaminase The method according to claim 1, characterized in that it comprises.

6. The contacting with the curing solution is - at a temperature in the range of 15 to 40 °C, and / or - for a period in the range of 30 minutes to 6 hours The method according to claim 1, characterized in that it is carried out.

7. The method according to claim 1, characterized in that the hydrogel is shaped before its curing.

8. Use of a solution comprising a divalent cation and transglutaminase for curing a hydrogel comprising alginate and gelatin.

9. A hydrogel comprising cured alginate and gelatin, intended to be implanted in a human subject, obtainable by the curing method according to claim 1.

10. A method for curing a hydrogel comprising alginate and gelatin, comprising the following steps: - a step of preparing the hydrogel, - a step of shaping the hydrogel, - a step of contacting the hydrogel with at least one divalent cation The method comprising in sequence.

11. The method according to claim 10, characterized in that the preparation of the hydrogel does not require the shaping of one or more components prior to said preparation of the hydrogel.

12. The method according to claim 10, characterized in that the preparation of the hydrogel does not require the addition of fibers.

13. The method according to claim 10, characterized in that the divalent cation(s) is selected from the group consisting of calcium, strontium and barium.

14. A hydrogel comprising a cured alginate and gelatin, intended to be implanted in a human subject, obtainable by the curing method according to claim 10.

15. The hydrogel according to claim 14, further comprising viable cells.

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