METHOD FOR CONSOLIDATING AN ALGINATE / GELATIN HYDROGEL

Abstract

The present invention relates to a method for consolidating a hydrogel comprising alginate and gelatin, which includes a step of consolidating the hydrogel by crosslinking it with a suitable solution, in order to impart particularly advantageous mechanical properties. The use of this crosslinking solution to consolidate the structure of a hydrogel comprising alginate and gelatin is also another aspect of the present invention. In all these aspects of this invention, the hydrogel may further comprise fibrinogen and / or living cells.

Description

Title of the invention: METHOD FOR CONSOLIDATING AN ALGINATE / GELATIN HYDROGEL FIELD OF THE INVENTION

[0001] The present invention relates to the field of manufacturing materials in general, and in particular bio-materials or bio-compatible materials.

[0002] The present invention relates to a method for consolidating a hydrogel comprising alginate and gelatin, which includes a step of consolidating by crosslinking the hydrogel using a suitable solution, in order to give it particularly advantageous mechanical properties. The use of this crosslinking solution to consolidate the structure of a hydrogel comprising alginate and gelatin also constitutes another aspect of the present invention. In all aspects of this invention, the hydrogel may further comprise fibrinogen and / or living cells. STATE OF THE ART

[0003] Hydrogel-based structures comprising alginate and gelatin are known from the state of the art, but they lack satisfactory mechanical strength, because these constituents most often have limited elasticity (low Young's modulus, in particular), which makes the resulting structures difficult to handle.

[0004] International application published under number WO2017115056 describes the manufacture of hydrogel-based body substitutes comprising alginate, gelatin and fibrinogen, which are then treated with a crosslinking solution containing calcium and thrombin.

[0005] The mechanical properties of the structures obtained in the prior art nevertheless remain insufficient to make them suitable for manipulation. Furthermore, in the case of structures intended to be implanted in the human or animal body, and where appropriate sutured, the methods of the prior art do not make it possible to obtain structures which have adequate mechanical properties, and whose degradability in contact with living cells or tissues is not too rapid.

[0006] Furthermore, when the structures are likely to be cellularized during their manufacture, there is a need to be able to maintain the survival of the cells. Thus, the process for preparing such structures must be compatible with the presence of living cells and their maintenance in life. Statement of the invention

[0007] One of the aims of the invention is to overcome these drawbacks of the prior art, and to in particular to enable the production of hydrogels, cellularized or not, which have particularly advantageous mechanical characteristics never described in the prior art, in particular in terms of (i) the mechanical resistance of the constituents, (ii) stability over time, (iii) flexibility and (iv) resistance to tearing and shocks.

[0008] To this end, it is proposed to provide, according to a first aspect of the present invention, a method for consolidating a hydrogel comprising alginate and gelatin, said method comprising preparing the hydrogel, then bringing it into contact with a consolidation solution comprising at least calcium and transglutaminase, and optionally one or more other divalent cations.

[0009] Optionally, the consolidation solution may also contain thrombin if, in addition to alginate and gelatin, the hydrogel contains fibrinogen. Furthermore, the consolidation step is compatible with the fact that the hydrogel may contain living cells.

[0010] According to a second aspect of the present invention, there is described the use of a solution as described above for consolidating the structure of a hydrogel comprising alginate and gelatin, which has a very particular advantage when it comes to preparing a hydrogel intended to be implanted in the human or animal body, i.e. as a body implant.

[0011] This use can also be implemented to consolidate a hydrogel further comprising fibrinogen. Furthermore, the consolidation can also be carried out on a hydrogel further comprising living cells.

[0012] In summary, the present invention relates to a method for consolidating a hydrogel comprising alginate and gelatin, said method comprising preparing the hydrogel and then contacting it with a consolidating solution comprising at least calcium and transglutaminase, and optionally one or more other divalent cations.

[0013] Preferably, the contacting of the hydrogel with the consolidation solution is carried out by immersion, preferably total, of the hydrogel in the consolidation solution, or by spraying the hydrogel with the consolidation solution.

[0014] Preferably, the hydrogel comprises from 0.5 to 3% alginate and from 1 to 17.5% gelatin.

[0015] Preferably, the hydrogel further comprises fibrinogen and the consolidation solution further comprises thrombin.

[0016] Preferably, the hydrogel comprises up to 2% fibrinogen.

[0017] Preferably, the consolidation solution comprises: - from 0.003 to 30% divalent cation, - from 0.004 to 16% transglutaminase, and possibly - from 2 to 25 U / ml of thrombin.

[0018] Preferably, the hydrogel further comprises living cells integrated during the preparation of the hydrogel before its consolidation.

[0019] Preferably, the contact with the consolidation solution is carried out: - at a temperature ranging from 15 to 40°C, and / or - for a period ranging from 30 minutes to 6 hours.

[0020] Preferably, the hydrogel is shaped, prior to its consolidation, by a molding or 3D printing technique.

[0021] The present invention also relates to the use of a solution comprising calcium and transglutaminase, and optionally one or more other divalent cations, for consolidating a hydrogel comprising alginate and gelatin. DESCRIPTION OF THE FIGURES

[0022] Other characteristics, aims and advantages of the invention will emerge from the following description, which is purely illustrative and non-limiting, and which must be read in conjunction with the appended drawings in which:

[0023] [Fig. 1] [Fig. 1] represents the comparison of the Young's modulus and the viscosity of AG and FAG hydrogels which constitute the implants according to the invention.

[0024] [Fig.2] [Fig.2] represents the comparison of the Young's moduli E0 (Pa) of an AG hydrogel in which the gelatin is crosslinked with and without transglutaminase and stored for up to 7 days at 37°C.

[0025] [Fig.3] [Fig.3] represents the comparison of the Young's moduli E0 (Pa) of an AG hydrogel and commercial hydrogels crosslinked or not with transglutaminase.

[0026] [Fig.4] [Fig.4] represents the viability and cell growth measured kinetically on FAG and AG hydrogels which constitute the implants according to the invention, and which have been colonized in vitro by fibroblasts, after their manufacture.

[0027] [Fig.5] [Fig.5] represents the viability and cell growth measured kinetically on FAG and AG hydrogels which constitute the implants according to the invention, and which have been colonized in vitro by adipose tissue stem cells, after their manufacture.

[0028] [Fig.6] [Fig.6] represents the metabolic activity of AG implants according to the invention at different culture points following their in vitro colonization by a fraction of purified adipose tissue, after their manufacture.

[0029] [Fig.7] [Fig.7] represents the histological analyses by Hematoxylin, Phloxine, Saffron (HPS) staining of AG implants according to the invention after 2 days (4 images on the left) or 7 days (2 images on the right) of in vitro incubation with a fraction of purified adipose tissue, after their manufacture (Top: external edges of the matrices; Bottom: internal pores of the matrices; images taken in white light; magnification 100X; scale 100 pm).

[0030] [Fig.8] [Fig.8] represents the immunostaining of perilipin-1 and the staining cell nuclei with Dapi, on AG implants according to the invention after 2 days (top image) or 7 days (bottom image) of in vitro incubation with a purified adipose tissue fraction, after their manufacture (fluorescence imaging; magnification 200X; scale 50 pm).

[0031] [Fig.9] [Fig.9] represents the comparison of Young's moduli of AG implants for crosslinking at variable times at 21 and 37°C.

[0032] [Fig. 10] [Fig. 10] represents the comparison of the Young's moduli E0 and the Viscosities of AG and FAG implants after crosslinking with different concentrations of CaCl2, T AG and thrombin.

[0033] [Fig. 11] [Fig. 11] represents the comparison of the Young's moduli E0 and the Viscosities of AG and FAG implants after sequential or concomitant crosslinking with CaCl2, T AG and thrombin.

[0034] [Fig. 12] [Fig. 12] represents the comparison of the Young's moduli E0 and the Viscosities of AG and FAG implants after crosslinking with a solution containing calcium chloride or barium chloride.

[0035] [Fig.l3A] [Fig.l3A] illustrates the study of the variation in the dimensions and pores of AG and FAG implants according to the invention before and after crosslinking (A) and [Fig.l3B] [Fig.l3B] the impact of sterilization on the dimensions and Young's modulus of these implants (B).

[0036] [Fig. 14] [Fig. 14] illustrates the repeatability of AG implant production according to the invention in terms of dimensions, volume and porosity.

[0037] [Fig. 15] [Fig. 15] illustrates the repeatability of AG implant retraction according to the invention after consolidation.

[0038] [Fig. 16] [Fig. 16] illustrates the repeatability of AG implant retraction according to the invention depending on the sterilization method.

[0039] [Fig.l7A] [Fig.l7A] illustrates the repeatability of the extrusion diameter (A) and [Fig.l7B] [Fig.l7B] of the pore length (B) of AG implants according to the invention.

[0040] [Fig. 18] [Fig. 18] shows images of pores of varying size in an implant AG according to the invention.

[0041] [Fig. 19] [Fig. 19] represents the surgical plan (left) of the implantation in vivo subcutaneous (right) of AG and FAG implants according to the invention.

[0042] [Fig.20] [Fig.20] represents the histological analyses after staining with Masson trichrome on sections of AG implants according to the invention, after subcutaneous implantation in vivo in rat back sites for 3 weeks (low, medium and high magnification images).

[0043] [Fig.21A] [Fig.21A] represents the 28-day analysis of cell survival measured by lactate accumulation (A) and [Fig.21B] [Fig.21B] represents the 28-day analysis of cell growth followed by calcein labeling (B) in a cellularized FAG hydrogel.

[0044] [Fig.22] [Fig.22] represents the measurement of Young's moduli (E0) as a function of the initial cell concentration present in a cellularized FAG hydrogel.

[0045] [Fig.23] [Fig.23] represents the evolution over 21 days of the lactate concentration measured in the culture supernatants of cellularized and consolidated AG or FAG hydrogels.

[0046] [Fig.24] [Fig.24] represents the histological analyses after HPS staining of the bioprinted constructs, solid (left panel) or porous (right panel), of FAG fibroblast / endothelial cell hydrogel after 21 days of culture.

[0047] [Fig.25] [Fig.25] represents the histological analyses after HPS staining of the bioprinted constructs, solid (left panel) or porous (right panel), of AG fibroblast / endothelial cell hydrogel after 21 days of culture.

[0048] [Fig.26] [Fig.26] represents CD31-DAB immunostaining (black) on solid bioprinted constructs of FAG fibroblast / endothelial cell hydrogel after 21 days of culture.

[0049] [Fig.27] [Fig.27] represents CD31-DAB immunostaining (black) on porous bioprinted constructs of FAG fibroblast / endothelial cell hydrogel after 21 days of culture.

[0050] [Fig.28] [Fig.28] represents the histological analysis by HPS staining of the skins reconstructed from 3D bioprinted FAG hydrogel and consolidated with T AG. DETAILED DESCRIPTION OF THE INVENTION

[0051] The present invention provides a process for preparing hydrogels comprising alginate and gelatin, cellularized or not. By acting on the crosslinking of the constituents of the hydrogel using a particular and suitable solution, the structure of the hydrogel will be consolidated, so as to give it particularly advantageous mechanical characteristics, in particular in terms of the mechanical resistance of the constituents, stability over time and remarkable resistance to tearing and shocks.

[0052] In the present context, the expressions “consolidation solution” and “crosslinking solution” are equivalent and designate the solution used in the method according to the invention to consolidate the hydrogel by crosslinking.

[0053] Thus, in a first aspect, the present invention relates to a method for consolidating a hydrogel comprising alginate and gelatin, said method comprising preparing the hydrogel, then bringing it into contact with a consolidation solution comprising at least calcium and transglutaminase, and optionally actually one or more other divalent cations.

[0054] Alginate is a linear polysaccharide extracted from marine algae, primarily from the brown algae species Phaeophyceae. This biocompatible polymer is composed of homopolymeric blocks of 1,4 [3 -D mannuronic acid] (M) and its epimer C-5 aL guluronic acid (G). This biopolymer consists of sequences of M-blocks, G-blocks, intercalated with sequences of MG-blocks. Only the G units appear to be involved in intermolecular cross-linking during polymerization. Sodium alginate is widely used as a hydrogel.

[0055] Gelatin is a macromolecule derived from collagen that contains bioactive sequences such as the RGD (arginine-glycine-aspartic acid) motif for cell adhesion. It is obtained by denaturing the native triple helix structure of collagen via acid (gelatin type A), or alkaline (gelatin type B) treatment. The amino acid composition of gelatin is similar but different from that of collagen following denaturation (deamination of glutamine to glutamic acid in the manufacturing process of gelatin type B). The structure of gelatin changes during gelation.

[0056] The preparation of hydrogels is well known in the art (EM Ahmed; Journal of Advanced Research, 2015, 6, 105-121), as well as the polymerization and crosslinking of alginate and gelatin (Chen Q, Tian X, Fan J, Tong H, Ao Q, Wang X. An Inter-penetrating Alginate / Gelatin Network for Three-Dimensional (3D) Cell Cultures and Organ Bioprinting. Molecules. 2020;25(3):756.)).

[0057] Concerning the alginate, and according to what is mentioned above, an alginate enriched in M ​​unit will be more flexible because the chain will have a more linear configuration, while a gel containing more G units will be more rigid because it is more polymerized. In the context of the present invention, the alginate used preferably has an M / G ratio of 1.9.

[0058] Preferably, the gelatin contained in the hydrogel is of type A.

[0059] In the context of the present invention, the hydrogel is preferably prepared from an alginate solution to which a gelatin solution is added, or conversely from a gelatin solution to which an alginate solution is added, so as to obtain a hydrogel comprising from 0.5 to 3% alginate and from 1 to 17.5% gelatin, and even more preferably from 1 to 2.5% alginate and from 2 to 10% gelatin. Advantageously, the hydrogel comprises 2% alginate and 5% gelatin.

[0060] Unless otherwise indicated, the percentages mentioned in the present description are expressed in weight / volume of the total composition.

[0061] Preferably, when the hydrogel to be consolidated contains alginate and gelatin, these constituents are present in a mass ratio ranging from 1:0.3 to 1:35, and very particularly in a mass ratio of 1:2.5, respectively.

[0062] With regard to the consolidation solution, this preferably comprises: - from 0.003 to 30% of divalent cation(s), - from 0.004 to 16% transglutaminase.

[0063] More preferably, the consolidation solution comprises from 1 to 6% of divalent cation(s), and advantageously 3% of divalent cation(s).

[0064] More preferably, the consolidation solution comprises from 1 to 10% of transglutaminase, and advantageously 4% of transglutaminase.

[0065] The enzyme transglutaminase (TAG), is an extracellular aminoacyltransferase. It is a monomeric protein that has a single catalytic cysteine ​​residue (active site).

[0066] In the context of the invention, the consolidation solution preferably contains type 2 transglutaminase. This TAG is notably produced commercially as a recombinant microbial protein by fermentation of the microorganism Streptoverticillium moboarense. The consolidation solution used in the context of the invention may also contain several TAGs.

[0067] In addition to calcium, any other divalent cation, in particular non-toxic, can be used in the context of the method according to the invention.

[0068] However, it is preferably chosen from strontium and barium. It may also be envisaged to use several divalent cations in a mixture. Furthermore, the divalent cation(s) are present in the form of salts in the consolidation solution. Any salt may be used, and preferably anhydrous salts.

[0069] Preferably, the consolidation solution comprises only calcium and transglutaminase. Advantageously, if one or more divalent cation(s) other than calcium were present, it would preferably be a single other divalent cation, and in particular barium.

[0070] According to a preferred embodiment, the consolidation solution contains calcium chloride as the only divalent cation.

[0071] According to the present invention, the simultaneous crosslinking of the components of the hydrogel, i.e. the transformation into a three-dimensional polymer from a linear polymer, takes place by contacting with a consolidation solution in which (i) the calcium or the other divalent cation(s) allows the crosslinking of the alginate and (ii) the transglutaminase induces the enzymatic crosslinking of the gelatin.

[0072] Preferably, during the consolidation method according to the invention, the contacting of the hydrogel with the consolidation solution is carried out by immersion, during which the hydrogel is immersed in its entirety in the consolidation solution. This is also referred to as a consolidation bath. The contacting of the hydrogel with the consolidation solution can also be carried out by imbibition, by spraying, using a drip, trickle, or similar system. The contacting contact preferably relates to the entire hydrogel. According to a particularly advantageous embodiment, the hydrogel is completely immersed in the consolidation solution.

[0073] The hydrogel to be consolidated according to the present invention may further comprise fibrinogen, in addition to alginate and gelatin.

[0074] The fibrinogen monomer is composed of two repeats of three α, β and γ chains linked by a central domain E and two fibrinopeptides A and B (FpA, FpB) linking the α chains to the E domain. It has a large number of cell adhesion motifs and thus allows increased development of cells within the hydrogel.

[0075] According to a particularly preferred embodiment, the hydrogel which is prepared to be consolidated according to the method of the invention is composed of alginate and gelatin, or else of alginate, gelatin and fibrinogen, without any other constituent capable of forming a gel.

[0076] Preferably, when the hydrogel comprises fibrinogen, in addition to alginate and gelatin, it is prepared so as to contain up to 6% fibrinogen, and in particular from 0.0001% to 6% fibrinogen, and in particular 2% fibrinogen.

[0077] Also preferably, when the hydrogel contains alginate, gelatin and fibrinogen, these constituents are present in a mass ratio ranging from 1:0.3:0.00003 to 1:35:12, and very particularly in a mass ratio of 1:1:2.5, respectively.

[0078] When the hydrogel contains fibrinogen in addition to alginate and gelatin, the consolidation solution used in the method according to the invention further comprises thrombin, i.e. thrombin in addition to calcium and TAG, and optionally one or more other divalent cations.

[0079] Indeed, and in addition to what is mentioned above on the crosslinking of alginate and gelatin, thrombin in this case crosslinks fibrinogen into fibrin. Fibrin is a natural biological polymer resulting from a polymerization mimicking the last step of the coagulation cascade by the action of thrombin on fibrinogen. Thrombin will initially cleave fibrinopeptide A, leading to the formation of a protofibril. The cleavage of fibrinopeptide B leads to a release of the α chains and subsequently the lateral polymerization of the fibrinogens forming fibrin.

[0080] Since the hydrogel to be consolidated by the method of the invention comprises fibrinogen, the consolidation solution used comprises thrombin, in addition to calcium and TAG, and optionally one or more other divalent cations. In this case and preferably, the consolidation solution comprises: - from 2 to 25 U / ml of thrombin, - from 0.003 to 30% of divalent cation(s), and - from 0.004 to 16% transglutaminase.

[0081] As above, more preferably, the consolidation solution comprises from 1 to 6% of divalent cation(s), and advantageously 3% of divalent cation(s).

[0082] More preferably, the consolidation solution comprises from 1 to 10% of transglutaminase, and advantageously 4% of transglutaminase.

[0083] More preferably, when the consolidation solution also comprises thrombin, the latter is preferably present from 2 to 10 U / ml.

[0084] Still in this particular embodiment and even more preferably, the consolidation solution comprises 4 U / ml of thrombin, 3% of calcium, and 4% of transglutaminase.

[0085] In the context of the invention, the alginate, gelatin and, where appropriate, fibrinogen, which are used are chosen from those which have characteristics most similar to the following: - alginate: viscosity of 130-300 mPa.s for a 2% solution; - gelatin: type A, porcine, bloom degree 280 (strength or resistance to penetration); - fibrinogen: human, coagulable protein level >91 mg / mL; - thrombin: human, activity > 500 U / mL.

[0086] The hydrogels advantageously contain alginate, gelatin and optionally fibrinogen as natural constituents of the hydrogel. However, other natural components may also be present in the hydrogels, among which we can notably cite: chitin, chitosan, cellulose, agarose, chondroitin sulfate, hyaluronic acid, glycogen, starch, pullulan, carrageenan, heparin, collagen, albumin, fibrin, fibroin, dextran, xanthan, gellan, any component extracted from extracellular matrix such as collagens, laminin, proteoglycans of the Matrigel type, methacrylate gelatin of the GelMa type.

[0087] In addition to the constituents of natural origin, and in particular those listed above, the hydrogels of the present invention may also contain synthetic components, such as polyolefins (PE, PP, PTFE, PVC), silicone (PDMS), polyacrylates (PMMA, pHEMA), polyester (PET, dacron, PGA, PLLA, PLA, PDLA, PDO, PCL), polyethers (PEEK, PES), polyamides, polyurethanes, PEG, pluronic F127.

[0088] Textile fibers of natural or synthetic origin may also be present in the composition of hydrogels.

[0089] According to another preferred embodiment of the method of the invention, the hydrogel further comprises living cells.

[0090] These living cells can be of any type, except stem cells human embryonic stem cells obtained by destruction of an embryo, and preferably several types of living cells can coexist. The living cells are preferably chosen from cells of epithelial tissue, connective tissue, adipose tissue, endothelial tissue, and in particular from fibroblasts, keratinocytes, adipose tissue stem cells, adipocytes, melanocytes, endothelial cells, macrophages, leukocytes, etc. In this implementation of the method of the invention, the cells are therefore manipulated under conditions that the person skilled in the art is able to determine in the art, to allow the maintenance of their viability and proliferation, and ideally their differentiation.

[0091] These cells can be integrated during the preparation of the hydrogel itself when the alginate, gelatin, and optionally fibrinogen solutions are mixed to prepare the hydrogel, before its consolidation. This is then referred to as a cellularized hydrogel. They can also be added to the consolidation solution or even be added following the consolidation of the hydrogel. Preferably, the hydrogel to be consolidated according to the invention comprises living cells which are integrated during the preparation of the hydrogel before its consolidation. For example, the living cells can be suspended in the fibrinogen solution, to which the alginate and gelatin are added, in one or more steps, so as to obtain a hydrogel comprising the quantities of alginate / gelatin / fibrinogen mentioned above.

[0092] To illustrate different implementations of the present invention, the following sequences are preferably used to prepare the hydrogel to be consolidated according to the method of the invention: (i) alginate is added to the living cell suspension, then gelatin is added; (ii) alginate is added to fibrinogen, then gelatin is added; (iii) the living cells are suspended in fibrinogen, then alginate is added, and then gelatin. Advantageously, sequence (iii) is used to prepare a cellularized hydrogel according to the invention.

[0093] In these particular sequences, the range preferences for each of the constituents mentioned above also apply. It is also possible to prepare the hydrogel to be consolidated in a single step, i.e. by mixing all the constituents at the same time, whether alginate and gelatin, and possibly fibrinogen and / or living cells, if applicable.

[0094] According to a preferred embodiment of the method of the invention, the consolidation step which consists of bringing the hydrogel into contact with the consolidation solution, is carried out at a temperature ranging from 15 to 40°C, and preferably from 20 to 40°C and even more preferably from 21 to 37°C.

[0095] According to another preferred embodiment, but which can also be combined with the one above concerning the temperature conditions, this consolidation step is carried out for a duration ranging from 30 minutes to 6 hours, and ideally for 1 hour to 3 hours.

[0096] Thus, according to an advantageous embodiment of the method of the invention, the consolidation step is carried out at 37°C for 1 hour 30 minutes.

[0097] According to another advantageous embodiment of the method of the invention, the hydrogel is made up of alginate and gelatin, or of alginate, gelatin and fibrinogen, and the consolidation step is carried out at 37°C for 1 hour 30 minutes.

[0098] According to yet another advantageous embodiment of the method of the invention, the hydrogel is made up of alginate and gelatin, or of alginate, gelatin and fibrinogen, and the consolidation step is carried out by total immersion of the hydrogel in the consolidation solution at 37°C for 1 hour 30 minutes.

[0099] In the context of the consolidation method of the invention, this may comprise a step of shaping the hydrogel once prepared, and prior to its consolidation.

[0100] This step of shaping the hydrogel consists of giving it a particular shape and structure or architecture, and in particular adapted to the destination of the hydrogel once consolidated.

[0101] The hydrogels as described in detail above, once prepared, can therefore be "shaped", before consolidation, by different methods well known to those skilled in the art allowing volume structuring (3D in particular), and in particular by addition or agglomeration of material by stacking of layers or successive deposition. Among these methods, mention may in particular be made of injection methods, extrusion methods, and in particular molding, 3D printing. In particular, the person skilled in the art will take care to choose a method which allows the shaping of high viscosity materials, since a hydrogel consisting solely of alginate and gelatin can have a viscosity ranging from 50 to 6000 Pa.s., for a measurement at a temperature of 5 to 45°C.

[0102] In the context of the invention, the hydrogel which is consolidated according to the consolidation method is preferably shaped before consolidation, by a 3D printing technique.

[0103] As indicated previously, the implementation of the method according to the invention makes it possible to confer on the hydrogel, once consolidated, advantageous mechanical properties, which are particularly suitable for the provision of a hydrogel intended to be implanted in the human or animal body, that is to say as a body implant.

[0104] For this, the shaping of the hydrogel, prior to its consolidation, by the use of 3D printing has the advantage of being able to prepare a “custom-made” structured hydrogel whose dimensions and / or filling / porosity are defined. with regard to the needs of the body of the organism intended to receive the bodily implant and the role / function it will have to play in this recipient organism. Indeed, the porosity of the implant is a key parameter to be adjusted according to the tissues or organs concerned which are in particular to be replaced and / or augmented. Porosity reflects the empty space present in the implant which can be adapted in order to provide more or less material and thus to confer a certain mechanical resistance approaching as closely as possible that of the native tissue of the implantation area. It can in particular be expressed in two different but correlated and therefore equivalent or alternative ways: the pore size expressed in micrometers and / or the hydrogel filling rate expressed as a percentage (hydrogel volume / total volume of the implant).

[0105] To consolidate a hydrogel containing living cells (cellularized hydrogel or cellularization subsequent to the manufacture of the hydrogel) having such a destination, it is moreover particularly advantageous in terms of safety that the living cells are autologous cells, that is to say cells originating from the recipient organism.

[0106] According to a second aspect, the present invention relates to the use of a solution comprising calcium and transglutaminase, and optionally one or more other divalent cations, for consolidating a hydrogel comprising alginate and gelatin.

[0107] This consolidation solution can also be used to consolidate a hydrogel comprising, in addition to alginate and gelatin, fibrinogen and therefore, said solution will comprise thrombin in addition to calcium and transglutaminase, and the possible other divalent cation(s).

[0108] All the above-mentioned preferences concerning the consolidation method apply mutatis mutandis to the use of the consolidation solution according to the invention. EXAMPLES

[0109] Materials & Methods

[0110] Protocol #1 Preparation of an AG hydrogel: In order to prepare the AG hydrogel, 2 g of alginate (very low viscosity, Alpha Aesar, France), 5 g of gelatin (Sigma-Aldrich, France) are dissolved at 37°C for 12 hours in 100 mL of a 0.1M NaCl solution (Labelians, France).

[0111] Protocol #2 Preparation of a FAG hydrogel: In order to prepare the FAG hydrogel, 2 g of alginate (very low viscosity, Alpha Aesar, France), 5 g of gelatin (Sigma-Aldrich, France) and 2 g of fibrinogen (Sigma-Aldrich, France) are dissolved at 37°C for 12 hours in 100 mL of a 0.1 M NaCl solution (Labelians, France).

[0112] Protocol #3 Molding of an AG or FAG hydrogel: 1.8mL of the prepared hydrogel according to protocol #1 or #2 are deposited in the wells of a 6-well culture plate and incubated at 21°C for 30 minutes.

[0113] Protocol #4 Crosslinking of an AG hydrogel: A crosslinking solution is prepared by dissolving 4 g of Transglutaminase (Ajinomoto, Japan), 3 g of CaCl2 (Sigma Aldrich, France) in 100 mL of a 0.1 M NaCl solution (Labelians, France). The crosslinking solution is then brought into contact with the hydrogel for 1 hour 30 minutes at 37°C (unless otherwise indicated).

[0114] Protocol #5 Crosslinking of a FAG hydrogel: A crosslinking solution is prepared 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 a 0.1 M NaCl solution. The crosslinking solution is then brought into contact with the hydrogel for 1 hour 30 minutes at 37°C (unless otherwise indicated).

[0115] Protocol #6 Dynamic mechanical analysis (DMA) in compression: The mechanical properties of the FAG and AG hydrogels are measured in triplicate with a rotational rheometer (DHR2, TA Instrument, France), a Peltier plane (TA Instrument, France) and a notched 8mm geometry (TA Instrument, France). Three 8mm diameter discs are cut from the molded hydrogels according to protocol #3. The disc is placed on the lower geometry at 37°C for 60 seconds then an oscillatory lOpm compression procedure is carried out from 0.1 to 10Hz at lOOpm / s and at 37°C. The values ​​of the Young's modulus E0 (Pa) and the viscosity qO (Pa.s) of the hydrogel are obtained from a modeling of the visco-hyperelastic solid using the E' and E” values ​​acquired during the test.

[0116] Protocol #7 Preparation of a cellularized hydrogel: In order to prepare the cellularized FAG hydrogel, 0.12 g of alginate (very low viscosity, Alpha Aesar, France), 0.3 g of gelatin (Sigma-Aldrich, France) are dissolved in 6 mL of DMEM culture medium (Gibco Cell Culture, Invitrogen, France). The freshly trypsinized cells are resuspended in 2 mL of an 8% fibrinogen solution (Sigma-Aldrich, France). This cell suspension is then added to the previous solution in order to form the cellularized FAG. To prepare the cellularized AG hydrogel, 0.12 g of alginate (very low viscosity, Alpha Aesar, France), 0.3 g of gelatin (Sigma-Aldrich, France) are dissolved in 6 mL of DMEM culture medium (Gibco Cell Culture, Invitrogen, France). The freshly trypsinized cells are resuspended in 2 mL of 0.1 M NaCl solution (Labelians, France).This cell suspension is then added to the previous solution in order to form the cellularized AG.

[0117] Protocol #8 3D printing of hydrogels: The hydrogels prepared according to protocol #1, #2 are transferred into a 30 mL cartridge (Nordson EFD) equipped with a 410 pm diameter extrusion nozzle (Nordson EFD). The cartridge assembly The nozzle is then placed in a 3D printer (BioassemblyBot, Advanced Solution Lifescience, USA) allowing constant pressure to be applied to the cartridge while moving in all three directions of space. The printing parameters are a speed of 10mm / sec, a pressure of 25-35PSI and a temperature of 21°C. Obtaining different filling rates is carried out by the internal slicer of the printer control software (Tsim, Advanced Solution Lifescience, USA).

[0118] Protocol #9 In vivo implantation in rats: In vivo implantation studies in rats were conducted on the BIOVIVO preclinical research technical platform - Institut Claude Bourgelat (Lyon, France). The experiments were conducted in accordance with European Directives 2010 / 63 / EU. The 16 animals (Sprague Dawley rat, 250-300g) were anesthetized by inhalation (oxygen and 5% isoflurane). The dorsal implantation sites were shaved and disinfected with povidone and sterile gauze, and sterile drapes were placed to delimit the surgical area. General anesthesia was maintained by isoflurane (2%) and oxygen inhalation. Pre-surgical analgesia was performed subcutaneously with meloxicam and morphine at 1mg / kg, respectively. The rats' body temperature and pulse rate were monitored during the operation. Two 2-3 cm skin incisions were made in the dorsal region. A bioprosthesis was implanted in the subcutaneous dorsal region of each animal. The control group was created by performing only the incision and dissection. In one animal per group, 4 surgical sites were made, three bioprostheses and one control sample. The surgical site was closed in layers using subcutaneous and cutaneous sutures with absorbable braided sutures (PDS® polidioxanone, 4 / 0 and Nylon 3 / 0, Ethicon J&J). After surgery, animals were monitored for signs of distress, and surgical wounds were inspected daily for skin healing and infection. Explantation took place 21 days after implantation.

[0119] Protocol #10 Histological analysis: The implants are fixed for 24 hours in a 4% formalin solution (Alphapat, France) then dehydrated by successive baths of absolute ethanol (vwr Chemicals, France) and methylcyclohexane (vwr Chemicals, France) with an STP 120 dehydrator (Myr, Spain) then embedded in paraffin (Sakura, Japan). Sections of 5qm thickness are made with a HM 340e microtome (Microm, France). Hematoxylin Phloxine Safran (HPS), Masson's Trichrome staining and DAPI labeling were performed.

[0120] Example 1 - Mechanical properties of alginate / gelatin (AG) and fibrinogen / alginate / gelatin (FAG) hydrogels

[0121] AG and FAG hydrogels were prepared from protocols #1 and #2, molded according to protocol #3, then crosslinked using protocols #4 and #5 in order to study their DMA mechanical properties using protocol #6.

[0122] The results are grouped in [Fig.l]. The measured Young's modulus and viscosity values ​​are similar between the AG hydrogel and the FAG hydrogel following their crosslinking by the method of the invention. The Young's moduli under the precise conditions of this study are around 68 OOOPa.

[0123] Example 2 - Impact of crosslinking with transglutaminase on the mechanical properties of alginate / gelatin (AG) hydrogel

[0124] The molded AG samples were prepared from protocols #1 and #3 and crosslinked from a variant of protocol #4. In this variant, the crosslinking solution is composed of a 30mg / mL calcium chloride solution only or a 30mg / mL calcium chloride solution and 40mg / mL transglutaminase. 4 gels of each condition were molded and then tested in DMA on the same day and respectively after 1, 4 and 7 days of storage at 37°C in order to mimic physiological conditions.

[0125] The samples were then studied by DMA using protocol #6.

[0126] The results are grouped in [Fig.2]. This study shows the advantageous effect of the use of transglutaminase during crosslinking on the mechanical properties of hydrogels. This effect is even greater when the gels are kept at 37°C, justifying the particular interest of crosslinking according to the invention for hydrogels intended to be implanted.

[0127] Example 3 - Impact of crosslinking with transglutaminase on the mechanical properties of commercial hydrogels based on gelatin and / or collagen

[0128] The molded AG samples were prepared from protocols #1 and #3 and crosslinked from protocol #4. The commercial hydrogel samples listed in Table 1 below were prepared according to the protocols provided by the suppliers and molded according to protocol #3.

[0129] [Tables 1] Hydrogel Trade Name Reference Supplier Gel4Cell BIS-101 Bioink Solution Gel4Cell-VEGF BIS-103 Bioink Solution Col4Cell BIS-108 Bioink Solution GelMA VL5000000010 Cellink Lifeink 200 Type I Collagen 5278-5ML CellSystems BiogelX B5X-0000 Biogelx Rat Tail Collagen A10483-01 Sigma

[0130] The hydrogels were crosslinked with a variation of protocol #4, using either a solution comprising only 30mg / mL calcium (no TAG), or a solution of 30mg / mL calcium and 40mg / mL transglutaminase, in order to observe the impact of TAG.

[0131] The non-crosslinked and TAG-crosslinked samples were then studied by DMA using protocol #6.

[0132] The results are grouped in [Fig.3]. 6 of the 7 commercial hydrogels studied were crosslinked by transglutaminase. The collagen-based hydrogels (Col4Cell, Rat Collagen) are not rigid enough to be analyzed by DMA but the gelatin-based hydrogels (Gel4cell, Gel4cell-VEGF and GelMa) have a significantly higher Young's modulus after crosslinking by transglutaminase (7.3, 9.9 and 50 kPa respectively). This study shows the effect of crosslinking with transglutaminase on the rigidity of commercial hydrogels.

[0133] Example 4 - Influence of the amount of alginate and gelatin in a fibrinogen / alginate / gelatin (FAG) hydrogel on the mechanical properties

[0134] FAG hydrogels were prepared from a variant of protocol #2, molded according to protocol #3, then crosslinked using protocol #5, then their mechanical properties were studied by DMA using protocol #6. In this variant, we studied these mechanical properties by preparing the FAG hydrogel, with 1 or 3 or 2 g of alginate, and 10 or 7.5 or 5 g of gelatin, respectively, and 2 g of fibrinogen.

[0135] The results are grouped in Table 2 below. Young's moduli under the precise conditions of this study range from 200 to 800 kPa.

[0136] [Tables2] Gelatin (%) Alginate (%) Fibrinogen (%) Young's modulus (kPa) 10 1 2 200 7.5 3 2 600 5 2 2 800

[0137] Example 5 - Evaluation of colonization of fibrinogen / alginate / gelatin (FAG) and alginate / gelatin (AG) hydrogels by fibroblasts

[0138] AG and FAG hydrogels were prepared using protocols #1 and #2. Square implants with sides of 1.5 cm and thickness of 0.2 cm were then printed using protocol #8 and crosslinked using protocols #4 or #5. The printed implants were produced with a 50% filling rate and an extrusion nozzle with an internal diameter of 410 μm. A negative control (empty well) was also used.

[0139] Normal human fibroblasts in passage 6 were thawed and amplified in 175cm2 culture flasks in culture medium containing DMEM supplemented with 10% calf serum and 1% antibiotics. Each implant was seeded on its surface with a cell suspension of normal human fibroblasts at a concentration of 4,000,000 fibroblasts / ml. 250 μl of this suspension were deposited drop by drop on each implant, i.e. 1,000,000 fibroblasts / implant. After 1 hour of adhesion, the implants were immersed with culture medium. The implants were cultured in culture medium composed of DMEM containing 10% calf serum supplemented with vitamin C and EGF (Epidermal Growth Factor) at 37°C, 5% CO2. The implants were cultured with this same medium for 21 days, renewed every 3 days.

[0140] The metabolic activity of fibroblasts within the implants was studied by colorimetric analysis with Alamar Blue on culture days 3, 5, 8, 10, 14 and 21 after seeding. The solution was made by diluting a solution of Alamar Blue (DAL 1100, Invitrogen) to 10th in DMEM. After 19 hours of incubation at 37 °C, 100 μl of the supernatants were collected and their absorbance at 570 nm and 600 nm was measured using a spectrophotometer (NanoQuant® infinité M200PRO, TECAN).

[0141] Cell viability and growth were thus monitored over 21 days of culture using 6-point kinetics on days 3, 5, 8, 10, 14 and 21. The results are grouped in [Fig.4].

[0142] The results confirmed that all implants allowed fibroblast adhesion and survival from the 3rd day of culture. Cell growth was observable for each porous implant over the 21 days of culture, on both types of hydrogels (FAG and AG) as well as for each porosity used.

[0143] Example 6: Evaluation of the colonization of fibrinogen / alginate / gelatin (FAG) and alginate / gelatin (AG) hydrogels by adipose tissue stem cells (ASC).

[0144] The AG and FAG hydrogels were prepared using protocols #1 and #2. Square implants with sides of 1.5 cm and thickness of 0.2 cm were then printed using protocol #8 and crosslinked using protocols #4 or #5. The printed implants were produced with a filling rate of 50% and 75% and an extrusion nozzle with an internal diameter of 410 pm. Sterilization was carried out by the company IONISOS (France) by irradiation of the implants with a dose of 30 kGy of gamma rays.

[0145] Normal human adipocyte stem cells in passages 2 to 5 are thawed and amplified in 175cm2 culture flasks in culture medium containing DMEM supplemented with 10% serum and 1% antibiotics. Each implant was seeded on its surface with a cell suspension of ASC at a concentration of 6, 12 or 24 million ASC / ml. 250 μl of these suspensions were deposited drop by drop on each implant, i.e. 1.5, 3 or 6 million ASC / implant. After 1 hour of adhesion, the implants were immersed with culture medium. The implants were cultured in culture medium containing DMEM supplemented with 10% serum and 1% antibiotics for 7 days and then in medium containing DMEM supplemented with 10% serum, insulin, rosiglitasone and 1% antibiotics for 14 days. The culture media were renewed every 3 days.

[0146] The metabolic activity of fibroblasts within the implants was studied by colorimetric analysis with Alamar Blue on culture days 3, 5, 7, 14 and 21 after seeding. The solution was made by diluting a solution of Alamar Blue (DAL 1100, Invitrogen) to 10th in DMEM. After 5 hours of incubation at 37 °C, 100 μl of the supernatants were collected and their absorbance at 570 nm and 600 nm was measured using a spectrophotometer (NanoQuant® infinité M200PRO, TECAN).

[0147] Cell viability and growth were thus monitored over 21 days of culture using 6-point kinetics on days 3, 5, 7, 14 and 21. The results are grouped in [Fig.5].

[0148] The results confirmed that all implants allowed the adhesion and survival of adipocyte stem cells from the 3rd day of culture. Cell growth was observable for each porous implant over the 21 days of culture, on both types of hydrogels (FAG and AG) as well as for each seeding density.

[0149] Example 7: Evaluation of the colonization of alginate / gelatin (AG) hydrogels in contact with a fraction of purified adipose tissue.

[0150] AG hydrogels were prepared from protocol #1. Cubic implants of 1.5 cm side and 0.8 cm thickness were then printed according to the protocol #8 and crosslinked using protocol #4. The printed implants are produced with a 50% filling rate and an extrusion nozzle with an internal diameter of 410 pm.

[0151] The lipoaspirate is centrifuged at 1500 RPM for 2 minutes and then rinsed with PBS 1X. The lipoaspirate was centrifuged again at 1500 RPM for 30 seconds and then the PBS 1X was removed. The lipoaspirate is considered purified.

[0152] Each implant is then immersed in 6mL of purified lipoaspirate, then the whole was placed in a culture insert in a 6-well plate with incubation in medium containing DMEM supplemented with 10% serum and 1% antibiotics at 37°C 5% CO2 for 2 days or 7 days.

[0153] Following contact with the lipoaspirate, the implants were cultured in 6-well plates in culture medium containing DMEM supplemented with 10% serum, insulin, rosiglitasone and 1% antibiotics, with 3 medium renewals per week for up to 21 days.

[0154] The cellular metabolic activity within the implants was studied by colorimetric analysis with Alamar Blue on culture days 2, 7 and 21 after seeding. The solution was made by diluting to 10th a solution of Alamar Blue (DAL 1100, Invitrogen) in DMEM. After 5 hours of incubation at 37 °C, 100 μl of the supernatants were collected and their absorbance at 570 nm and 600 nm was measured with a spectrophotometer (NanoQuant® infinité M200PRO, TECAN).

[0155] Cell viability and growth were thus monitored over 21 days. The results are grouped in [Fig.6].

[0156] A metabolic activity much higher than the negative control was observed in the implants which had been in contact with a purified lipoaspirate.

[0157] Histological analyses were carried out to complete this study according to protocol #10. The results are grouped in [Fig.7].

[0158] The images reveal the presence of agglomerated, polygonal, uniform, unilocular and voluminous adipocytes. These morphological characteristics are those of healthy adipocytes, which can be found in adipose tissue.

[0159] Immunostaining for perilipin-1 was also performed. Samples were embedded in OCT (CellPath, KMA-0100-00A), then stored at -80 °C. Sections 16 μm thick were made for each sample with a cryostat (Microm, HM 520). The sections were then fixed in an acetone / methanol (v / v) solution for 20 minutes and rinsed 3 times in 1X PBS. A one-hour incubation at room temperature in a 4% PBS-BSA solution was performed to saturate the nonspecific sites. The sections were then incubated overnight at room temperature with a primary antibody solution specific for perilipin-1. The next day, the sections were rinsed three times in 1X PBS and then incubated 45 minutes with a secondary antibody solution coupled to Alexa fluor 568, at room temperature. Then the sections were rinsed 3 times with 1X PBS and mounted between slide and coverslip with Dapi fluoromount-G® mounting medium (SouthernBiotech). The images obtained are grouped together [Fig.8].

[0160] The images show adipocytes with large spherical or polygonal vacuoles depending on the grouping of the cells. The adipocytes appear as unilocular and their size is also physiological since it is between 50 and 200 qm.

[0161] All of these results confirm the adhesion, survival and regeneration of human adipose tissue brought into contact with the implants. The particular structure and composition of the implants thus form an environment favorable to the regeneration of healthy adipose tissue.

[0162] Example 8 - Influence of temperature and crosslinking time on the mechanical properties of an alginate / gelatin (AG) hydrogel

[0163] The molded AG samples were prepared from protocols #1 and #3 and crosslinked from a variant of protocol #4. In this variant, the crosslinking times and temperatures were modified, from 10 minutes to 14 hours and from 37°C to 21°C.

[0164] The samples were then studied by DMA using protocol #6.

[0165] The results are grouped in [Fig.9]. The crosslinking times as well as the tem Crosslinking temperature has very little influence on the final mechanics (Young's modulus) of the hydrogels. However, it seems that an optimum can be found around 1h30, whatever the temperature.

[0166] These Young's moduli are also very stable over 7 days after crosslinking at 37°C. The crosslinking of the gelatin was effective since there is no loss of dissolved gelatin in the medium.

[0167] Example 9 - Impact of the concentration of the components of the crosslinking solution on the mechanical properties of alginate / gelatin (AG) and fibrinogen / alginate / gelatin (FAG) hydrogels once crosslinked

[0168] The molded samples of AG and FAG were prepared from protocols #1, #2 and #3, crosslinked from a variant of protocols #4 and #5. In this variant, the concentrations of the components of the crosslinking solution were modified (transglutaminase, calcium chloride and thrombin).

[0169] The samples were then studied by DMA using protocol #6.

[0170] The results are grouped in [Fig. 10]. Over this range of concentrations of reagents, no significant variations were observed (E0 all very similar).

[0171] Example 10 - Impact of sequential or concomitant crosslinking of alginate / gelatin (AG) and fibrinogen / alginate / gelatin (FAG) hydrogel

[0172] The molded samples of AG and FAG were prepared from protocols #1, #2 and #3, crosslinked from a variant of protocols #4 and #5. In this variant, we studied a sequential crosslinking on FAG and AG, which consists of crosslinking the hydrogel in several stages. Each stage lasted 1 h and three rinses with a 0.1 M NaCl solution were carried out between each stage in order to remove the residual crosslinking agents.

[0173] The samples were then studied by DMA using protocol #6.

[0174] The results are grouped in [Fig. 11]. All sequential crosslinks (FAG and AG) generate hydrogels with lower Young's moduli than single-step crosslinking.

[0175] It can be observed that very flexible and fragile gels are obtained if calcium is not added first, in fact TAG and thrombin are calcium-dependent their activity is therefore largely reduced without the addition of CaCl2. The gels are therefore difficult to handle without calcium crosslinking. When thrombin is added first, the gels have very little mechanical strength and holes appear.

[0176] Example 11 - Impact of the nature of the divalent cation for the crosslinking of alginate / gelatin (AG) and fibrinogen / alginate / gelatin (FAG) hydrogels

[0177] The molded samples of AG and FAG were prepared from protocols #1, #2 and #3, crosslinked from a variant of protocols #4 and #5. In this variant, We studied crosslinking in the presence of 30 mg / mL barium chloride.

[0178] The samples were then studied by DMA using protocol #6.

[0179] The results are grouped in [Fig. 12]. Crosslinking in the presence of barium allows to obtain gels with Young's moduli very similar to those obtained with CaCl2. However, since barium allows to increase the viscosity of the gels, we can assume the formation of additional pendant chains.

[0180] Example 12 - Maintenance of the three-dimensional structure and mechanical properties of alginate / gelatin (AG) and fibrinogen / alginate / gelatin (FAG) hydrogel implants after sterilization

[0181] The AG and FAG hydrogels were prepared from protocols #1, #2 and #3, crosslinked using protocols #4 and #5, observed optically and then studied by DMA using protocol #6. The printed shapes are half-spheres of 2 cm diameter produced with variable filling rates (30, 50 and 75%).

[0182] Sterilization was carried out by the company IONISOS (France) by irradiation of the implants with a variable dose (30 kGy and 40 kGy) of Gamma rays.

[0183] The impact of the crosslinking step on the dimensions of alginate / gelatin and fibrinogen / alginate / gelatin hydrogel implants was studied. These dimensions were measured from macroscopic images.

[0184] The dimensions of the pores obtained as a function of the filling rate also have were studied. These dimensions were measured from images taken using a microscope (Olympus, x4 magnification).

[0185] The results are grouped in FIG. 13. The implants shrink on average by 10% following the crosslinking step. The pore size, however, does not vary significantly ([Fig.l3A]).

[0186] Regarding sterilization, it appears that the 40 kGy dose causes a greater shrinkage of the constructs than the 30 kGy dose. Regarding E0, sterilization does not cause any change in the mechanics of the material for both doses ([Fig.l3B]).

[0187] Example 13: Production quality of large-sized implant based on an alginate / gelatin (AG) hydrogel: repeatability of printing dimensions, dimensions after consolidation and sterilization of the implant according to several methods.

[0188] AG hydrogels were prepared using protocol #1. Hemisphere-shaped implants of 6cm diameter and 2cm thickness were then printed using protocol #8 and crosslinked using protocol #4, then optically observed and measured. The printed shapes are produced with variable filling rates (25 to 65%) and extrusion nozzles of 410 or 840 pm internal diameter. Sterilization was carried out by IONISOS (France) by irradiation of the implants with 2 doses (30 kGy and 40 kGy) of Beta rays or a dose of gamma rays of 30 kGy.

[0189] The impact of the crosslinking and sterilization step on the dimensions of large alginate / gelatin hydrogel implants was studied. These dimensions were measured from macroscopic images.

[0190] The dimensions of the pores obtained as a function of the filling rate were also studied. These dimensions were measured from images taken using a microscope (Olympus, x4 magnification).

[0191] The results after printing are grouped together [Fig.14]. These results show a high repeatability of the dimensions of the large-size 3D-printed implants, reflecting a high production quality.

[0192] The results after consolidation of the implants are grouped [Fig. 15]. This graph shows a high repeatability of the retraction of large implants after the consolidation step.

[0193] The results after sterilization of implants according to 3 methods (rays [3 doses 40 and 30 kGy and rays y 30 kGy) are grouped [Fig. 16]. These results show a lower retraction of large implants with rays [3 30 and 40 kGy.

[0194] Large-size implants were printed with 2 extrusion nozzles with internal diameters of 410 and 840 pm with filling rates of 25 to 65%. The repeatability of the extrusion diameter as well as the length of the pores obtained were measured. results are grouped in FIG 17.

[0195] [Fig. 17A] shows the high repeatability of the extruded bead size.

[0196] Images of pores of varying size were taken and are grouped in [Fig. 18]

[0197] These data show the wide range of pores that can be obtained for the implants and their high repeatability and production quality.

[0198] Example 14 - In vivo implant resistance studies

[0199] AG and FAG hydrogels were prepared from protocols #1, #2 and #8, crosslinked using protocols #4 and #5. The printed shapes are 1 cm diameter half-spheres, produced with variable filling rates (30, 50 and 75%).

[0200] The porous half-spheres were sterilized with the dose of 30kGy then implanted subcutaneously in rats according to protocol #9.

[0201] The details of the implantation groups are described in the following Table 3, which refers to the surgical implantation plan described in [Fig. 19].

[0202] [Tables3] Group (hydrogel / filling rate) animal number implant number implant position FAG 30% 1 1 A 2 2 A 3 3 A 4 4 A 5 B 6 C AG 30% 5 7 A 6 8 A 7 9 A 8 10 A 11 B 12 C AG 50% 9 9 A 10 14 A 11 15 A 12 16 A 17 B 18 C AG 75% 13 19 A 14 20 A 15 21 A 16 22 A 23 B 24 C

[0203] Histological analyses were carried out using protocol #10, and the results are grouped in [Fig.20]. Explantation allowed us to validate the resistance of the implants to skin tension. Histological analyses allowed us to evaluate the colo cellular nization, vascularization, extracellular matrix synthesis as well as the presence of areas of inflammation.

[0204] Example 15 - Cell viability and growth and mechanical properties of fibrinogen / alginate / gelatin (FAG) hydrogel

[0205] Cellularized FAG hydrogels were prepared using protocol #7 in the presence of different concentrations of human dermal fibroblasts (0.5 / 0.25 / 0.125 million cells / mL of hydrogel). Slabs of 1cm2, 0.2cm thick and 100% filling were prepared using protocol #8 and then crosslinked using protocol #5.

[0206] The different slabs were then cultured for 28 days at 37°C and 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM) / Glutamax medium (Gibco Cell Culture, Invitrogen, France), supplemented with 10% (v / v) bovine calf serum (Gibco Cell Culture, Invitrogen, France), 0.5% (v / v) amphotericin B (Gibco Cell Culture, Invitrogen, France) and 1% Penicillin / Streptomycin.

[0207] In order to monitor cell growth, assays of the L-lactic acid produced were carried out every two 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 using an INFINITE plate reader (TECAN, France).

[0208] In order to observe the presence of living cells within the hydrogel slabs throughout the culture, viability marking was carried out every 5 days in the presence of IpM of Calcein-AM (Thermofisher, France).

[0209] The results are grouped in [Fig.21A]. Lactate synthesis is an indicator of cellular activity. A clear increase in the amount of lactate is observed from D15 at all cell concentrations, indicating cell growth in the consolidated bioprinted hydrogels according to the invention.

[0210] The images of the calcein-AM labeling grouped in [Fig.21B] show a viable and increasing cell presence from D11 and a spreading of the cells from D17 at 0.25M cells / mL and from D22 for the other cell concentrations.

[0211] Cellularized FAG hydrogels prepared and cultured as described above were studied according to protocol #6.

[0212] The results are grouped in [Fig.22]. The Young's modulus of the cellularized hydrogels after 28 days of culture is not dependent on the cell concentration used during production. These Young's moduli are, however, lower than those obtained from non-cellularized hydrogels, which denotes a remodeling of the hydrogel by the cells.

[0213] Example 16 - Bioprinting of equivalent dermis in consolidated alginate / gelatin (AG) and fibrinogen / alginate / gelatin (FAG) hydrogels

[0214] Cellularized AG and FAG hydrogels were prepared from protocol #7 in the presence of a mixture of human dermal fibroblasts (0.25 million cells / mL of hydrogel) and human dermal microvascular endothelial cells (1 million cells / mL of hydrogel). Slabs of 2.25cm2, 0.2cm thick and 50 and 100% filling were prepared using protocol #8 and then consolidated using protocols #4 and #5.

[0215] The different constructs were then cultured for 21 days at 37°C and 5% CO2 in a culture medium adapted to the culture of human dermis composed of DMEM supplemented with 10% calf serum, 1% antibiotics, vitamin C and EGF.

[0216] In order to monitor cell growth, assays of the L-lactic acid produced were carried out every two 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 using an INFINITE plate reader (TECAN, France).

[0217] The results are grouped in [Fig.23]. An increase in lactate production by cells is observed from D11 under all conditions. Lactate production is higher in AG-based hydrogels and in porous constructs. These results indicate cell proliferation in the consolidated hydrogels for 21 days.

[0218] Histological analyses were performed using protocol #10. HPS staining was performed for all conditions. To assess the presence of endothelial cells in the hydrogels, CD31 immunohistochemical staining specific for endothelial cells with DAB revelation was performed on 5qm thick paraffin sections.

[0219] The results are grouped in [Fig.24] to [Fig.27]: [Fig.24]: Images of the full FAG constructs show that a large number of cells are present in the gel and appear to proliferate in the form of an agglomerate with adhesion sites on the gel. For the porous constructs, proliferation clusters are also observed in the gel, as well as a proliferative layer on the surface of the pores. The beginning of degradation of the hydrogel is also observed around the cells in the gel. [Fig.25]: Images of the full AG constructs show that a large number of cells are present in the gel. For the porous constructs, proliferation clusters are also observed in the gel, as well as a proliferative layer on the surface and areas of high cell density in the corners of the pores. Degradation of the hydrogel around the cells in the gel is also observed. [Fig.26]: Images of the full FAG constructs reveal the presence of substantial fibroblast cell clusters in the gel with small endothelial cell clusters located around and within the fibroblast clusters. [Fig.27]: Images of the porous FAG constructs reveal the presence of fibroblast cell clusters in the gel with endothelial cell clusters located around and within the fibroblast clusters. A proliferative layer of fibroblasts is also observed on the surface.

[0220] Example 17 - Obtaining equivalent skins by epidermization of bioprinted constructs in consolidated fibrinogen / alginate / gelatin (FAG) hydrogels.

[0221] Cellularized FAG hydrogels were prepared from protocol #7 in the presence of a mixture of human dermal fibroblasts. Two distinct conditions were tested: bilayer bioprinting at 1,000,000 fibroblasts / ml and hybrid bilayer bioprinting of an acellular lower layer and a cellularized upper layer at 2,000,000 fibroblasts / ml. For the cellularized bilayer constructs, slabs of dimensions 2.2 cm x 2.2 cm x 0.2 cm (two layers) and 100% infill were printed using protocol #8 and then consolidated using protocols #5. For the hybrid cellularized bilayer constructs, a first acellular layer with dimensions of 2.2 cm x 2.2 cm x 0.1 cm and then a second cellularized layer (2,000,000 fibroblasts / ml) with dimensions of 2.2 cm x 2.2 cm x 0.1 cm and 100% filling were printed using protocol #8 and then consolidated using protocols #5.

[0222] The different constructs were then cultured for 21 days at 37°C and 5% CO2 in a culture medium adapted to the culture of human dermis composed of DMEM supplemented with 10% calf serum, 1% antibiotics, vitamin C and EGF. After three weeks of culture, a suspension of normal human keratinocytes at 4,000,000 c / ml was prepared in 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 were deposited on each construct for a seeding of 250,000 keratinocytes / cm2. After seeding, the different constructs were cultured for 21 days at 37°C and 5% CO2 in culture media adapted to the culture of equivalent human skin composed of DMEM / HAMF12 supplemented with insulin, hydrocortisone, vitamin C and 1% antibiotics.

[0223] The collagen content of the bioprinted constructs was evaluated under the different conditions. Quantitative analysis of the collagen present in the supports was performed using a Sircol assay (Kit S1000, Biocolor). The control for this study was an acellular FAG construct made using the same methodology as the bioprinted and surfaced samples. The pellets resulting from collagen degradation were dissolved in 250 μl of basic reagent solution (kit). The absorbance was then measured at 555 nm ((NanoQuant® infinité M200PRO, TECAN) and the results compared to those of a standard range to allow the evaluation of the collagen concentration of the digestion supernatants. This concentration was then reported to the mass of each sample to assess their collagen concentration.

[0224] The results are grouped in Table 4 below, in which the concentration of neosynthesized collagen is indicated by type of construct. These results were obtained by subtracting the collagen concentration per mg of sample from the control from the concentrations of the different constructs studied (mass in pg of collagen per mg of sample weighed wet at the end of cell culture):

[0225] [Tables4] Reconstruction conditions Concentration of newly synthesized collagen in the constructs (pg of collagen / mg of sample) Conventional bilayer bioprinting 0.12044817 Hybrid bilayer bioprinting 0.17182893

[0226] Since the value corresponding to the control sample is non-zero, it can be deduced that the sircol test highlights the gelatin of the FAG hydrogel as collagen. The constructs showed a higher collagen concentration than the control, thus confirming the presence of neosynthesized collagen.

[0227] At the end of culture, histological analyses were carried out. The constructs were cut in two. The first half was fixed in 4% formalin for 24 hours, then dehydrated by successive baths of absolute ethanol and methylcyclohexane with an STP 120 dehydrator (Microm) and then embedded in paraffin. Sections 5 μm thick were made using an HM 340e microtome (Microm). Hematoxylin Phloxine Saffron (HPS) staining was then carried out on these sections.

[0228] The results are grouped in [Fig.28], in which the images reveal that the two conditions tested supported the establishment of a thick and healthy dermis with synthesis of extracellular matrix and a multi-stratified and differentiated epidermis.

[0229] This study demonstrated the feasibility of producing reconstructed tissues from FAG hydrogel supports consolidated with TAG. The different conditions led to the production of reconstructed skins (thick equivalent dermis and suitably differentiated epidermis). The two methods studied here made it possible to produce mature dermo-epidermal assemblies demonstrating collagen neosynthesis.

Claims

Claims

1. A method of consolidating a hydrogel comprising alginate and gelatin, said method comprising preparing the hydrogel and then contacting it with a consolidating solution comprising at least calcium and transglutaminase, and optionally one or more other divalent cations.

2. Method according to claim 1, characterized in that the contacting of the hydrogel with the consolidation solution is carried out by immersion, preferably total, of the hydrogel in the consolidation solution, or by spraying the hydrogel with the consolidation solution.

3. A method according to any one of claims 1 and 2, characterized in that the hydrogel comprises from 0.5 to 3% mass / volume of alginate and from 1 to 17.5% mass / volume of gelatin.

4. Method according to any one of claims 1 to 3, characterized in that the hydrogel further comprises fibrinogen and in that the consolidation solution further comprises thrombin.

5. A method according to claim 4, characterized in that the hydrogel comprises up to 2% mass / volume of fibrinogen.

6. Method according to any one of claims 1 to 5, characterized in that the consolidation solution comprises: - from 0.003 to 30% mass / volume of divalent cation, - from 0.004 to 16% mass / volume of transglutaminase, and optionally - from 2 to 25 U / ml of thrombin.

7. Method according to any one of claims 1 to 6, characterized in that the hydrogel further comprises living cells integrated during the preparation of the hydrogel before its consolidation.

8. Method according to any one of claims 1 to 7, characterized in that the contact with the consolidation solution is carried out: - at a temperature ranging from 15 to 40°C, and / or - for a period ranging from 30 minutes to 6 hours.

9. Method according to any one of claims 1 to 8, characterized in that the hydrogel is shaped, prior to its consolidation, by a molding or 3D printing technique.

10. Use of a solution comprising calcium and transglutaminase, and optionally one or more other divalent cations, for consolidating a hydrogel comprising alginate and gelatin. 30

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