3D intracorporeal implant

Abstract

The present invention relates to a three-dimensional body implant, in particular a breast implant, comprising a hydrogel comprising cross-linked alginate and gelatin. The hydrogel of the implant according to the invention may further comprise fibrinogen. The implant according to the invention is acellular, i.e. does not contain cells during production.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to the general field of biomaterials, and in particular to implants intended to be introduced into the human body / living organisms to replace and / or augment most soft tissues and / or to fill spaces between the skeleton and the skin or to be sutured to the skin.

[0002] The present invention relates to three-dimensional body implants, in particular temporary or permanent implants, comprising a hydrogel comprising cross-linked alginate and gelatin. The implants of the present invention have a given particularly advantageous porosity and mechanical strength. These implants are also acellular, i.e. do not contain cells, in particular no viable cells are incorporated into the implant during manufacture. In all aspects of the present invention, the hydrogel may further comprise fibrinogen. [Background technology]

[0003] cutting edge technology Hydrogel-based structures comprising alginate and gelatin are known from the state of the art, but these components usually have limited elasticity (in particular a low Young's modulus), which means that they lack sufficient mechanical strength and make the resulting structures difficult to handle.

[0004] Recent scientific reviews by Armin Vedadghavami et al., 2017, Acta Biomaterialia 62, 42-63, Marta Calvo Catoira et al., 2019, Journal of Materials Science: Materials in Medicine, 30:115, and Gils Jose et al., 2020, Current Medicinal Chemistry, 27, 2734-2776 highlight the biocompatible properties of natural hydrogels, especially alginate and gelatin-based hydrogels, but also their limitations in terms of mechanical properties. In fact, these natural polymers are not suitable for the manufacture of steerable and / or implantable devices, as well as for the construction of complex structures and structures up to 5 cm. 3 For the implementation of products with larger dimensions, they have too low mechanical resistance, thus limiting the clinical application of this technology. The mechanical properties of the structures obtained in the prior art are still insufficient to make them suitable for operation. Moreover, for structures intended to be implanted in the human or animal body and sutured as necessary, it is necessary to obtain structures that have suitable mechanical properties and whose degradability when in contact with living cells or tissues is not too rapid.

[0005] To realize a regenerative, resorbable implant, a structure with macroscopic pore sizes (e.g., larger than 100 μm) is highly advantageous. Moreover, this type of medical application often requires implants with large volumes.

[0006] To date, the literature does not describe any macroporous implants composed of hydrogels, especially those of large volume, because the fabrication of large porous objects from hydrogels is limited by their poor mechanical properties. Summary of the Invention [Problem to be solved by the invention]

[0007] overview One of the aims of the present invention is to overcome the drawbacks of prior art implants and to make it possible to provide a biocompatible implant whose main components are of natural origin.

[0008] Indeed, the implants of the present invention have particularly advantageous and innovative characteristics, in particular with regard to (i) mechanical strength of the components similar to that of natural tissue when introduced, (ii) stability over time, (iii) flexibility, (iv) outstanding tear and impact resistance, and (v) engraftment by cells of the host organism. [Means for solving the problem]

[0009] According to a first aspect, the present invention provides a three-dimensional intracorporeal implant comprising a hydrogel comprising cross-linked gelatin and cross-linked alginate, said hydrogel having a mechanical strength between 1 kPa and 1000 kPa, said implant having at least one porous section, the porous section comprising a plurality of pores, each of the pores having a pore size, the porous section having a total porosity between 100 μm and 10,000 μm, the total porosity corresponding to the average value of the pore sizes measured within the porous section.

[0010] The pores of the porous section may have uniform pore sizes, ie, may differ from each other by no more than 15%.

[0011] The pores of the porous section may be uniformly, i.e. evenly distributed.

[0012] The pores of the porous section may each extend along a central axis having a uniform orientation, ie, orientations differing from each other by no more than 20°.

[0013] The central axes of the holes in the porous section may be uniformly spaced, i.e., not differing from each other by more than 15%.

[0014] The pores of the porous section may each have a uniform geometric structure, ie their contours may overlap by more than 50% with overlapping or parallel parts.

[0015] 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%.

[0016] The gelatin may be cross-linked by an enzyme, preferably transglutaminase.

[0017] The implant may include multiple porous sections.

[0018] The plurality of porous sections may include at least two porous sections in which the pores have different pore sizes and / or shapes.

[0019] The porous sections may be arranged to form a gradient of pore size distributed throughout the implant, with the porous sections joining one another along the gradient direction in an order selected from ascending and descending pore size.

[0020] Implants are a first porous section forming a base, which represents between 5% and 40%, preferably between 20% and 40%, of the total volume of the implant and has a pore size 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 between 500 micrometers and 2500 micrometers, in particular between 100 micrometers and 250 micrometers; a third porous section forming an outer shell, representing 5% to 40%, preferably 10% to 40%, of the total volume of the implant and having a pore size between 1000 micrometers and 10000 micrometers, in particular between 1000 micrometers and 2500 micrometers; may include:

[0021] The implant may include at least one non-porous section, the non-porous section having a fill factor of greater than 99%.

[0022] At least one non-porous region may include a perimeter surrounding the porous region.

[0023] Said at least one porous section 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%.

[0024] The implant may be made of multiple layers, each having a mesh of multiple meshes, stacked on top of each other such that the meshes form the pores.

[0025] The mesh in each layer may have a uniform mesh size, i.e. mesh sizes differing from each other by no more than 15%.

[0026] The mesh in each layer may be uniformly, i.e. evenly distributed.

[0027] The meshes of each layer may extend about a central mesh axis having uniform orientations, i.e. orientations that differ from each other by no more than 20°.

[0028] 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.

[0029] 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.

[0030] 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%.

[0031] The implant may have a volume in the range of 0.05 mL to 3 L, preferably 100 mL to 600 mL.

[0032] The implant may be a breast implant.

[0033] According to another aspect, the present invention provides a method for producing a method for manufacturing a pharmaceutical composition comprising: - preparing a hydrogel comprising gelatin and alginate; - shaping the hydrogel in three dimensions to form at least one porous section, the porous section having a plurality of pores, each of the pores having a pore size, the porous section having a total porosity between 100 μm and 10,000 μm, the total porosity corresponding to an average value of the pore sizes measured within the porous section; - crosslinking the hydrogel with at least one divalent cation, preferably calcium, and transglutaminase, said hydrogel having a mechanical strength of between 1 kPa and 1000 kPa. The present invention proposes a three-dimensional intracorporeal implant, in particular as defined above, obtainable by a manufacturing process comprising in succession:

[0034] During the cross-linking step, the divalent cation and transglutaminase may be added simultaneously.

[0035] The hydrogel may contain 0.5% to 3% alginate, and 1% to 17.5% gelatin.

[0036] The hydrogel may further comprise cross-linked fibrinogen, preferably up to 2% cross-linked fibrinogen.

[0037] The manufacturing process may further provide for the use of thrombin during the cross-linking step.

[0038] During the three-dimensional shaping step, the manufacturing process may provide for carrying out an additive manufacturing process, in particular 3D printing.

[0039] The manufacturing process may further include a sterilization step.

[0040] According to another aspect, the present invention relates to a method of performing an implant as defined above in the context of reconstructive or cosmetic surgery, comprising the step of implanting an implant, in particular a breast implant, in the body of a subject, in particular in the breast of the subject.

[0041] definition For purposes of the present invention, the following terms are defined as follows: - "crosslinking agent" in the context of the present invention means an agent capable of crosslinking the hydrogel components, in particular alginate, gelatin and fibrinogen. "Biodegradable" means capable of being broken down by a living organism. In particular, when an implant is implanted into a host, said implant is biodegradable if said host is capable of breaking it down. "Fiber" in the context of the present invention refers to any element of thread-like appearance, generally present in bundles. - "Gradient" in the context of the present invention means a gradual transition, either increasing or decreasing, from one pore size to another. - "host" and "recipient" in the context of the present invention are equivalent terms and are used interchangeably to refer to an organism into which an implant according to the invention can be introduced. "Shaping" in the context of the present invention consists in giving the hydrogel a particular shape and structure or configuration that is specifically adapted to the final goal of the cured hydrogel. - "total porosity" or "total pore size" in the context of the present invention refers to the average value of the pore size measured in the porous section(s) of the implant. It does not refer to the porosity of the hydrogel itself. - "Pore size" in the context of the present invention refers to the maximum distance between two opposing beads of material.

[0042] Detailed Description The invention makes it possible to provide three-dimensional internal implants having particularly advantageous mechanical characteristics similar to those of natural tissue, in particular with regard to the mechanical strength of the components, their stability over time, and their remarkable tear and impact resistance.

[0043] The implants according to the invention can be used either permanently or temporarily in place of (total or partial replacement) or in addition to (augmentation) various organs or tissues of the animal body, more particularly the human body. By definition, the implants according to the invention are suitable for contact with living body fluids or living tissues. In particular, they are intended to be implanted subcutaneously or even epicutaneously, in particular for skin regeneration and / or healing.

[0044] The implants of the present invention are therefore substitutes or additions for replacing and / or augmenting and / or reinforcing soft or flexible tissues, and sometimes elastic tissues. They are preferably used to replace in whole or in part, augment or reinforce connective tissues, skin and adipose tissues.

[0045] The implant according to the invention is therefore intended for plastic, reconstructive or regenerative surgery.

[0046] All components of the implant of the invention described below must meet the regulatory requirements specific to the device in which it is intended to be implanted, particularly with regard to purity.

[0047] An implant according to the invention is therefore an internal implant, such as for example a breast implant, a pectoral implant, a buttock implant, a facial implant or any other implant which compensates for a tissue volume defect.

[0048] According to a particularly preferred embodiment, the implant of the present invention is a breast implant.

[0049] The implants of the invention can be large, both in terms of volume, since they can reach volumes of 0.05 mL to 3 L, preferably 100 mL to 600 mL, and in terms of size, which can also be in the range of 0.5x0.5x0.2 to 20x15x15, i.e. in the range of the following length x width x thickness: length 0.5 cm to 20 cm x width 0.5 cm to 15 cm x thickness 0.2 cm to 15 cm. The size of implants, for example for tissue filling, generally does not exceed 20x15x15. For breast implants, the size is preferably around 12x12x3 or 12x12x4.

[0050] Thus, according to one embodiment, the implant of the invention has a volume of more than 0.05 mL, preferably a volume of between 0.05 mL and 3L.

[0051] The implant may be of any shape that relates to the volumes mentioned herein, for example, the implant may be in the form of a hemisphere, a hemidrop, or any other shape that may be customized to suit the subject matter of the invention.

[0052] In one embodiment, the implants according to the invention are temporary since due to their composition they are resorbable and disappear over time after implantation in the body, leaving in their place neotissue naturally vascularized by cells and the living host organism. These temporary implants are therefore more precise implants that can be engrafted by the cells of the host organism, in particular due to the presence of a defined porosity (with a maximum value) and / or the use of materials constituting the implant that preserve cell viability and encourage cell proliferation. These implants therefore define an internal space that is a kind of skeleton / framework / backbone / matrix (or in English "scaffold"), allowing engraftment by cells, in particular the cells of the recipient organism.

[0053] In one embodiment, the implants according to the invention are biodegradable due to their composition and therefore suitable for implantation in an animal body, in particular the human body.

[0054] In one embodiment, the implant according to the invention comprises at least one porous section.

[0055] In one embodiment, the at least one porous section, i.e., the porous section if only one is present, or all the porous sections if more than one is present, represents about 5% to 100% of the implant, preferably about 50% to 100% of the implant, more preferably about 90% to 100% of the implant. According to one embodiment, the porous section or all the porous sections constitute more than 90% of the implant. According to one embodiment, the porous section or all the porous sections constitute about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the implant.

[0056] In one embodiment, the porous section or all of the porous sections comprise the entire implant.

[0057] The porosity of the porous section of the implant is a crucial parameter that is adjusted in particular according to the associated tissue or organ to be replaced and / or augmented. In practice, the porosity is interpreted as the cavities present in the porous section of the implant and can be adapted in order to bring in the most material and thus give a specific mechanical resistance as close as possible to that of the natural tissue of the implanted section.

[0058] The total porosity corresponds to the average value of the pore size measured in each porous section.

[0059] In the context of the present invention, implants are characterized at the level of their structure by the porosity of the porous fraction, expressed herein in two different but correlated and therefore equivalent or alternative ways, namely the pore size expressed in micrometers and / or the porosity of the hydrogel expressed as a percentage (volume of hydrogel / total volume of implant).

[0060] According to one embodiment, the porous section includes a plurality of pores, each of the pores having a pore size.

[0061] In one embodiment, in soft tissues, a large overall pore size in the porous zone of the implant in the range of 1000 μm to 10000 μm, especially 1000 μm to 5000 μm, and / or a filling rate of the porous zone of the implant of 5% to 50% is preferred, the resulting implant containing less material and being more flexible. Preferably the porous section of the implant will have a filling rate of 5% to 50%, even more preferably 15% to 50%.

[0062] In one embodiment, for hard tissues, a low total pore size in the porous section of the implant, in particular less than 1000 μm, and / or a filling rate of the porous section of the implant of 50% to 99%, in particular 50% to 95%, is preferred to provide said implant with high mechanical strength for the hard tissue. Preferably, the porous section of the implant has a filling rate of 50% to 99%, even more preferably 50% to 90%.

[0063] In addition, the pore size can be adjusted to suit the different cell types present in the tissue. Again, a dense low-porosity environment, especially with implant pore sizes smaller than 1000 μm and / or a high implant loading rate of 50% to 99%, especially 50% to 95%, is favorable for osteoblast-type cells developing in very rigid matrices, while a flexible, more porous environment, especially with implant pore sizes lying between 1000 μm and 5000 μm and / or an implant loading rate of 5% to 50%, favors the survival, proliferation and metabolism of fibroblast and adipocyte types developing in flexible matrices.

[0064] Finally, the choice of pore size allows to adjust the degradation time of the implant in the body. Implants with small pore sizes, especially less than 1000 μm, and / or a filling rate of 50% to 99%, especially 50% to 95%, will be composed of more material, which will result in a total degradation that will be more or less long depending on the implant size. On the other hand, if a more rapid degradation of the implant is desired, for example less than 12 months, it is preferable to have more pores of large size in the implant, especially between 1000 μm and 5000 μm in size, and / or a filling rate of the implant of 5% to 50%.

[0065] When the implant according to the invention is prepared using 3D printing techniques, the pore sizes disclosed above correspond to the length between the stacked hydrogel filaments, in particular the gap length between these filaments.

[0066] The present disclosure therefore relates to a three-dimensional intracorporeal implant comprising a hydrogel comprising cross-linked alginate and cross-linked gelatin, characterized in that said hydrogel has a mechanical strength of 1 kPa to 1000 kPa, and that said implant has at least one porous section, the porous section comprising a plurality of pores, each of the pores having one pore size, and the porous section has a total porosity of up to 5000 μm.

[0067] The invention also relates to a three-dimensional intracorporeal implant comprising a hydrogel comprising cross-linked alginate and cross-linked gelatin, characterized in that said hydrogel has a mechanical strength between 1 kPa and 1000 kPa, and that said implant has at least one porous section, the porous section comprising a plurality of pores, each of the pores having one pore size, the porous section having a total porosity between 100 μm and 10000 μm, in particular up to 5000 μm, the total porosity corresponding to the average value of the pore sizes measured in the porous section.

[0068] The hydrogel has a mechanical strength of 1 kPa to 1000 kPa.

[0069] The implant according to the invention in terms of its components and structure preferably has an apparent mechanical strength of 10 kPa to 800 kPa, more preferably 10 kPa to 300 kPa, or more preferably 50 to 300 kPa.

[0070] The implants of the present invention therefore have mechanical properties similar to those of the natural tissue they seek to replace or augment.

[0071] By way of example, the average mechanical strengths of different natural tissues are given in Table 1 below (as cited in Guimaraes C. et al., Nature Reviews Materials volume 5, pages 351-370 (2020)): [Table 1]

[0072] The mechanical strength discussed herein may also be referred to as elasticity or Young's modulus, by which we mean the longitudinal elastic or tensile modulus, which is a constant related to the onset of tensile (or compressive) stress and deformation of an isotropic elastic material.

[0073] Young's modulus is the mechanical stress that causes a material to elongate to 100% of its original length, i.e., double in length.

[0074] This Young's modulus is determined by Hooke's Law: σ=Eε, where: σ = mechanical stress (in units of pressure); E = Young's modulus (in units of pressure); ε = relative elongation or strain (dimensionless); (ε = l-l0 / l0; l0 is the initial length and l is the length after deformation) is governed by.

[0075] In addition to the individual mechanical strength imparted by their components, the implants of the present invention have at least one porous section containing a plurality of pores, each of the pores having one pore size.

[0076] In one embodiment, the porous section has a total porosity of at most 10000 μm. According to one embodiment, the porous section has a total porosity of at most 5000 μm.

[0077] The porous section has a total porosity of at least 10 micrometers, such as at least 50 micrometers. According to one embodiment, the porous section has a total porosity of at least 100 micrometers, more preferably at least 500 micrometers.

[0078] Thus, according to one embodiment, the total porosity of the porous section of the implant according to the invention may be in the range of 10 micrometers to 1000 micrometers, or 20 micrometers to 1000 micrometers, or 1000 micrometers to 5000 micrometers.

[0079] Thus, according to one embodiment, the total porosity of the porous section of the implant according to the invention may be in the range of 100 micrometers to 10000 micrometers, preferably 500 micrometers to 2500 micrometers, more preferably 500 micrometers to 1000 micrometers, or 1000 micrometers to 2500 micrometers, or 2500 micrometers to 5000 micrometers.

[0080] In this respect, the implant according to the invention may have a plurality of porous sections, said porous sections having different pore sizes within the three-dimensional structure, for example in the form of a gradient of porosity distributed throughout the implant. The porous sections are thus successive to one another along the gradient direction in a sequence selected from ascending and descending pore sizes. The gradient of pore sizes allows for example the selection of cell types to be engrafted on the implant.

[0081] In one embodiment, the implant according to the present invention comprises multiple porous sections.In one embodiment, the implant according to the present invention comprises at least two porous sections, preferably three porous sections, with different pore sizes, each porous section having a defined total pore size, for example in the form of a porosity gradient.This allows the most rigid sections to be defined, for example according to the type of tissue to be regenerated or to be in contact with the implant in the host organism.The porous sections can also comprise different pore shapes.

[0082] In one embodiment, the pore size of the porous section is preferably in the range of 100 micrometers to 7000 micrometers, in particular 100 micrometers to 3000 micrometers.

[0083] When producing an implant having sections of different pore sizes, for example in the form of a porosity gradient, these sections may each be defined as having a subrange of pore sizes, so long as the resulting pore sizes in all of the porous sections remain within the range of 100 micrometers to 10,000 micrometers, preferably 100 micrometers to 3,000 micrometers.

[0084] In one embodiment, the pore size subranges are preferably in the ranges 100 micrometers to 250 micrometers, 250 micrometers to 800 micrometers, and 1000 micrometers to 2500 micrometers, if the implant contains three different pore size segments, or in the ranges 100 micrometers to 250 micrometers, 250 micrometers to 3000 micrometers, if the implant contains only two different pore size segments. According to one embodiment, these subranges constitute a gradient from 100 micrometers to 3000 micrometers.

[0085] In one embodiment, the pore size subranges are preferably within the ranges of 500 micrometers to 2500 micrometers, 500 micrometers to 5000 micrometers, and 1000 micrometers to 10000 micrometers if the implant contains three different pore size segments, or within the ranges of 500 micrometers to 5000 micrometers, 1000 micrometers to 10000 micrometers if the implant contains only two different pore size segments.

[0086] The construction of the implant according to the present invention can be broken down into three separate sections.

[0087] The base of the implant (5% to 40%, preferably 20% to 40% of the total volume of the implant), which is preferably placed in direct contact with the muscle tissue, has an intermediate pore size (500 micrometers to 5000 micrometers, in particular 250 micrometers to 800 micrometers) that favors engraftment by endothelial cells and the vascular structures surrounding it. Endothelial cells easily migrate through this pore size and organize the cells into vascular / microvascular structures, allowing vascularization of the implant and thus a better integration with the adjacent tissue. The easy vascularization of the structure also limits the risk of necrosis of the tissue engrafted on the implant.

[0088] The core of the implant (20%-70%, preferably 30%-50% of the total implant volume) is not in direct contact with the host tissue of the implanted section. This section has a fine pore size (500 micrometers-2500 micrometers, in particular 100 micrometers-250 micrometers) and plays a role in supporting tissue regeneration. This area is composed of more material than other areas, so it biodegrades slower in the body and provides a support matrix for cells to grow on.

[0089] The outer shell of the implant (5% to 40%, preferably 10% to 40% of the total volume of the implant) is arranged to be the first part to come into contact in case of impact and / or compressive stress. The outer shell has a large pore size (1000 micrometers to 10000 micrometers, in particular 1000 micrometers to 2500 micrometers) allowing easy migration of cells towards the core of the implant. This outer shell acts as a mechanical protection for the implant core.

[0090] The pore size subranges may be combined to form a gradient with one pore size across all porous sections in the range of 100 micrometers to 10,000 micrometers. The gradient is preferably in the range of 500 μm to 7,000 μm.

[0091] When the implant of the invention can be prepared using additive manufacturing techniques, in particular 3D printing, in particular by extrusion of a viscoelastic material, the presence of pores in the implant can be related to a three-dimensional structure in the form of a "lattice", preferably a gyroid, a cube or a hexagon, whose size in the XY plane is given by the pore diameter and whose height is given by the diameter of the printing filament, in particular between 200 micrometers and 1500 micrometers, preferably between 200 micrometers and 1000 micrometers.

[0092] Thus, according to one embodiment, the pores in the porous section(s) have a gyroid, cubic or planar hexagonal shape. According to one embodiment, the pores in the porous section(s) have the same shape as each other within each porous section.

[0093] The pores within each of the porous sections may have uniform pore sizes, ie, pore sizes that differ from one another by no more than 15%.

[0094] In one embodiment, the pores are uniformly and evenly distributed, i.e., located equidistant from one another throughout the volume of the porous section(s).

[0095] More particularly, the holes of the porous section may each extend along a central axis having a uniform orientation, i.e., orientations that differ from each other by no more than 20°. The central axes of the holes of the porous section may be uniformly spaced apart, i.e., spaced apart from each other by no more than 15%.

[0096] In addition, the pores of the porous section may each have a uniform geometric structure, ie, their contours may overlap by more than 50% with overlapping or parallel portions.

[0097] The holes in the porous section may be separated from one another by stacks of material each having a uniform thickness, i.e. thicknesses that differ from one another by no more than 15%.

[0098] In a porous section of defined pore size and shape, the pore organization is characterized by the repetition of the same pattern, which is organized into one or more meshes through the translation of this same pattern along at least one direction in space.

[0099] In one embodiment, an implant according to the present invention further comprises one or more non-porous sections, known as dense sections.

[0100] In one embodiment, a dense section is a section with a filling rate (pore size 0 μm) of more than 99%, in particular 100%, and can in particular be obtained by using manufacturing techniques such as, for example, moulding.

[0101] In one embodiment, the non-porous segment represents about 0%-50% of the implant, preferably about 0%-25% of the implant, more preferably about 0%-10% of the implant, by volume. According to one embodiment, the non-porous segment represents less than 10% of the implant. According to one embodiment, the non-porous segment is 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% of the implant.

[0102] One or more perimeters (dense structures surrounding the entire perimeter of the implant) may be present in one or more layers of thickness within the structure, this addition limits irritation and inflammation in the body that may occur in the event of fracturing the edges of the implant.

[0103] Dense regions forming passageways through the implant may also be present in the structure to impart additional mechanical resistance to the implant. These passageways act as mechanical reinforcement for the structure. In the context of breast reconstruction, these passageways are largely inspired by Cooper's ligaments in biomimetic thinking.

[0104] The porosity and the specific structure of the implant therefore allow vascularization of the neoplastic and / or transplanted tissue, favor the diffusion of nutrients and metabolic products, provide a support and mechanical environment compatible with the cells, and thus limit the phenomena of ischemia and necrosis of the neoplastic and / or transplanted tissue, thereby generating a favorable environment for engraftment and tissue regeneration.

[0105] The implant may also include a void section, i.e. a volume having a filling factor of 0. According to one embodiment, the void section represents about 0% to 25% of the implant, preferably about 0% to 10% of the implant, by volume.

[0106] According to one embodiment, this void section allows for the injection of cells from the subject upon implantation of the implant in the subject, which cells can then engraft on the implant.

[0107] In one embodiment, the invention relates to an implant, in particular a breast implant, comprising a porous section. According to one embodiment, said porous section represents the entire implant.

[0108] In one embodiment, the present invention relates to an implant, in particular a breast implant, comprising a porous section and a non-porous section, for example the periphery as defined above. According to one embodiment, said porous section represents more than 90% of the implant by volume. According to one embodiment, said non-porous section represents less than 10% of the implant by volume.

[0109] In one embodiment, the present invention relates to an implant, in particular a breast implant, comprising two porous sections. In one embodiment, the present invention relates to an implant, in particular a breast implant, comprising three porous sections. According to one embodiment, said implant further comprises a non-porous section, for example a periphery as defined above.

[0110] In one embodiment, the present invention relates to implants, particularly breast implants, having different pore sizes distributed across three sections, e.g., in the form of a porosity gradient, as set forth in Table 2 below and illustrated in FIG. [Table 2]

[0111] The implants according to the invention have a particular advantage in the context of breast reconstruction, since they must be sufficiently resistant to withstand high compressive stresses in this anatomical area that is subject to this type of stress very regularly. Due to their mechanical characteristics, in particular their elasticity and flexibility, the implants according to the invention make it possible to generate less mechanical stresses on the host tissues in direct contact, thus reducing inflammatory phenomena.

[0112] Various methods known to those skilled in the art can be used to measure pore size. Among them, we can mention optical microscopy and electron microscopy. The void volume (the inverse of the packing fraction) of the implant can be measured by weighing (using the specific gravity of the material), drainage volume (Archimedes' method), etc.

[0113] Preferably, in the context of the present invention, the ranges disclosed herein correspond to pore size measurements by optical microscopy. Thus, according to one embodiment, the porosity or pore size is measured by optical microscopy. According to one embodiment, the porosity or pore size is measured by electron microscopy.

[0114] Since variations in the fill ratio can affect the porosity of the implant and vice versa, the porosity of the porous section of the implants of the present invention can also be characterized by the fill ratio of the hydrogel implant structure, which can be obtained, for example, by measuring the volume of the implant and measuring the void volume.

[0115] As shown in the examples, the selected packing parameters can result in a given range of pore sizes, which in turn correlates with specific packing parameters.

[0116] The implants of the present invention may have a hydrogel fill rate ranging from 5% to 99% of the total volume of the implant. The more the implant is filled (less than 50% filled), the more flexible the implant will be, and the flexibility / hardness ratio can be adjusted depending on the final goal in the body. Conversely, the more the implant is filled (greater than 50% filled), the more rigid the implant will be.

[0117] The porosity of the implant of the present invention favors the engraftment by cells. Thus, the three-dimensional structure combined with variable pore size or filling ratio makes it possible to obtain an implant containing multiple cavities, which, once placed, can be engrafted by the cells / tissues of the host organism and subsequently proliferate and differentiate in situ.

[0118] The three-dimensional structure and the particularly advantageous mechanical properties of the implant according to the invention are maintained even after sterilization, in particular after sterilization by radiation or plasma.

[0119] Various methods well known to those skilled in the art can be used to measure the mechanical strength of the implant, such as dynamic mechanical analysis (DMA) or compression, tensile and / or flexural testing. Examples of methods for measuring the mechanical strength of the implant are described in the Examples.

[0120] In one embodiment, the mechanical strength of the implant is measured by dynamic mechanical analysis (DMA). According to one embodiment, the mechanical strength of the implant is measured by compression, tensile and / or bending tests.

[0121] As mentioned, the main components of the implant according to the invention are components of natural origin.

[0122] 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 1,4β-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.

[0123] With regard to alginates and according to 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, for example, an M / G ratio 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, for example, an M / G ratio of 1.9.

[0124] Preferably the gelatin contained in the hydrogel is of type A.

[0125] 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 native triple helical structure of collagen through acid (type A gelatin) or alkaline (type B gelatin) treatment. 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.

[0126] The preparation of hydrogels (E.M. Ahmed; Journal of Advanced Research, 2015, 6, 105-121) and 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.

[0127] Preferably, the alginate is crosslinked with a crosslinking agent selected from divalent cations, particularly non-toxic cations.According to one embodiment, the divalent cation is selected from the group comprising or consisting of calcium, strontium, barium, zinc, copper, iron and nickel.According to one embodiment, the divalent cation is selected from the group comprising or consisting of calcium, strontium and barium.Preferably, the divalent cation is calcium.

[0128] Preferably the gelatin is crosslinked by any enzymatic, physical, such as UV light, or chemical method, in particular by an enzymatic method carried out with an agent capable of forming covalent bonds between lysine and glutamine residues, most preferably by transglutaminase.

[0129] The transglutaminase enzyme (TAG) is an extracellular aminoacyltransferase. It is a monomeric protein that contains a single cysteine ​​catalytic residue (active site). In the context of the present invention, the gelatin of the hydrogel is preferably crosslinked with type 2 transglutaminase. In particular, this TAG is commercially produced as a recombinant microbial protein by fermentation of the microorganism Streptoverticillium moboarense.

[0130] According to the invention, the alginate and gelatin present in the hydrogel contained in the implant are crosslinked, ie converted from linear polymers into three-dimensional polymers through the action of the crosslinking agents mentioned above.

[0131] In a particularly preferred embodiment, the implant according to the invention comprises a hydrogel comprising 0.5% to 3% alginate and 1% to 17.5% gelatine, more preferably 1% to 2.5% alginate and 2% to 10% gelatine. Advantageously the hydrogel comprises 2% alginate and 5% gelatine.

[0132] Unless otherwise indicated, percentages referred to herein are expressed by weight / volume of the total composition and are relative to the total composition.

[0133] Preferably, in the hydrogel of the implant of the invention, the cross-linked alginate and gelatin are present in a weight ratio ranging from 1:0.3 to 1:35, respectively, most particularly in a weight ratio of 1:2.5.

[0134] The hydrogel of the implant of the present invention may, in addition to alginate and gelatin, also contain fibrinogen, which is also cross-linked.

[0135] The fibrinogen monomer is composed of three α, β and γ chains linked by a central E domain and two repeats of two fibrinopeptides A and B (FpA, FpB) that link the α chains to the E domain. It has numerous cell adhesion motifs and can therefore increase cell development within the hydrogel.

[0136] In this case, the hydrogel will preferably contain 0.0001% to 6% cross-linked fibrinogen, in particular 2% cross-linked fibrinogen.

[0137] According to a particularly preferred embodiment, the hydrogel of the implant of the invention is composed of cross-linked alginate and gelatin, or of cross-linked alginate, gelatin and fibrinogen, without any other components capable of forming a gel.

[0138] Preferably, the hydrogel of the implant of the invention comprises cross-linked alginate, gelatin and fibrinogen in a weight ratio within the range of 1:0.3:0.00003 to 1:35:12, respectively, most particularly in a weight ratio of 1:1:2.5.

[0139] The implant of the invention advantageously contains, as natural constituents of the hydrogel, alginate, gelatin and optionally fibrinogen, but other natural components may also be present in the implant of the invention, such as in particular 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, proteoglycans such as Matrigel, methacrylate gelatin of the GelMa type, etc.

[0140] In one embodiment, the natural ingredients are present in a concentration in the range of 0.001% to 50%, preferably 0.01% to 25%, or more preferably 0.1% to 10%.

[0141] In addition to components of natural origin and especially those listed above, the implants of the invention may 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, etc.

[0142] In one embodiment, the synthetic components are present at a concentration in the range of 0.001% to 50%, preferably 0.01% to 25%, or more preferably 0.1% to 10%.

[0143] Woven fibers of natural or synthetic origin may also be present in the implant composition.

[0144] Examples of fibers of natural origin include, but are not limited to, cellulosic fibers.

[0145] Examples of fibers of synthetic origin include, but are not limited to, polyester fibers, nylon fibers, polyethylene fibers, polypropylene fibers, and acrylic fibers.

[0146] In one embodiment, said fibres are present at a concentration of less than 20%, preferably less than 10%, most preferably less than 5%.According to one embodiment, the implant of the invention is free of fibres, whether of natural or synthetic origin.

[0147] The implants according to the invention are acellular, i.e. they do not contain any cells, in particular any viable cells, during production. Nevertheless, the implants of the invention can be engrafted with viable cells after production, thereby making it possible to avoid any production constraints related to maintaining cell survival, proliferation and / or differentiation, as well as to carry out in vitro engraftment of the implant before it is produced and implanted in the host organism, in order to optimize uptake.

[0148] Thus, examples of specific embodiments of the implant according to the invention include: - hydrogels consisting only of alginate and gelatin and not containing fibrinogen; - hydrogels consisting of alginate, gelatin and fibrinogen; - hydrogels consisting of alginate, gelatin and collagen; - a hydrogel consisting of alginate, gelatin, collagen and fibrinogen, The implant comprises:

[0149] Particularly preferred embodiments within the scope of the present invention relate to implants as defined in Table 3 below. [Table 3]

[0150] In one embodiment, the implant of the invention is obtained by a manufacturing process in which alginate and gelatin are hardened by crosslinking with at least one dication, preferably calcium, and transglutaminase.

[0151] In one embodiment, the curing is carried out sequentially, i.e., the crosslinking agents mentioned above are not added simultaneously during curing.

[0152] In one embodiment, the prepared hydrogel is contacted with a solution containing a divalent cation, preferably calcium, and then with a solution containing transglutaminase. According to another embodiment, the prepared hydrogel is contacted with a solution containing transglutaminase and then with a solution containing a divalent cation, preferably calcium.

[0153] In one embodiment, the curing is carried out simultaneously, i.e., the crosslinking agents mentioned above are added at the same time during curing.

[0154] In a particularly advantageous embodiment, the implant of the invention is obtained by a manufacturing process in which alginate and gelatine are hardened by crosslinking with a solution comprising at least one divalent cation, preferably calcium, and transglutaminase.

[0155] Within the scope of the present invention, said solution may be obtained by alternative but equivalent processes: the hardening solution may be obtained by adding different elements, namely at least one divalent cation, preferably calcium, and transglutaminase, to the same solution, or by mixing at least two solutions, namely a solution containing at least one divalent cation, preferably calcium, and a solution containing at least transglutaminase.

[0156] In one embodiment, during curing, contacting the hydrogel with the above-mentioned solution(s) is performed by immersion, where the hydrogel is totally immersed in the above-mentioned solution(s), which can also be performed by dipping, spraying, dripping, trickling, or the like.

[0157] For example, the prepared hydrogel is contacted with a hardening solution containing at least one divalent cation, preferably calcium, and transglutaminase. During hardening, contacting the hydrogel with the hardening solution can be performed by immersion, in which the hydrogel is entirely immersed in the hardening solution. It can also be performed by immersion, spraying, dripping, trickling, or the like.

[0158] If the hydrogel contains fibrinogen in addition to alginate and gelatin, the 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.

[0159] In one embodiment, when the hydrogel contains fibrinogen in addition to alginate and gelatin, the prepared hydrogel is contacted with a solution containing a divalent cation, preferably calcium, followed by contact with a solution containing transglutaminase, followed by contact with a solution containing thrombin.

[0160] In one embodiment, when the hydrogel contains fibrinogen in addition to alginate and gelatin, the prepared hydrogel is contacted with a solution containing a divalent cation, preferably calcium, followed by contact with a solution containing thrombin, followed by contact with a solution containing transglutaminase.

[0161] In one embodiment, when the hydrogel contains fibrinogen in addition to alginate and gelatin, the prepared hydrogel is contacted with a solution containing transglutaminase, then with a solution containing a divalent cation, preferably calcium, and then with a solution containing thrombin.

[0162] In one embodiment, when the hydrogel contains fibrinogen in addition to alginate and gelatin, the prepared hydrogel is contacted with a solution containing transglutaminase, then with a solution containing thrombin, and then with a solution containing a divalent cation, preferably calcium.

[0163] In one embodiment, when the hydrogel contains fibrinogen in addition to alginate and gelatin, the prepared hydrogel is contacted with a solution containing thrombin, then with a solution containing transglutaminase, and then with a solution containing a divalent cation, preferably calcium.

[0164] In one embodiment, when the hydrogel contains fibrinogen in addition to alginate and gelatin, the prepared hydrogel is contacted with a solution containing thrombin, then with a solution containing a divalent cation, preferably calcium, and then with a solution containing transglutaminase.

[0165] In one embodiment, when the hydrogel contains fibrinogen in addition to alginate and gelatin, the setting solution includes at least one divalent cation, preferably calcium, transglutaminase, and thrombin.

[0166] In a preferred embodiment, the implant of the invention is obtained by a manufacturing process in which the curing step consisting of contacting the hydrogel with the curing solution(s) is carried out at a temperature in the range of 15° C. to 40° C., preferably 20° C. to 40° C., more preferably 21° C. to 37° C. According to another embodiment, which is preferred but which can be combined with the above embodiment regarding the temperature conditions, the implant of the invention is obtained by a manufacturing process in which the curing step consisting of contacting the hydrogel with the curing solution(s) is carried out for a period in the range of 10 minutes to 6 hours, in particular 30 minutes to 6 hours, ideally 1 hour to 3 hours. Thus, according to an advantageous embodiment, the implant of the invention is obtained by a manufacturing process in which the curing step is carried out at 37° C. for 1 hour and 30 minutes.

[0167] In one embodiment, the hydrogel is molded prior to curing.

[0168] The implant of the invention can be manufactured and shaped simultaneously, in particular by any volumetric construction process (in particular in 3D) and in particular by adding or agglomerating material by layer-by-layer or successive deposition. Thus, according to one embodiment, the implant of the invention is obtained by additive manufacturing.

[0169] Among these processes, mention may especially be made of methods by injection, by extrusion, in particular moulding, by 3D printing.According to one embodiment, the implant of the invention is thus obtained by extrusion of material, preferably by 3D printing.

[0170] The implant may then be made up of multiple layers, each layer having one mesh made up of multiple meshes, and the layers may be stacked on top of each other such that the meshes form holes. According to one embodiment, the implant is made up of a large number of layers, between 2 and 3000.

[0171] According to the aforementioned pore characteristics, the meshes of each layer may have a uniform mesh size, i.e. mesh sizes that differ from each other by no more than 15%.

[0172] In addition, the mesh in each layer may be uniformly, i.e. evenly distributed.

[0173] More particularly, the meshes of each layer may extend about central mesh axes that are uniformly oriented, i.e., differing from each other by no more than 20°. The central mesh axes of the meshes of each layer may be uniformly spaced apart, i.e., differing from each other by no more than 15%.

[0174] The meshes of each layer may each have a uniform geometric structure, i.e. their contours may overlap by more than 50% with overlapping or parallel parts.

[0175] The meshes of each layer may be separated from one another by strands of material each having a uniform thickness, i.e. thicknesses that differ from one another by no more than 15%.

[0176] In particular, hydrogels consisting only of alginate and gelatin may have viscosities in the range of 50 Pa.s to 6000 Pa.s when measured at temperatures between 5°C and 45°C, so the skilled artisan will be careful to select a process that allows for molding of highly viscous materials.

[0177] In the context of the present invention, the implant is preferably obtained by a 3D printing process. This technique can also give the implant a suitable shape. In fact, as previously indicated, the implant according to the invention has advantageous mechanical properties, making it particularly suitable for these purposes.

[0178] With this technique, the implant can be "molded" to match the appearance and / or desires of the host. Thus, the implant of the present invention provides a "custom-made" composed solution, whose dimensions and / or fill rate / porosity are defined with respect to the requirements within the host body that is intended to receive the internal implant, and the role / function it is to fulfill in this recipient's organ.

[0179] The present invention therefore also relates to a three-dimensional internal implant obtainable by the manufacturing process described above. In particular, the internal implant comprises: - preparing a hydrogel comprising gelatin and alginate; - a three-dimensional shaping step of the hydrogel to form at least one porous section, the porous section comprising a plurality of pores, each of the pores having one pore size, the porous section having a total porosity between 100 μm and 10,000 μm, the total porosity corresponding to the average value of the pore sizes measured within the porous section, said shaping step can for example comprise the implementation of an additive manufacturing process, in particular 3D printing; - crosslinking a hydrogel comprising at least one divalent cation, preferably calcium, and transglutaminase, said hydrogel having a mechanical strength between 1 kPa and 1000 kPa. The method can be obtained by a manufacturing process including the steps of:

[0180] The process may further include a sterilization step.

[0181] According to specific applications, the present disclosure also relates to implants that may have one or more of the following features: - a three-dimensional intracorporeal implant, which may have a total porosity of up to 5000 μm, comprising a hydrogel comprising cross-linked alginate and cross-linked gelatin, said hydrogel having a mechanical strength, called elastic modulus or Young's modulus, of between 1 kPa and 1000 kPa; - three-dimensional intracorporeal implants which may have sections of different porosity within their three-dimensional structure, in particular in the form of a gradient distributed over more than one section of the implant; the three-dimensional body implant may also contain cross-linked fibrinogen; a three-dimensional body implant, which may preferably be a breast implant having at least two, in particular three, sections, each of which has a different porosity; a three-dimensional intracorporeal implant, which may comprise a hydrogel comprising cross-linked gelatin and cross-linked alginate, said hydrogel having a mechanical strength of between 1 kPa and 1000 kPa, said implant having a total porosity of up to 5000 μm, In this case, the gelatine can be crosslinked by an enzyme, preferably a transglutaminase, - three-dimensional intracorporeal implants which may have a porosity gradient distributed in more than one section of the implant; - a three-dimensional body implant, which may have a volume ranging from 0.05 mL to 3 L, preferably from 100 mL to 600 mL; - the three-dimensional body implant may be a breast implant; In this case, the hydrogel of the implant may contain 0.5% to 3% alginate and 1% to 17.5% gelatin, in this case, the hydrogel of the implant may further comprise cross-linked fibrinogen, preferably between 0.0001% and 6% of fibrinogen, a three-dimensional body implant obtainable by a process comprising a step of crosslinking a hydrogel with a solution containing a divalent cation, preferably calcium, and an agent capable of forming a covalent bond between lysine and glutamine residues, such as an enzyme, for example transglutaminase; - if the hydrogel comprises fibrinogen, said solution may further comprise thrombin; - Three-dimensional internal implants that can be obtained by 3D printing.

[0182] The present invention also relates to methods of implementing the implants described above in the context of reconstructive or cosmetic surgery, comprising the step of implanting the implant within the body of a subject.

[0183] Preferably, said implant is a breast implant.The invention therefore relates to a method for breast reconstruction comprising implanting an implant according to the invention into the body of a subject in need thereof.

[0184] The method may further comprise the step of injecting cells, preferably autologous cells, into the implant prior to implantation in the subject.

[0185] According to one embodiment, the subject is female. According to one embodiment, the subject is a woman who has had a mastectomy.

[0186] Further features, 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]

[0187] [Figure 1] 1 is a schematic representation of a breast-shaped implant according to the invention having a pore size gradient distributed in three sections. [Diagram 2] 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 3] Figure 1 shows a comparison of Young's modulus E (Pa) of AG hydrogels when gelatin was crosslinked in the presence and absence of transglutaminase and stored at 37°C for up to 7 days. [Figure 4] Comparison of Young's modulus E0 (Pa) of AG hydrogels cross-linked with or without transglutaminase and commercial hydrogels. *: Liquid compound at 37°C. +: Polymerization is visible at 37°C by DMA measurement, but the gel is not stiff enough. [Diagram 5] FIG. 1 represents the viability and cell growth measured by kinetics in FAG and AG hydrogels constituting an implant according to the invention and engrafted with fibroblasts in vitro after production. [Figure 6] FIG. 1 represents the viability and cell growth measured by kinetics on FAG and AG hydrogels constituting the implants according to the invention and engrafted in vitro by adipose tissue stem cells after production. [Figure 7] FIG. 1 represents the metabolic activity of an AG implant according to the invention at different culture times after in vitro engraftment of purified adipose tissue fractions after production. [Figure 8] Figure 1. Histological analysis by hematoxylin-phloxine-saffron (HPS) staining of AG implants according to the invention after 2 days (four images on the left) or 7 days (two images on the right) of in vitro incubation with purified adipose tissue fraction after production (top: outer edge of the matrix; bottom: inner hole of the matrix; images taken under white light; magnification x100; scale 100 µm). [Figure 9] Figure 2 shows perilipin 1 immunostaining and Dapi staining of cell nuclei on AG implants 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 10]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 11] FIG. 11 presents a comparison of Young's modulus E0 and viscosity of AG and FAG implants after crosslinking with different concentrations of CaCl2 (A, D), TAG (B, E) and thrombin (C, F). [Figure 12] 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 13] 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 14A] 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 14B] The effect of sterilization on the dimensions (B1-B2) and Young's modulus (B3-B4) of these implants is shown. [Figure 15] 1 shows the reproducibility of the production of AG implants according to the invention in terms of dimensions (A), volume (B) and pore size (C). [Figure 16] 1 shows the reproducibility of shrinkage of an AG implant according to the invention after curing. [Figure 17] 1 shows the reproducibility of shrinkage of an AG implant according to the invention as a function of sterilization method. [Figure 18A] The reproducibility of the extrusion diameter is shown. [Figure 18B] 1 shows the reproducibility of the hole length (B1-B2) of the AG implant according to the present invention. [Figure 19] 1 shows images of various pore sizes in an AG implant according to the present invention. [Figure 20] 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 21]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 22] 1 depicts the average pore length of implants produced with different pore sizes. [Diagram 23] 1 represents the average pore length of implants produced with an increasing pore size gradient from base to top. [Figure 24] 1 depicts the apparent Young's modulus values ​​of different sub-portions of implants produced with different pore sizes. [Diagram 25] 4 shows compression tests on complete dentures with different configurations and stress displacement curves. [Figure 26] Microscopic observation of implant bases with (left) or without (right) peripheral appendages is shown. [Figure 27A] 1 represents images of 3D printing of a large volume A / G implant (implant 9 cm long, 7 cm wide, and 2.7 cm thick), the implant obtained after crosslinking, and the large holes obtained within the structure. [Figure 27B] 1 represents images of 3D printing of a large volume A / G implant (12.6 cm diameter implant, and 5.3 cm thickness), the implant obtained after crosslinking, and the large holes obtained within the structure. [Figure 28] 13 depicts macroscopic observations of the pores of implants with different filling rates. [Figure 29] 1 represents the average distance between the centers of the pores of implants with different filling rates. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0188] Working Example The invention will be better understood on reading the following examples which illustrate the invention in a non-limiting manner.

[0189] 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.

[0190] 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 (Labelians, France) for 12 h at 37 °C.

[0191] Protocol #3 Molding of AG and FAG hydrogels: Deposit 1.8 mL of hydrogels prepared according to protocols #1 and #2 into wells of a 6-well culture plate and incubate at 21 °C for 30 min.

[0192] 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 (Labelians, France). The crosslinking solution is then placed in contact with the hydrogel for 1 h 30 min at 37 °C (unless otherwise stated).

[0193] 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).

[0194] Protocol #6 Dynamic Mechanical Analysis (DMA) in Compression: The mechanical properties of FAG and AG hydrogels are measured in triplicate on a rotational rheometer (DHR2, TA Instrument, France), on a Peltier plane (TA Instrument, France) and in an 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.

[0195] Protocol #7 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) to apply a constant pressure 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).

[0196] Protocol #8 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 subcutaneously with 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 area. The bioprosthesis was implanted in the dorsal subcutaneous area of ​​each animal. The control group underwent incision and dissection only. Four surgical sites were performed in one animal per group: three bioprostheses 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, animals were monitored for signs of morbidity and the surgical wounds were examined daily for skin healing and absence of infection. Explants were performed 21 days after implantation.

[0197] Protocol #9 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.

[0198] Protocol #10 Dynamic Mechanical Analysis (DMA) under Compression: The mechanical properties of FAG and AG hydrogels are measured in triplicate on a rotational rheometer (DHR2, TA Instrument, France), a Peltier plane (TA Instrument, France) and a 25 mm geometric structure (TA Instrument, France). A 25 mm diameter punch is cut out of the implants produced according to protocol #9. The punch is placed on the lower geometric structure for 60 s at 37 °C, after which a 10 μm oscillatory compression procedure is carried out at 0.1-10 Hz and 37 °C at 100 μm / s. 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.

[0199] Protocol #11 Total mechanical analysis of implants under compression: The implants are mounted on a Lloyd tension / compression machine equipped with a 1 kN load cell and compression plate, using a test speed of 10 mm / min.

[0200] Example 1 – Mechanical properties of alginate / gelatin (AG) and fibrinogen / alginate / gelatin (FAG) hydrogels AG and FAG hydrogels were prepared from protocols #1 and #2, molded according to protocol #3, then crosslinked using protocols #4 and #5, and their DMA mechanical properties were tested using protocol #6.

[0201] The results are shown in Figure 2 (A-B). The measured Young's modulus and viscosity values ​​are similar for 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.

[0202] Example 2 - Effect of cross-linking with transglutaminase on the mechanical properties of alginate / gelatin hydrogels (AG) Molding samples of AG were prepared from protocols #1 and #3 and crosslinked from a variation of protocol #4, where the crosslinking solution consisted of 30 mg / mL calcium chloride solution alone or 30 mg / mL calcium chloride and 40 mg / mL transglutaminase solution. Four gels of each condition were cast and examined by DMA on the same day and after 1, 4 and 7 days of storage at 37° C., respectively, to mimic physiological conditions.

[0203] The samples were then tested by DMA using protocol #6.

[0204] The results are shown in Figure 3. This study shows the beneficial effect of the use of transglutaminase in cross-linking on the mechanical properties of the hydrogel, which is even greater when the gel is transformed at 37°C, justifying the special interest of cross-linking according to the invention for hydrogels intended to be implanted.

[0205] Example 3 - Effect of cross-linking with transglutaminase on the mechanical properties of commercial gelatin and / or collagen hydrogels Molded samples of GA were prepared from protocols #1 and #3 and crosslinked from protocol #4. Commercially available hydrogel samples listed in Table 4 below were prepared according to the protocols provided by the supplier and molded according to protocol #3. [Table 4]

[0206] Hydrogels were crosslinked using a modification of protocol #4 with 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.

[0207] Uncrosslinked or crosslinked samples with TAG were then tested by DMA using protocol #6.

[0208] The results are summarized in Figure 4. 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 transglutaminase cross-linking (7.3, 9.9 and 50 kPa, respectively). This study shows the effect of cross-linking with transglutaminase on the stiffness of commercial hydrogels.

[0209] Example 4 - Effect of the amount of alginate and gelatin in fibrinogen / alginate / gelatin (FAG) hydrogels on the mechanical properties FAG hydrogels were prepared from 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.

[0210] The results are summarized below in Table 5. Young's modulus under the specific conditions of this test is in the range of 200-800 kPa. [Table 5]

[0211] Example 5 – Evaluation of fibrinogen / alginate / gelatin (FAG) and alginate / gelatin (AG) hydrogel engraftment by fibroblasts AG and FAG hydrogels were prepared from protocols #1 and #2. Square implants with sides of 1.5 cm and thickness of 0.2 cm were then printed using protocol #7 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.

[0212] Normal human fibroblasts of 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 dropped onto each implant. After 1 hour of adhesion, the implants were immersed in culture medium. The implants were cultured at 37° C., 5% CO2 in a culture medium composed of DMEM containing 10% bovine serum and supplemented with vitamin C and EGF (epidermal growth factor). The implants were cultured for 21 days with the same medium replaced every 3 days.

[0213] 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).

[0214] Cell viability and growth was monitored over 21 days of culture using six-point kinetics on days 3, 5, 8, 10, 14 and 21. The results are shown in FIG.

[0215] 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 hydrogel types (FAG and AG) and each total porosity used.

[0216] Example 6: Evaluation of engraftment of fibrinogen / alginate / gelatin (FAG) and alginate / gelatin (AG) hydrogels by adipose tissue stem cells (ASCs) AG and FAG hydrogels were prepared from protocols #1 and #2. Then, square implants with sides of 1.5 cm and thickness of 0.2 cm were printed using protocol #7 and crosslinked using protocols #4 or #5. The printed implants were produced with a 410 μm inner diameter extrusion nozzle at a filling rate of 50% and 75%. Sterilization was performed by irradiating the implants with a dose of 30 kGy of gamma radiation by IONISOS (France).

[0217] Normal human adipocyte stem cells from pathways 2-5 were thawed and expanded in 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 dropped 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.

[0218] 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).

[0219] 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.

[0220] 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 at each seeding density for both types of hydrogels (FAG and AG).

[0221] Example 7: Evaluation of engraftment of alginate / gelatin (GA) hydrogels in contact with purified adipose tissue fragments GA hydrogels were prepared from protocol #1. Then, cubic implants with sides of 1.5 and thickness of 0.8 cm were printed using protocol #7 and crosslinked using protocol #4. The printed implants were produced with a 410 μm inner diameter extrusion nozzle at 50% fill factor.

[0222] The lipoaspirate was centrifuged at 1500 rpm for 2 min and then rinsed with 1× PBS. The lipoaspirate was centrifuged again at 1500 rpm for 30 s, after which the 1× PBS was removed. The lipid aspirate was considered purified.

[0223] 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.

[0224] 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.

[0225] The cell metabolic activity in the implants was tested by colorimetry with Alamar Blue on the 2nd, 7th and 21st culture days 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 in a spectrophotometer (NanoQuant® infinite M200PRO, TECAN).

[0226] Cell viability and growth was monitored over a period of 21 days and the results are shown in Figure 7. Significantly higher metabolic activity was observed in implants contacted with purified lipoaspirate than in the negative control.

[0227] Histological analysis was performed and this study was completed according to protocol #9. The results are shown in FIG. 8. The images reveal the presence of aggregated polygonal uniform unilocular bulky adipocytes. These morphological characteristics are those of healthy adipocytes that can be found in adipose tissue.

[0228] Immunostaining with 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 with 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 non-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 images obtained are summarized in FIG.

[0229] The images show adipocytes with large spherical or polygonal vacuoles depending on the clustering of the cells. The adipocytes appear unilocular and are also physiological in size, ranging from 50 to 200 μm.

[0230] Taken together, these results confirm the attachment, survival and regeneration of human adipose tissue in contact with the implant. The unique structure and composition of the implant therefore creates a favorable environment for the regeneration of healthy adipose tissue.

[0231] Example 8 - Effect of temperature and crosslinking time on the mechanical properties of alginate / gelatin hydrogels (AG) Molding samples of AG were prepared from Protocol #1 and Protocol #3 and crosslinked from a variation of Protocol #4, in which the crosslinking time and temperature were changed from 10 minutes to 14 hours and from 37°C to 21°C.

[0232] The samples were then tested by DMA using protocol #6.

[0233] The results are shown in Figure 10 (A-B). The crosslinking time and temperature have very little effect on the final mechanical properties (Young's modulus) of the hydrogel. However, it appears that an optimum can be found around 1 hour 30 minutes, regardless of the temperature.

[0234] 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.

[0235] Example 9 - Effect of crosslinking solution component concentrations on the mechanical properties of crosslinked alginate / gelatin (AG) and fibrinogen / alginate / gelatin (FAG) hydrogels AG and FAG molding samples were prepared from protocols #1, #2, and #3 and cross-linked from a variation of protocols #4 and #5, in which the concentrations of the components of the cross-linking solution were altered (transglutaminase, calcium chloride, and thrombin).

[0236] The samples were then tested by DMA using protocol #6.

[0237] The results are summarized in Figure 11 (A-F). No significant changes were observed over this range of reagent concentrations (all of the E0s were very similar).

[0238] 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 from protocols #1, #2, and #3 and crosslinked from a variation of protocols #4 and #5. In this variation, we examined sequential crosslinking with FAG and AG, which involves crosslinking the hydrogel in multiple steps. Each step took 1 h, and three rinses with 0.1 M NaCl solution were performed between each step to remove residual crosslinker.

[0239] The samples were then tested by DMA using protocol #6.

[0240] The results are shown in Figure 12 (A-D). The sequential crosslinking set (FAG and AG) produces hydrogels with lower Young's modulus than single step crosslinking.

[0241] It can be observed that if calcium is not added first, a very soft and fragile gel is obtained; 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 little mechanical strength and holes appear.

[0242] Example 11 - Effect of the nature of the divalent cation for crosslinking of alginate / gelatin (AG) and fibrinogen / alginate / gelatin (FAG) hydrogels Molding samples of AG and FAG were prepared from protocols #1, #2, and #3 and crosslinked from a variant of protocols #4 and #5, in which we tested crosslinking in the presence of 30 mg / mL barium chloride.

[0243] The samples were then tested by DMA using protocol #6.

[0244] The results are shown in Figure 13 (A-B). Cross-linking in the presence of barium leads to a gel with a 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 assumed.

[0245] 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 from protocols #1, #2 and #3, crosslinked using protocols #4 and #5, optically observed and then tested by DMA using protocol #6. The printed geometries were 2 cm diameter hemispheres, generated with variable filling fractions (30, 50 and 75%).

[0246] Sterilization was performed by ionisos (France) by irradiating the implants with various doses of gamma radiation (30 kGy and 40 kGy).

[0247] The effect of the cross-linking step on the dimensions of alginate / gelatin and fibrinogen / alginate / gelatin hydrogel implants was studied, these dimensions were measured from macroscopic images.

[0248] 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).

[0249] The results are shown in Figure 14 (A-B). The implants shrink by an average of 10% after the crosslinking step, but the pore size does not change significantly (Figure 14A (A1-A4)).

[0250] 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 for either dose (FIG. 14B (B1-B4)).

[0251] Example 13: Production quality of large alginate / gelatin (AG) hydrogel implants: Reproducibility of implant hole dimensions by multiple methods, i.e. dimensions after curing and sterilization AG hydrogels were prepared from protocol #1. Hemispherical implants with a diameter of 6 cm and a thickness of 2 cm were then printed according to protocol #7, crosslinked using protocol #4, and then optically observed and measured. The printed shapes were produced using an extrusion nozzle with an inner diameter of 410 or 840 μm with variable fill factor (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.

[0252] The effect of cross-linking and sterilization steps on the dimensions of large alginate / gelatin hydrogel implants was studied, these dimensions being measured from macroscopic images.

[0253] 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).

[0254] The post-printing results are shown in Figure 15 (A-C). These results show high reproducibility of the dimensions of the 3D printed large implants, reflecting high production quality.

[0255] The results after curing of the implants are summarized in Figure 16. This graph shows the high reproducibility of the shrinkage of the large implants after the curing phase.

[0256] The results after sterilization of the implants by the three methods (beta radiation at doses of 40 and 30 kGy, and gamma radiation at 30 kGy) are summarized in Figure 17. The results show that beta radiation at 30 and 40 kGy results in less shrinkage of the larger implants.

[0257] 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 18 (A-B).

[0258] Figure 18A shows the high reproducibility of the size of the extruded beads, and Figure 18B (B1-B2) shows the variation of the pore length with the filling rate of the hydrogel.

[0259] Images of various pore sizes were taken and are summarized in FIG.

[0260] These data demonstrate the wide range of pores that can be obtained in the implants, as well as their high reproducibility and production quality.

[0261] Example 14 – In vivo implant resistance testing GA and FAG hydrogels were prepared from protocols #1, #2 and #7 and crosslinked through protocols #4 and #5. The printed geometries were 1 cm diameter hemispheres and were generated with various filling fractions (30, 50 and 75%).

[0262] The porous hemispheres were sterilized with a dose of 30 kGy and then implanted subcutaneously in rats according to protocol #8.

[0263] Details of the implantation groups are set forth in Table 6 below, which references the surgical implantation plan set forth in FIG. [Table 6]

[0264] Histological analysis was performed using protocol #9 and the results are summarized in Figure 21. Explants were used to verify the resistance of the implants to skin tension. Histological analysis was used to evaluate cell engraftment, angiogenesis, extracellular matrix synthesis, and the presence of inflammatory areas.

[0265] Example 15 - Study of the production quality of implants with various large pore sizes and the effect of these porosities on the mechanical properties of said implants Semi-anatomical prosthetic breast-shaped implants (height: 8.83 cm; width: 6.37 cm; height: 2.86 cm) are produced using Protocol #1 and a variant of Protocol #7 (using a nozzle with an internal diameter of 840 μm) and then crosslinked using Protocol #4. These implants are produced with different internal porosities: - an implant having one pore size throughout its volume, - an implant with two pore sizes distributed according to the idea that a first portion at the base of the implant has one pore size and a second portion at the top of the implant has another pore size; - an implant with three pore sizes distributed according to either: a first part at the base of the implant (called the base) has one pore size, a second separate part in the middle and on the top of the base of the implant (called the core) has another pore size, and a third separate part at the surface and on the top of the implant (called the shell) has another pore size; - Implants with a gradient of increasing pore size from the base have also been produced.

[0266] The dimensions of the pores obtained were examined. These dimensions were measured from images taken under a microscope (Olympus, magnification x4). The results of these measurements are shown in Figures 22 and 23. Pores of different sizes and very reproducible can be obtained in the different parts of the implant. A reproducible gradient of increasing pore size from the base to the top can also be obtained.

[0267] The mechanical properties of the subsections of these implants were tested by DMA according to protocol #11, and the mechanical properties of the implants were tested by total mechanical analysis according to protocol #12. The results of these measurements are shown in Figures 24 and 25. The observed Young's modulus varies inversely with the pore size. Thus, for example, a higher Young's modulus can be obtained by decreasing the pore size of the pores in the center of the implant, reflecting a higher mechanical resistance. Thus, varying the pore size and the distribution of pore size fractions allows a wide range of Young's modulus and therefore a stronger or weaker implant. Regarding the compression tests of the entire prosthesis, the inventors have observed that each configuration of pores results in different mechanical properties for the implant. In fact, applying -35N, the prosthesis with only one porosity fraction deformed less than the more deformed prosthesis with three porosity fractions. The inventors are also able to identify different fracture behaviors by the curves. Whereas the prosthesis with only one porosity section deforms gradually before fracture, the prosthesis with two porosity sections fractures gradually, but then becomes brutal at -217N and recovers from the stress. Thus, by varying the pore size and distribution of pore size sections, implants with different mechanical properties can be obtained, which allows the mechanical properties of the implant to be tailored depending on the desired application.

[0268] Example 16 - Adding a perimeter around the base of the implant Semi-anatomically sized artificial breast implants (height: 8.83 cm; width: 6.37 cm; height: 2.86 cm) are produced from Protocol #1 and a variation of Protocol #7 (using a nozzle with an internal diameter of 840 μm, a perimeter is added to the base of some implants) and then crosslinked from Protocol #4. All of these implants have a single pore size, and a perimeter is added to some implants. This perimeter is characterized by the addition of continuous filaments around the implant to all filaments located tangentially around the implant. This perimeter is added to the first three layers of the implant.

[0269] The resulting configuration was examined using a microscope (Olympus, magnification x4) and the results of these observations are shown in Figure 26.

[0270] The addition of a perimeter to the base of the implant makes it possible to obtain a more coherent base with less peripheral irregularities, thus limiting inflammatory friction in vivo.

[0271] Example 17 – Creation of voluminous porous implants from alginate / gelatin hydrogels Implant 1: 200 mL of AG hydrogel was prepared from protocol #1, then 12 cm long, 10 cm wide and 5 cm thick anatomical breast-like implants were printed with a single porosity area from a variant of protocol #7 (840 μm inner diameter extrusion nozzle and 30 mm / s print speed) and then crosslinked with protocol #4 (200 mL instead of 100 mL of hardening solution for larger implants). The average pore size of the resulting implants was measured by optical microscopy and the dimensions of the implants were measured with calipers. After crosslinking, 9 cm long, 7 cm wide and 2.7 cm thick implants are obtained with an average pore size of 1380 ± 57 μm.

[0272] Implant 2: 500 mL of AG hydrogel was prepared from protocol #1, then a hemispherical breast-like implant with a 7 cm radius and 6 cm thickness was printed with a single hole area from a variant of protocol #7 (840 μm inner diameter extrusion nozzle and 30 mm / s print speed) and then crosslinked with protocol #4 (700 mL instead of 100 mL of hardening solution for the larger implant). The average pore size of the resulting implant was measured by optical microscopy and the dimensions of the implant were measured with a caliper. After crosslinking, a 12.5 cm diameter and 5.3 cm thick implant is obtained with an average pore size of 3354 ± 273 μm.

[0273] Images of these implants are shown in Figures 27A and 27B.

[0274] Example 18 – Measurement of spatial distribution of pores in the same section of the implant Semi-anatomically sized artificial breast-shaped implants (height: 8.83 cm; width: 6.37 cm; height: 2.86 cm) are generated from protocol #1 and protocol #7, followed by crosslinking from protocol #4. These implants are generated with different fill rates (45, 50 and 55%).

[0275] The distance separating the centers of the resulting pores (square shaped) was tested. These dimensions were measured from images taken with a microscope (Olympus, magnification x4). The results of these measurements are shown in Figures 28 and 29. The distance separating the centers of the pores was found to be reproducible for each filling rate and varies between the different filling rates. These observations reflect a uniform pore distribution within the sections of the implant with the defined filling rate.

Claims

1. A three-dimensional in vivo implant comprising a hydrogel containing crosslinked gelatin and crosslinked alginate, wherein the hydrogel has a mechanical strength of 1 kPa to 1000 kPa, and the implant has at least one porous section, the porous section containing a plurality of pores each having one pore diameter, the porous section having a total porosity between 100 μm and 10,000 μm, and the total porosity corresponding to an average value of the pore diameters measured within the porous section. A three-dimensional in vivo implant characterized by this.

2. The pores of the porous section are having a uniform pore diameter, or uniformly distributed, or extending along a central axis each having a uniform orientation, or each having a uniform geometric structure, or separated from each other by bundles of a material each having a uniform thickness, or having a gyroidal, cubic or planar hexagonal shape, The implant according to claim 1.

3. The implant according to claim 1, wherein the gelatin is crosslinked by an enzyme.

4. The implant according to claim 1, comprising a plurality of porous sections.

5. The implant according to claim 4, wherein the plurality of porous sections includes at least two porous sections having different pore diameters and / or shapes.

6. The implant according to claim 5, wherein the porous sections are arranged to form a gradient of pore diameters distributed throughout the implant, and the porous sections are connected to each other along the gradient direction in an order selected from ascending and descending order of pore diameters.

7. - A first porous section forming a base, representing 5% to 40% of the total volume of the implant and having a pore diameter between 500 micrometers and 5000 micrometers, - A second porous section forming a core, representing 20% to 70% of the total volume of the implant and having a pore diameter between 500 micrometers and 2500 micrometers, - A third porous section forming an outer shell, representing 5% to 40% of the total volume of the implant and having a pore diameter between 1000 micrometers and 10,000 micrometers, The implant according to claim 5, comprising.

8. The implant according to claim 1, having at least one non-porous section, the non-porous section having a filling rate greater than 99%.

9. Comprising a plurality of layers each having one mesh composed of a plurality of meshes, the layers being stacked on top of each other such that the meshes form the holes, the implant according to claim 1.

10. The meshes of each layer are having a uniform mesh size, or uniformly distributed, or extending around a central mesh axis each having a uniform orientation, or arranged at uniform intervals, or separated from each other by bundles of a material each having a uniform thickness, the implant according to claim 9.

11. The implant according to claim 1, characterized in that it has a volume in the range of 100 mL to 600 mL.

12. The implant according to claim 1, characterized in that it is a breast implant.

13. - preparing a hydrogel comprising gelatin and alginate; - three-dimensionally shaping the hydrogel to form at least one porous section, the porous section having a plurality of pores each having one pore diameter, the porous section having a total porosity between 100 μm and 10,000 μm, the total porosity corresponding to the average value of the pore diameters measured within the porous section; - crosslinking the hydrogel with at least one divalent cation and transglutaminase, the hydrogel having a mechanical strength of 1 kPa to 1000 kPa A three-dimensional in-vivo implant obtainable by a manufacturing process continuously comprising.

14. During the crosslinking step, the divalent cation and the transglutaminase are added simultaneously, or during the crosslinking step, thrombin is further used, or during the three-dimensional shaping step, a stereolithography process is carried out, or further comprising a sterilization step, the three-dimensional in-vivo implant according to claim 13, obtainable by the manufacturing process.

15. the hydrogel comprises 0.5% to 3% alginate and 1% to 17.5% gelatin, or the hydrogel further comprises crosslinked fibrinogen, the three-dimensional in-vivo implant according to claim 13 or 14, obtainable by the manufacturing process.

Images