Porous collagen material and process for preparing the same by topotactic fibrillogenesis

Abstract

The present invention relates to a process for preparing a porous collagen material by topotactic fibrillogenesis. The obtained porous collagen material presents properties similar to native fibrillar collagen tissues, being thereby useful as three-dimensional scaffold for in vitro cell culture or for tissue engineering.

Description

POROUS COLLAGEN MATERIAL AND PROCESS FOR PREPARING THE SAME BY TOPOTACTIC FIBRILLOGENESISFIELD OF INVENTION

[0001] The present invention relates to a process for preparing a porous collagen material by topotactic fibrillogenesis. The obtained porous collagen material presents properties similar to native fibrillar collagen tissues, being thereby useful as three- dimensional scaffold for in vitro cell culture or for tissue engineering.BACKGROUND OF INVENTION

[0002] Standard in vitro cultures of cells are usually performed on two-dimensional substrates (2D cell cultures). Nevertheless, it is commonly admitted that 2D cell cultures lack the capacity to reproduce the native cellular microenvironment and may thus provide biological responses in disagreement with in vivo observations.

[0003] Substrates for three-dimensional (3D) cell cultures have been developed, including for example hydrogel scaffolds, which enable to provide in vitro systems of increased physiological relevance.

[0004] Nevertheless, there is an ongoing need to develop improved substrates for 3D cell cultures that can mimic the native cellular microenvironment. Especially, there is a need for materials combining extensive macroporosity, favoring cell migration and nutrient exchanges, and that mimic locally the extracellular matrix.

[0005] Further to in vitro cell culture, this type of 3D porous materials is of interest as scaffold for tissue engineering. In this context, the structural and mechanical properties of the 3D porous materials need to be finely tuned, depending on the targeted tissue.

[0006] Whether for cell culture or tissue engineering, the composition of the porous materials is of importance. Collagen, and especially type I collagen, is a candidate ofchoice since it is the most abundant protein in connective tissues and presents the advantage to be non-immunogenic and highly conserved across species.

[0007] In vitro, under a narrow window of physicochemical conditions, type I collagen self-assembles to form complex supramolecular porous architectures similar to those found in native extracellular matrix (ECM). Nevertheless, coupling such delicate collagen fibrillogenesis events with a controlled shaping process in non-denaturating conditions is a major challenge to provide porous collagen-based materials that reproduce in vivo architectures.

[0008] Several attempts have led to the provision of macroporous collagen matrices that need to be crosslinked in order to maintain the 3D structure. Nevertheless, crosslinking requires the use of toxic molecules and modifies the cellular response to the matrices.Closest to native fibrillated collagen, Rieu et al. (ACS Applied Material and Interfaces, 2019, 11, 14672-14683) and Parisi et al. (Biomaterials Sciences, 2022, 10, 6939-6950) disclose fibrillated porous collagen materials, without the need to reticulate the material. In these references, a porous collagen material is obtained by a two-steps process comprising a first step of ice-templating of an aqueous solution collagen that promotes the structuration of collagen by the ice crystals; followed by a second step of topotactic fibrillogenesis comprising the pre-fibrillogenesis of the ice-templated collagen solution by exposure to ammonia vapors at 0°C, followed by the consolidation of the fibrillogenesis in a PBS buffer 5X at room temperature after removal of ammonia. These collagen-only, non-cross-linked scaffolds exhibit interesting mechanical properties in the wet state, with a Young’s modulus of 33 ± 12 kPa, an ultimate tensile stress of 33 ± 6 kPa, and a strain at failure of 105 ± 28%.

[0009] Nevertheless, this method of topotactic fibrillogenesis by exposure to ammonia vapors might be difficult to implement due to the use of ammonia vapors. Moreover, this method only enables to provide porous surfaces, i.e. surfaces displaying open pores, while in some instances it might be interesting to obtain non-porous surfaces and / or a mix of porous and non-porous surfaces, for example in tissue engineering of arteries of tracheas. Another concern it that the thickness of the walls between the pores is very thin in thecollagen materials obtained by topotactic fibrillogenesis by exposure to ammonia vapors, while thicker walls would be preferable for the overall resistance of the material and / or for cells’ colonization.

[0010] There is thus a need for other processes of preparing porous collagen materials, whether anisotropic or not, in order to achieve the above properties, while being versatile and easy to implement.

[0011] In Rieu et al. (ib.), it was unsuccessfully attempted to perform the pre-fibrillogenesis of the ice-templated collagen solution by directly dipping it in PBS buffer at room temperature, since it resulted in a loose material with occluded pores, indicating an unfavorable competition between collagen fibrillogenesis and dissolution.

[0012] Despite this negative incentive, the Applicants further explored conditions of pre- fibrillogenesis of the ice-templated collagen solution in presence of PBS-derived buffer and it was unexpectedly found that conducting this step in a closed range of temperature, namely - 5°C to 0°C, enables to achieve an efficient process of preparing porous collagen materials with targeted properties.

[0013] The present invention thus refers to a process of preparing porous collagen materials by a two-steps process as detailed hereafter, comprising a first step of ice- templating of an aqueous solution collagen, followed by a second step of topotactic fibrillogenesis comprising the pre-fibrillogenesis of the ice-templated collagen solution by exposure to a first buffer at a temperature ranging from - 5°C to 0°C, followed by the consolidation of the fibrillogenesis in a second buffer at room temperature, wherein the first and second buffers can be the same of different.

[0014] The process of the invention thus mainly differs from previously disclosed methods in that the pre-fibrillogenesis of the ice-templated collagen solution is performed by direct exposure to a buffer, and at a temperature ranging from - 5 °C to 0°C.

[0015] This difference of pre-fibrillogenesis conditions leads to materials presenting different structural and mechanical properties, as detailed in the experimental part hereafter.

[0016] Especially, the collagen material obtained by the method of the invention presents walls between pores that are thicker compared to those of collagen materials obtained by topotactic fibrillogenesis by exposure to ammonia vapors.

[0017] Further, with the method of topotactic fibrillogenesis in buffer according to the invention, it is possible to provide material having both porous and non-porous surfaces, which was not achievable by the methods of the prior art. This might be controlled by selecting specific materials for the mold, and by adjusting the composition of the buffer.

[0018] The material obtained by the process of the invention is similar to native fibrillar collagen tissues and presents a good stability overtime. Moreover, the structural and mechanical properties are suitable for living cell seeding and enable cellular colonization. Consequently, obtained materials are useful as three-dimensional scaffolds for in vitro cell culture and tissue engineering.

[0019] Moreover, the process of the invention is more versatile than previously known processes, especially by its ease of handling and implementing the protocol. The process of the invention also enables to control the properties of the obtained materials, including their compositions, the size and orientation of the pores, the mechanical properties, by adjusting the properties of the buffers used in the fibrillogenesis steps. Further, the process of the invention enables a control of the shape of the obtained material.SUMMARY

[0020] This invention thus relates to a process of preparing a porous collagen material, comprising: a first step of ice-templating an aqueous solution of collagen to form an ice-templated scaffold; and a second successive step of topotactic fibrillogenesis in buffer, comprising: a pre-fibrillogenesis step by contacting at least one surface of the ice- templated scaffold with a first buffer, at a temperature ranging from - 5 °C to 0°C, preferably - 3°C, for a period of time ranging from 24 hours to 72 hours;wherein the first buffer has an ionic composition which is preferably derived from phosphate buffer saline (PBS), which maintains a pH value above 7, and which osmolarity is above 0.2 Osm / L, preferably above 1.2 Osm / L; and then a consolidation step by maintaining the contact between the at least one surface of the ice-templated scaffold with a second buffer, at room temperature until the resulting collagen material presents a denaturation onset temperature of at least 45°C; wherein the second buffer has an ionic composition which is preferably derived from PBS buffer, which maintains a pH value above 7, and which osmolarity is above 0.1 Osm / L, preferably above 0.6 Osm / L; wherein the first buffer and the second buffer are the same or different.

[0021] In one embodiment, the collagen is type I collagen. Preferably, the aqueous solution of collagen has a concentration ranging from 1 mg.mL1to 120 mg.mL1, preferably from 10 mg.mL1to 60 mg.mL1, more preferably 40 mg.mL1. The aqueous solution of collagen may further comprise excipients, additives, active substances such as growth factors or interleukins, or mixtures thereof.

[0022] In one embodiment, the ice-templating of the aqueous solution of collagen is performed by a method selected from: a) dipping a mold comprising the aqueous solution of collagen in a cryogenic liquid, at a dipping-speed ranging from 1 mm.min1to 100 mm.min1, preferably 10 mm.min1; b) placing a mold comprising the aqueous solution of collagen on a thermally conductive surface and cooling said mold at a rate ranging from - l0C.min-1to - 50oC.min-1, preferably - 50C.min-1, from room temperature to - 80°C; and c) placing a mold comprising the aqueous solution of collagen in a constant temperature field at - 80°C.

[0023] In one embodiment, in the consolidation step, the contact at room temperature between the at least one surface of the ice-templated scaffold and the second buffer is maintained for at least 5 days, preferably at least 8 days, more preferably at least 10 days.

[0024] Advantageously, the obtained porous collagen material is stored in aqueous medium until use.

[0025] In one embodiment, the process according to the invention can further comprise a step of topotactic fibrillogenesis by exposure to ammonia vapors, comprising exposing at least one surface of the ice-templated collagen scaffold to ammonia vapors for a period of time ranging from 24 hours to 72 hours, followed by elimination of ammonia vapors; said further step of topotactic fibrillogenesis by exposure to ammonia vapors being conducted: i) between the first step of ice-templating and the second step of topotactic fibrillogenesis in buffer; ii) concomitantly with the second step of topotactic fibrillogenesis in buffer; or iii) successively to the second step of topotactic fibrillogenesis in buffer.

[0026] In one embodiment, the topotactic fibrillogenesis by exposure to ammonia vapors is performed concomitantly with the second step of topotactic fibrillogenesis in buffer, wherein one surface of the ice-templated collagen scaffold is exposed to ammonia vapors in the above conditions, while a different surface of the ice-templated collagen scaffold is contacted with the first buffer in the conditions of the pre-fibrillogenesis step.

[0027] In one particular embodiment, the ice-templated scaffold obtained in the first step has the shape of a tube, and the second step is performed on both the luminal and outer surfaces of the tube, leading to a buffer-topotactically-fibrillated tube; and further comprising: a third successive step comprising: deposing, at room temperature, an aqueous solution of collagen on one of the outer or luminal surfaces of the buffer-topotactically-fibrillated tube, to form a collagen-deposited surface; and contacting, at room temperature, the collagen-deposited surface with a third buffer, to form a collagen-dense surface; wherein the third buffer has an ionic composition which is preferably derived from PBS buffer, which maintains a pH value above 7, and which osmolarity is above 0.1 Osm / L, preferably above 0.6 Osm / L; andoptionally a fourth successive step of turning inward the outer surface of the tube obtained in the third step, so that the luminal surface of the tube be the former outer surface.

[0028] The invention further provides a porous collagen material obtained by the process according to the invention. The porous collagen material of the invention can have a shape being a planar shape or a three-dimensional shape; preferably a shape being a tube, a cylinder, or a sheet.

[0029] In one embodiment, the porous collagen material according to the invention is seeded with living cells.

[0030] The invention also relates to the use of a porous collagen material according to the invention as scaffold for in vitro cell culture.

[0031] The invention further relates to the use of a porous collagen material according to the invention, as scaffold for tissue engineering.DEFINITIONS

[0032] In the present invention, the following terms have the following meanings:

[0033] “About” preceding a figure means plus or less 10% of the value of said figure.

[0034] "Cryogenic liquid” refers to a liquid with a normal boiling point below -90°C. Examples of cryogenic liquids include liquid argon, liquid helium, liquid hydrogen, liquid nitrogen, and liquid oxygen.

[0035] “Ice-templating” refers to a technique that may be involved in the preparation of porous materials. It exploits the solidification behavior of water present in an aqueous solution or suspension, in order to controllably template a porous material. By subjecting an aqueous solution or suspension to a directional temperature gradient or even to a constant temperature field, ice crystals nucleate and grow directionally since the icecrystals redistribute the solute or the suspended particles as they grow, effectively templating the material.

[0036] “Porous collagen material” refers to a material comprising essentially collagen, and that is porous.

[0037] “Topotactic fibrillogenesis” refers in the present invention to the stabilization of the ice-templated scaffold via collagen fibrillogenesis using a topotactic process, that is, without changing the material’s texture (i.e. the structure and substructure of the walls and pores). Here, the term “topotactic” refers to above definition used in the field of chemistry and not to the single-cell sensitivity to the topography gradient phenomenon.DETAILED DESCRIPTIONProcess of preparing a porous collagen material

[0038] This invention relates to a process of preparing a porous collagen material comprising a step of topotactic fibrillogenesis in a buffer of an ice-templated aqueous solution of collagen.

[0039] A first object of the invention is thus a process of preparing a porous collagen material, comprising: a first step of ice-templating an aqueous solution of collagen to form an ice-templated scaffold; and a second successive step of topotactic fibrillogenesis in buffer, comprising: a pre-fibrillogenesis step by contacting at least one surface of the ice- templated scaffold with a first buffer, at a temperature ranging from - 5 °C to 0°C, preferably - 3°C, for a period of time ranging from 24 hours to 72 hours; wherein the first buffer has an ionic composition which is preferably derived from phosphate buffer saline (PBS), which maintains a pH value above 7, and which osmolarity is above 0.2 Osm / L, preferably above 1.2 Osm / L; and then a consolidation step by maintaining the contact between the at least one surface of the ice-templated scaffold with a second buffer, at roomtemperature until the resulting collagen material presents a denaturation onset temperature of at least 45°C; wherein the second buffer has an ionic composition which is preferably derived from PBS, which maintains a pH value above 7, and which osmolarity is above 0.1 Osm / L, preferably above 0.6 Osm / L; wherein the first buffer and the second buffer are the same or different.Collagen

[0040] In one embodiment, the collagen used in the process of the invention is type I collagen. Type I collagen is advantageous to form materials for in vitro cell culture or for tissue engineering, since it is the most abundant form of collagen, especially in the human body.

[0041] In one embodiment, the aqueous solution of collagen used in the first step of ice- templating has a concentration ranging from 1 mg.mL1to 120 mg.mL1, preferably from 10 mg.mL1to 60 mg.mL1, more preferably about 40 mg.mL1. Advantageously, when the aqueous solution of collagen has a high concentration in collagen, i.e. typically a concentration of at least 5 mg.mL1, the process of the invention provides a material with a good robustness, especially having elastic properties.

[0042] In one embodiment, the aqueous solution of collagen further comprises excipients, additives, biological substances, biocompatible substances, or mixtures thereof.

[0043] Examples of biological substances and biocompatible substances include, without being limited to, growth factors, interleukins, extracellular matrix molecules, peptides, nucleic acids, fluorescent dyes, or mixtures thereof.

[0044] Especially, the presence of active substances may be beneficial when the material formed by the process of the invention is intended to be used for in vitro cell culture or for tissue engineering. Especially, the presence of growth factors or interleukins in the porous collagen material of the invention may favor cell colonization of the material.Ice-templating

[0045] The first step of the process of the invention aims at inducing the self-organization of collagen molecules into liquid-crystal phases within the frozen material and creating pores.

[0046] The first step of the process of the invention is a step of ice-templating an aqueous solution of collagen. This method of freezing of an aqueous solution is advantageous since its mild conditions avoid collagen molecules to be denatured, while enabling to tailor the material’ s porosity (including pores’ size and direction) using simple parameters such as the ice front velocity or the processing temperatures.

[0047] The first step of ice-templating can be conducted according to any ice-templating method described in the art, which one skilled in the art will be able to select.

[0048] In one embodiment, in the first step of the process of the invention, the ice-templating of the aqueous solution of collagen is performed by a method selected from: a) dipping a mold comprising the aqueous solution of collagen in a cryogenic liquid, at a dipping-speed ranging from 1 mm.min1to 100 mm.min1, preferably 10 mm.min1; b) placing a mold comprising the aqueous solution of collagen on a thermally conductive surface and cooling said mold at a rate ranging from - l0C.min-1to - 50oC.min-1, preferably - 50C.min-1, from room temperature to - 80°C; and c) placing a mold comprising the aqueous solution of collagen in a constant temperature field at - 80°C.

[0049] In method a), the cryogenic liquid is preferably liquid nitrogen.

[0050] In method b), the thermally conductive surface has preferably a thermal conductivity of at least 20 W.m^.K1. In one embodiment, the thermally conductive surface is a copper surface. Preferably, the thermally conductive surface is cooled below 0°C in order to cool the mold and the solution comprised therein.

[0051] Above ice-templating methods a) and b) involve a directional temperature field, while method c) involves a constant temperature field. The selection of the method of ice- templating will depend on the targeted porosity of the material and the targeted orientation of the pores in the material. One skilled in the art will be able to select a suitable method of ice-templating to obtain the intended pore structure, either among methods a) to c), or among other methods known in the art.

[0052] The ice-templating step is preferably conducted in a mold. Advantageously, the mold has a shape adapted to the intended use of the material obtained by the process of the invention. The mold can be made in one or more materials, which properties, such as thermal conductivity, may influence the orientation, structuration and size of the pores, during the ice-templating step.

[0053] One skilled in the art will be able to select the suitable material(s) for the mold to be used in the process of the invention. Examples of suitable materials include, without being limited to, polystyrene, polytetrafluoroethylene (PTFE), polycarbonate (PC), aluminum, brass, copper, steel.

[0054] One skilled in the art will select the material(s) of the mold depending on the properties expected for the resulting collagen material. Especially, it is possible to provide a collagen material having porous and / or non-porous surfaces. This might be controlled by selecting mold made of materials having various conductivities.

[0055] In some embodiments, the mold may have a shape enabling to form a tubular material. In such case, when the internal and external materials of the mold are both insulating materials, then the ice-templating step leads to pores with a longitudinal distribution, and thereby, the external and luminal surfaces of the tube are non-porous, while the section of the tube is porous. On the contrary, when the internal and external materials of the mold have different conductivity properties, then the ice-templating step leads to pores with a radial distribution. Especially, when the internal material of the mold is an insulating material while the external material of the mold is a conductive material, or alternatively when the internal material of the mold is a conductive material while the external material of the mold is an insulating material, then the ice-templating step leadsto pores with a radial distribution, and thereby the external and luminal surfaces of the tube are porous, while the section of the tube is non-porous.

[0056] Alternatively, the mold may have a shape enabling to form a plane sheet material.Topotactic fibrillogenesis in buffer

[0057] The second step of the process of the invention aims at stabilizing the structure build in the ice-templating step, via collagen fibrillogenesis, using a topotactic process - that is, without changing the material’s texture (i.e. the structure and substructure of the walls and pores).

[0058] The second step of the process of the invention is a step of topotactic fibrillogenesis in buffer.

[0059] In this second step, the ice-templated scaffold of the first step is stabilized via collagen fibrillogenesis induced by contact with a first buffer at a temperature ranging from - 5°C to 0°C. This induction of the fibrillogenesis, also called “pre-fibrillogenesis step”, is maintained for a period of time ranging from 24 hours to 72 hours. Then, the collagen fibrillogenesis is consolidated at room temperature, in presence of a second buffer, until the resulting collagen material presents a denaturation onset temperature of at least 45°C (“consolidation step”), wherein the first and second buffers can be the same or different.

[0060] In one embodiment, the collagen fibrillogenesis is preferably induced by contact with the first buffer at a temperature of - 3 °C. Preferably, the induction of the collagen fibrillogenesis is maintained for 72 hours at - 3°C.

[0061] In one embodiment, the consolidation of the collagen fibrillogenesis by the contact at room temperature with the second buffer is maintained for at least 5 days, preferably at least 8 days, more preferably at least 10 days. The duration of this consolidation step is adapted to reach the targeted degree of fibrillogenesis for the resulting collagen material. The final degree of fibrillogenesis is such that the resulting collagen material presents a denaturation onset temperature of at least 45 °C. In a preferredembodiment, the resulting collagen material presents a denaturation onset temperature of about 50°C.

[0062] “Denaturation onset temperature” refers to the temperature at which a protein or a material exposed to heat begins to denature. Denaturation occurs when the protein or the material loses its normal three-dimensional shape, which happens when some of the hydrogen weak bonds are broken down. The denaturation onset temperature does not depend on the method of determination of the heat denaturation.

[0063] The denaturation onset temperature can be measured by differential scanning calorimetry (DSC). Typically, an aqueous solution or a gel of the protein or material of interest is heated up to 100 °C at a rate ranging from PC.min1to 10oC.min-1. The onset of the endothermic peak of denaturation indicates the limit temperature of operation of the protein.

[0064] In one embodiment, the first buffer, used in the pre-fibrillogenesis step, has an ionic composition which is preferably derived from phosphate buffer saline (PBS), which maintains a pH value above 7, and which osmolarity is above 0.2 Osm / L, preferably above 1.2 Osm / L. In one embodiment, the first buffer, used in the pre-fibrillogenesis step, has an ionic composition which is derived from phosphate buffer saline (PBS), which maintains a pH value above 7, and which osmolarity is above 0.2 Osm / L, preferably above 1.2 Osm / L.

[0065] In one embodiment, the second buffer, used in the consolidation step, has an ionic composition which is preferably derived from PBS buffer, which maintains a pH value above 7, and which osmolarity is above 0.1 Osm / L, preferably above 0.6 Osm / L. In one embodiment, the second buffer, used in the consolidation step, has an ionic composition which is derived from PBS buffer, which maintains a pH value above 7, and which osmolarity is above 0.1 Osm / L, preferably above 0.6 Osm / L.

[0066] In one embodiment, the first buffer is the same as the second buffer. In another embodiment, the first buffer is different from the second buffer.

[0067] By “ionic composition derived from PBS buffer” it is referred to an ionic composition that it substantially similar to the one of a PBS buffer, i.e. that comprises the same ionic species as a PBS buffer, but in different concentrations. A PBS buffer comprises NaCl, KC1, NaiPO4 and KH2PO4. In one embodiment, the composition of reference of a PBS IX buffer is: NaCl 137 mM, KC1 2.68 mM, Na2HPO4 8.07 mM, and NaH2PO41.47 mM.

[0068] In one embodiment, the first buffer comprises: NaCl 1.37 M, KC1 2.68 mM, Na2HPC>4 8.07 mM, and NalHLPCL 1.47 mM, leading to an osmolarity of 2.78 Osm / L.

[0069] In another embodiment, the first buffer comprises: NaCl 1.37 M, KC1 26.8 mM, Na2HPC>4 80.7 mM, and NalHhPC 14.7 mM, leading to an osmolarity of 3.14 Osm / L.

[0070] In one embodiment, the second buffer comprises: NaCl 685 mM, KC1 13.4 mM, Na2HPO440.35 mM, and NaH2PO47.35 mM, leading to an osmolarity of 1.57 Osm / L.

[0071] By slightly modifying the composition of the first and / or second buffers, one can made vary the speed of fibrillogenesis and thereby influence of porosity of the resulting collagen material.

[0072] This second step of topotactic fibrillogenesis in buffer presents the following advantages: preservation of the pore structure obtained in the first step of ice-templating; no need to freeze-dry the resulting material, stability in aqueous medium of the resulting material, provision of mechanical stability to the resulting material.Other optional stepsTopotactic fibrillogenesis by exposure to NH3

[0073] The process according to the invention can comprise, further to the step of topotactic fibrillogenesis in buffer, a step of topotactic fibrillogenesis by exposure to ammonia vapors. Combining the topotactic fibrillogenesis in buffer according to theinvention with topotactic fibrillogenesis by exposure to ammonia vapors can be useful to provide a porous collagen material having surfaces presenting different properties.

[0074] The topotactic fibrillogenesis by exposure to ammonia vapors can be conducted as described in Rieu et al. (ACS Applied Material and Interfaces, 2019, 11, 14672-14683) or Parisi et al. (Biomaterials Sciences, 2022, 10, 6939-6950).

[0075] In one embodiment, the step of topotactic fibrillogenesis by exposure to ammonia vapors comprises a pre-fibrillogenesis step, comprising exposing at least one surface of the ice-templated collagen scaffold to ammonia vapors for a period of time ranging from 24 hours to 72 hours, followed by elimination of ammonia vapors.

[0076] The elimination of ammonia vapor can be conducted by exposing the materials in an atmosphere with a high level of humidity, preferably at 37 °C, and usually for a period of time of about 24 hours.

[0077] Then, the fibrillogenesis induced by exposure to ammonia vapors can be consolidated by the contact of the surface with a “second buffer”, at room temperature, wherein the “second buffer” is as previously defined, i.e. has an ionic composition which is preferably derived from PBS buffer, which maintains a pH value above 7, and which osmolarity is above 0.1 Osm / L, preferably above 0.6 Osm / L. As above for the consolidation step in the topotactic fibrillogenesis in buffer, the duration of the consolidation step in the fibrillogenesis induced by exposure to ammonia vapors is adapted to the degree of fibrillogenesis targeted for the final material.

[0078] The step of topotactic fibrillogenesis by exposure to ammonia vapors can be conducted: i) between the first step of ice-templating and the second step of topotactic fibrillogenesis in buffer; ii) concomitantly with the second step of topotactic fibrillogenesis in buffer; or iii) successively to the second step of topotactic fibrillogenesis in buffer.

[0079] In one embodiment, the surface of the ice-templated collagen scaffold exposed to ammonia vapors is different from the surface of the ice-templated collagen scaffold exposed to buffer.

[0080] In another embodiment, the surface of the ice-templated collagen scaffold exposed to ammonia vapors is the same as the surface of the ice-templated collagen scaffold exposed to buffer. In this case, the two steps of topotactic fibrillogenesis are not conducted concomitantly.

[0081] In a specific embodiment, the topotactic fibrillogenesis by exposure to ammonia vapors is performed concomitantly with the second step of topotactic fibrillogenesis in buffer, wherein one surface of the ice-templated collagen scaffold is exposed to ammonia vapors in the conditions detailed above, while a different surface of the ice-templated collagen scaffold is contacted with the first buffer in the conditions of the pre- fibrillogenesis step of the process of the invention. Then, the consolidation steps of the two types of fibrillogenesis can be conducted simultaneously for both surfaces, in the same conditions, with a common “second buffer”. This embodiment is particularly interesting when the ice-templated collagen scaffold has a shape of tube: the outer and luminal surfaces can undergo independently one of the two topotactic fibrillogenesis (buffer and ammonia vapors) and thereby develop different properties.Formation of a collagen-dense surface

[0082] The process according to the invention can further comprise a step of formation of a collagen-dense surface. Generally, such additional step is performed successively to the second step of fibrillogenesis in buffer. When a fibrillogenesis by exposure to ammonia vapors is also conducted, then the step of formation of a collagen-dense surface is preferably performed successively to the two fibrillogenesis steps.

[0083] This additional step of formation of a collagen-dense surface may be particularly advantageous when the ice-templated collagen scaffold has a shape of tube, enabling to treat differently the outer and luminal surfaces. The topotactic fibrillogenesis in buffer, and optionally further in ammonia vapors, can lead to a surface with open pores, whilethe treatment to form a collagen-dense surface leads to a smoother surface. A tube having a porous external surface and a smooth luminal surface can be useful for example for tissue engineering of arteries or trachea.

[0084] In one embodiment, the step of formation of a collagen-dense surface comprises deposing, at room temperature, an aqueous solution of collagen on at least one surface of the material, to form a collagen-deposited surface; and contacting, at room temperature, the collagen-deposited surface with a “third buffer”, to form a collagen-dense surface; wherein the “third buffer” has an ionic composition which is preferably derived from PBS buffer, which maintains a pH value above 7, and which osmolarity is above 0.1 Osm / L, preferably above 0.6 Osm / L. The “third buffer” can be the same as the first and second buffers defined above, or it can be different.

[0085] In one embodiment, the “third buffer” comprises: NaCl 685 mM, KC1 13.4 mM, Na2HPO440.35 mM, and NaH2PO47.35 mM, leading to an osmolarity of 1573 mOsm / L.

[0086] Preferably the contact of the collagen-deposited surface with the “third buffer”, is maintained for at least 5 days, preferably at least 8 days, more preferably at least 10 days, for example during 14 days.

[0087] In one embodiment, in the process of the invention, the ice-templated scaffold obtained in the first step has the shape of a tube, and the second step is performed on both the luminal and outer surfaces of the tube, leading to a buffer-topotactically-fibrillated tube; and the process of the invention further comprises: a third successive step comprising: deposing, at room temperature, an aqueous solution of collagen on the outer or luminal surface of the buffer-topotactically-fibrillated tube, to form a collagen-deposited surface; and contacting, at room temperature, the collagen-deposited surface with a third buffer, to form a collagen-dense surface; wherein the third buffer has an ionic composition which is preferably derived from PBS buffer, which maintains a pH value above 7, and which osmolarity is above 0.1 Osm / L, preferably above 0.6 Osm / L; andoptionally a fourth successive step of turning inward the outer surface of the tube obtained in the third step, so that to luminal surface of the tube be the former outer surface.

[0088] In one embodiment, the third step is conducted on the outer surface of the tube, forming a collagen-dense outer surface and the fourth step is conducted by turning inward this collagen-dense outer surface so that to luminal surface of the tube be the collagen- dense surface. This is useful for example when the tube is intended to be used for tissue engineering of arteries or trachea. For this use, the third step can alternatively be conducted directly on the luminal surface and in such case, the fourth step is not necessary.

[0089] When the third step is performed on the luminal surface of the tube, the aqueous solution of collagen is preferably introduced between the luminal surface of the tube and a cylindrical mold of reduced diameter placed in the luminal area.

[0090] In one embodiment, the process of the invention does not comprise a step of cross-linking the collagen fibers.Porous collagen material

[0091] A second aspect of the invention is a porous collagen material obtained by the process according to the invention.

[0092] In one embodiment, the porous collagen material obtained by the process of the invention is stored in aqueous medium until use. Indeed, it is not necessary to dry the material at the end of the process of the invention. Even stored in aqueous medium, the properties of the material of the invention are maintained. This is advantageous compared to the collagen materials known in the art that are stored under freeze-dried form, and for which the rehydration step is usually a delicate step that may lead to the loss of material, a degradation of the structural and / or mechanical properties.

[0093] In one embodiment, the shape of the porous collagen material obtained by the process of the invention is a planar shape or a three-dimensional shape. For example, theshape of the material is a tube, a cylinder, or a sheet. The shape of the material can be obtained by using a suitable mold, especially during the first step of ice-templating.

[0094] In one embodiment, the porous collagen material of the invention in hydrated conditions has pores having a size ranging from 5 pm to 100 pm, preferably from 15 pm to 30 pm.

[0095] Advantageously, the porous collagen material of the invention is sufficiently porous, with large enough pores, to allow cells to infiltrate the material and grow in 3D culture. Nevertheless, the pore size should not be too large as cells will otherwise fall through the pores and the material will not be an effective scaffold.

[0096] The porous collagen material of the invention presents properties similar to native fibrillar collagen tissues, being thereby useful as biomaterials or as three-dimensional scaffold for in vitro cell culture. It may thus be useful to seed the material with living cells before such uses. Therefore, the invention also relates to the porous collagen material of the invention seeded with living cells.

[0097] A wide variety of cell types can be used with the material of the invention, including cell lines, stem cells and primary cells. Typically, the cells are mammalian cells.

[0098] Examples of cells that can be used with the material of the invention include, without being limited to, endothelial cells, mesenchymatous stem cells, muscular cells, epithelial cells, and fibroblasts.

[0099] The invention further relates to a multi- well assay plate comprising plurality of sample wells, and a material according to the invention in at least one of the sample wells. Preferably, the material of the invention is present in at least 50% of the sample wells, for instance in at least 90% of the sample wells, if not in all the sample wells.

[0100] The multi- well assay plate may for instance be a 96-wells plate, a 96-wells plate, a 384-wells plate, a 1536-wells plate, or a 3456-wells plate.

[0101] In one embodiment, the multi- well assay plate comprising the material of the invention further comprises a cell culture medium.Uses

[0102] The porous material of the invention can be used as scaffold for in vitro cell culture, advantageously as 3D scaffold for in vitro cell culture.

[0103] One further object of the invention is thus the use of a porous collagen material according to the invention, as scaffold for in vitro cell culture, preferably as 3D scaffold for in vitro cell culture.

[0104] The type of cells that can be cultured on the porous collagen material of the invention will be apparent to one skilled in the art and include, for example, endothelial cells, mesenchymatous stem cells, muscular cells, epithelial cells, and fibroblasts.

[0105] The porous material of the invention can also be used as scaffold for tissue engineering.

[0106] By “tissue engineering” it is herein referred to biomedical engineering that aims at restoring, maintaining, improving, or replacing different types of biological tissues. It involves the combined use of materials, cells, and suitable biochemical and physicochemical factors. Tissue engineering involves the use of tissue scaffolds in the formation of new viable tissue for a medical purpose, especially to repair or replace portions of or whole tissues.

[0107] By “scaffold for tissue engineering”, it is referred to a material that provides a structural support for cell attachment and subsequent tissue development.

[0108] One further object of the invention is thus the use of a porous collagen material according to the invention, as scaffold for tissue engineering. In one embodiment, the invention relates to the use of a porous collagen material according to the invention, as scaffold for in vitro tissue engineering or for ex vivo tissue engineering. In one embodiment, the invention is not directed to the in vivo use of the porous collagen material according to the invention.

[0109] In one embodiment, the invention provides a method for the in vitro production of a biological tissue comprising the use of a porous collagen material according to the invention as scaffold.

[0110] When the material of the invention is used as scaffold for tissue engineering, it can be seeded with various types of living cells, such as for example endothelial cells, mesenchymatous stem cells, muscular cells, epithelial cells, and fibroblasts.

[0111] The invention is also directed to regenerative medicine using the porous material according to the invention. Especially, the invention provides a method for growing a tissue in a patient, comprising the step of implanting a porous material according to the invention in the patient. In one embodiment, the implanted porous material can be seeded with living cells, preferably with living cells of the patient to whom the material will be implanted.

[0112] Examples of tissues that can be engineered from the porous material of the invention include, without being limited to, trachea and arteries.BRIEF DESCRIPTION OF THE DRAWINGS

[0113] Figure 1 is a scheme of a home-made set-up used for unidirectional controlled ice-templating that allows a continuous and regulated dipping of the sample in liquid nitrogen, (a) DC motor (12V), (b) shaft coupler, (c) lead crew, (d) 2 optical axes, (e) 4 linear rod rail support guides, (f) ABS 3D-printed part linking (c) and (e), (g) 4-tubular molds carrier, (h) set-up made of aluminum rods (h = 60 cm, w = 30 cm).

[0114] Figure 2 is a group of images obtained by confocal microscopy of: the luminal surface (Figure 2A) and the external surface (Figure 2B) of a tubular collagen material obtained by fibrillogenesis by exposure to ammonia vapors after ice-templating completed using internal and external molds being both made of ABS.

[0115] Figure 3 is a group of images obtained by confocal microscopy of the luminal surface (Figure 3A) and the external surface (Figure 3B) of a tubular collagen materialobtained by fibrillogenesis in buffer after ice-templating completed using an internal mold made of polytetrafluoroethylene and an external mold made of expanded polytetrafluoroethylene.

[0116] Figure 4 is a group of images of a PeP-buffer material showing: the wall thickness measurement with RidgeDetection (Figure 4A), the pore width measurement with RidgeDetection (Figure 4B), the surfacic porous fraction measurement (Figure 4C).

[0117] Figure 5 is a graph representing the Youngs’ moduli of AU-fNH3, VV-fNH3 and PeP-buffer materials, in the longitudinal direction (“long”) or in the circumferential direction (“circ”).

[0118] Figures 6A and 6B are DSC graphs of (A) UU-buffer and (B) UU-fNH3 materials, measured at “day 0”, i.e. just after the end of the pre-fibrillogenesis step, at the beginning of the consolidations step, and at “day 5”, i.e. during the course of the consolidation step.

[0119] Figure 7 is an image obtained by polarized light optical microscopy of the transversal view of a self-standing tubular collagen material obtained by a fibrillogenesis process combining a fibrillogenesis in buffer on the luminal side concomitant with a fibrillogenesis by exposure to ammonia vapors on the outer surface.

[0120] Figure 8 is a group of images obtained by polarized light optical microscopy (PLOM) (Figure 8A) and confocal microscopy (Figures 8B and 8C) of a self-standing tubular material obtained by a hybrid fibrillogenesis process. Figure 8A: PLOM - transversal sections at 0° analyzer / polarizer angle; Figures 8B and 8C: Confocal microscopy- transversal section of the entire thickness of the material (B) and a focus on the porous outer layer (C).EXAMPLES

[0121] The present invention is further illustrated by the following examples.Example 1: Manufacturing of porous collagen materials

[0122] This example aims at manufacturing porous collagen materials by the process of the invention. For comparison purposes, porous collagen materials were also manufactured by fibrillogenesis by exposure to ammonia vapors. The materials were manufactured in the form of tubes.Materials and Methods

[0123] Collagen extraction and concentration. Collagen I was extracted from young rat tails’ tendons. After thorough cleaning of tendons with phosphate buffered saline (PBS) IX and 4 M NaCl, tendons were dissolved in 3mM HC1. Differential precipitation with 300 mM NaCl and 600 mM NaCl, followed by redissolution and dialysis in 3 mM HC1, provided collagen of high purity. The final collagen concentration was determined using hydroxyproline titration with a spectrophotometer.

[0124] To attain a high fibrillar collagen density in the model, the remaining collagen solutions were concentrated at 40 mg.mL-1. The solutions were then transferred into Vivaspin tubes with 300 kDa filter and centrifugated at 3000g at 10°C, to reach this final concentration.

[0125] Ice-templating. Concentrated collagen is introduced in between two cylindrical molds, which ends are hermetically closed by caps. Using this type of molds enables to obtain a shape of tube. For removal of air bubbles, sample is centrifuged at 1 rpm, 10°C, for 10 minutes. Sample is placed onto a home-made set-up (Figure 1), which allows a continuous and regulated dipping of the sample in liquid nitrogen. It is composed of: 2 optical axis, 4 linear rod rail support guides, a lead screw, a DC motor (12V), a speed controller, a transformer and an ABS (Acrylonitrile Butadiene Styrene) 3D-printed part linking the support guide and the lead screw. Sample is ice-templated at a dipping speed of 10 mm.min1.

[0126] The two cylindrical molds can be made of the same material or of two different materials. In the present example, in conditions (i), the internal and external molds were both made of ABS. In conditions (ii), the internal mold was made of polytetrafluoroethylene, and the external mold was made of expanded polytetrafluoroethylene.

[0127] Topotactic fibrillogenesis. Once frozen, the outer mold is quickly removed and a topotactic fibrillogenesis condition is applied. At low temperature, two conditions are compared:(a) exposure to ammonia vapors (comparative conditions), and(b) buffer (according to the process of the invention).

[0128] In the present example, the fibrillogenesis conditions (a) were applied to a sample ice-templated in conditions (i), and the fibrillogenesis conditions (b) were applied to a sample ice-templated in conditions (ii).

[0129] A pre-fibrillogenesis step is first conducted:(a) exposing the sample to ammonia vapors at 0°C in an ice-water bath for 48h, followed by a removal of ammonia using distilled water vapors for 24h at 37 °C in a heat room, or(b) the sample is maintained in a “first buffer” at -3°C in a thermostatic bath for 72h, wherein the “first buffer” consist of NaCl 1.37 M, KC1 2.68 mM, NazHPO-t 8.07 mM, and NatCPO-i 1.47 mM, leading to an osmolarity of 2780 mOsm / L.

[0130] For both, the fibrillogenesis is then completed in a consolidation step, by keeping the sample in a “second buffer” for 14 days at room temperature, wherein the “second buffer” consist of NaCl 685 mM, KC1 13.4 mM, NaiHPO-t 40.35 mM, and NaH2PO47.35 mM, leading to an osmolarity of 1573 mOsm / L.

[0131] Images of both materials are then acquired by confocal microscopy.Results

[0132] Both pre-fibrillogenesis conditions (a) and (b) lead after consolidation to the formation of porous collagen materials under the shape of tubes. Images of the luminal and external surfaces of both materials are reported in Figures 2A and 2B and in Figures 3 A and 3B.

[0133] The external surfaces of both materials are porous. The luminal surface of the material obtained using the fibrillogenesis in buffer according to the invention is non- porous while the luminal surface of the comparative material exposed to ammonia vapor exhibits pores. Therefore, the material obtained by the method according to the invention might be particularly interesting for mimicking arteries, by promoting endothelialization on its smooth luminal surface and smooth muscle cells (SMCs) colonization on its porous external surface.Example 2: Characterization of the porous collagen materials

[0134] This example aims at providing physical and structural characteristics of the collagen materials obtained as described in example 1: porosity assessment, Young’s modulus measurement, and assessment of the efficacy of the consolidation step.Materials and Methods

[0135] Four types of materials were obtained as described in example 1: two samples were obtained using conditions (b), i.e. by pre-fibrillogenesis in buffer according to the invention, one from a sample ice-templated in conditions (i) (internal and external molds both made of ABS), referred to as UU -buffer, and the other from a sample ice-templated in conditions (ii) (internal mold made of polytetrafluoroethylene, and external mold made of expanded polytetrafluoroethylene), referred to as PeP-buffer, two samples were obtained using conditions (a), i.e. by fibrillogenesis by exposure to ammonia vapors, one from a sample ice-templated in conditions (i) (internal and external conductive molds both made of ABS), referred to as VV-fNH3, andthe other from a sample ice-templated in conditions (iii) (external conductive mold (aluminum) and internal conductive mold (ABS)), referred to as AV-fNH3.

[0136] Porosity assessment. Porosity was quantified using the ImageJ plugin RidgeDetection. This latter detects ridges or linear structures in images by analyzing intensity variations and identifying pixels with high gradient values along a specific direction. In the end, the mean width of the pores for each image can be retrieved. Pores orientation was quantified using ImageJ plugin OrientationJ. Pore surfacic porous fraction was measured by dividing the area of pores by the total area.

[0137] Axial and circumferential Young’s modulus measurement. The study focused on the characterization of the mechanical properties of porous collagen tubes obtained as detailed above. To do this, the tubes were first cut open and unfolded toward a flat geometry. Rectangular strips measuring 2 cm x 1 cm were then cut from the samples in both circumferential and longitudinal directions. To measure the thickness of the samples, a compression rheometer (MCR 302, Anton Paar) was used. The base of a cylindrical probe was approached at a constant speed toward the sample until a force was detected.

[0138] For the uniaxial tests, a homemade stage with a 10 N load cell (ME-MeBsysteme GmbH, Germany) was used to hold each strip in place with serrated jaws blocking the ends. The initial distance between the jaws was adjusted until the strip was flattened, and this distance was recorded as the unloaded sample length. Each strip was then subjected to cyclic testing, and data from the third cycle's loading phase were used for analysis. To ensure consistency and accuracy of results, three pre-conditioning cycles were performed to produce a consistent force-elongation curve for each strip. This protocol was followed for all tests. The resulting stress / strain curves were used to calculate the Young's modulus, and only the linear portion of these curves was utilized.

[0139] Differential scanning calorimetry (DSC). DSC was performed on materials UU-fNH3 and UU-buffer at “day 0”, i.e. just after the end of the pre-fibrillogenesis step, at the beginning of the consolidations step, and at “day 5”, i.e. during the course of the consolidation step.Results

[0140] Porosity assessment. The characteristics of porosity of the luminal and external surfaces of the materials PeP-buffer and VV-fNH3 are summarized in the recapitulative table below:

[0141] In the PeP-buffer tube, the luminal surface is non-porous, while the luminal surface of the VV-fNH3 tube comprises pores. For both samples, the size of the pores of the external surface is similar, and adapted to be colonized by cells. Nevertheless, the wall thickness is more important for the sample PeP-buffer than for VV-fNH3. This difference of wall thickness is linked to the use of the buffer during fibrillogenesis, instead of ammonia. Without willing to be linked by a theory, it appears that in presence of buffer, the ice crystals melt slower than in presence of ammonia, leading to a more substantial division between pores. In both materials, there is substantially the same quantity of collagen, but when fibrillated in presence of ammonia, the walls inside the obtained material are denser than when fibrillated in presence of buffer. The PeP-buffer material is thus more favorable to cell penetration.

[0142] Figures 4A-4C show examples of measurements of wall thickness, pore width and surfacic porous fraction for PeP-buffer material.

[0143] Young’s modulus measurement. Traction Young’s moduli were measured on AU-fNH3, UU-fNH3 and PeP-buffer samples. Results for longitudinal (“long”) and circumferential (“circ”) directions are provided in Figure 5. Maximal stiffness was observed in the longitudinal direction of VV-fNH3 and PeP-buffer samples, with a Youngs’ modulus (YM) of approximately 50-60 kPa. In the circumferential direction of the VV-fNH3 samples and for both directions of the AV-fNH3 samples, stiffness wasmuch lower, with a YM between 6 and 10 kPa. In the circumferential direction of the PeP-buffer samples, stiffness was intermediate, with a YM of approximately 30 kPa.

[0144] Differential scanning calorimetry (DSC). DSC analysis was performed to assess the efficacy of the consolidation step, after the pre-fibrillogenesis step, for the UU- buffer and VV-fNH3 samples. Results are provided in Figures 6A and 6B, which show, for both materials UU-buffer and UU-fNH3, the appearance of peaks characteristic of collagen ordered fibrils after 5 days of fibrillogenesis consolidation in the “second buffer”.Example 3: Preparation of a tubular collagen material by simultaneous different fibrillogenesis processes on each side of the materialMaterials and methods

[0145] A concentrated collagen solution (40 mg / mL) is introduced in between two cylindrical molds, the external one being made of expanded polytetrafluoroethylene (ePTFE) and the internal one of Acrylonitrile Butadiene Styrene (ABS). After ice templating in liquid nitrogen, the internal mold is retrieved and a first buffer (NaCl 1.37 M, KC1 26.8 mM, NazHPO-t 80.7 mM, and NalTPO-i 14.7 mM) is inserted inside the lumen space to perform the topotactic pre-fibrillogenesis step in buffer in the luminal surface. Meanwhile, the material is placed in a chamber at -3°C under ammonia vapors for 48h to perform concomitantly a topotactic pre-fibrillogenesis step on the outer surface of the tube. Ammonia is further removed using distilled water vapors for 24h at 37 °C in a heat chamber. The fibrillogenesis is then completed in a consolidation step, by keeping the sample in a second buffer (NaCl 685 mM, KC1 13.4 mM, NaiHPO-t 40.35 mM, and NaHiPO-t 7.35 mM) for 14 days at room temperature.Results

[0146] The self-standing material obtained by this concomitant fibrillogenesis process is observed by polarized light optical microscopy (PLOM), as shown in Figure 7.Example 4: Hybrid fibrillogenesis with exposure to ammonia vapor between the first step of ice-templating and the second step of topotactic fibrillogenesis in bufferMaterials and methods

[0147] A concentrated collagen solution (40 mg / mL) is introduced in between two cylindrical molds, the external one being made of brass and the internal one ofAcrylonitrile Butadiene Styrene (ABS). After ice templating in liquid nitrogen, the external mold is retrieved and the material is exposed to ammonia vapor at 0°C for 24h. The material is then dipped into a fibrillogenesis buffer for 48h (NaCl 1.37 M, KC1 26.8 mM, NaiHPO-t 80.7 mM, and NaH2PO4 14.7 mM). The fibrillogenesis is then completed in a consolidation step, by keeping the sample in a second buffer (NaCl 685 mM, KC113.4 mM, Na2HPC>440.35 mM, and NaH2PC>47.35 mM) for 14 days at room temperature.Results

[0148] The obtained self-standing material following this hybrid fibrillogenesis process is observed by polarized light optical microscopy (PLOM) (Figure 8A) and confocal microscopy (Figures 8B and 8C).

Claims

CLAIMS1. A process of preparing a porous collagen material, comprising: a first step of ice-templating an aqueous solution of collagen to form an ice-templated scaffold; and a second successive step of topotactic fibrillogenesis in buffer, comprising: a pre-fibrillogenesis step by contacting at least one surface of the ice- templated scaffold with a first buffer, at a temperature ranging from - 5 °C to 0°C, preferably - 3°C, for a period of time ranging from 24 hours to 72 hours; wherein the first buffer has an ionic composition which is preferably derived from phosphate buffer saline (PBS), which maintains a pH value above 7, and which osmolarity is above 0.2 Osm / L, preferably above 1.2 Osm / L; and then a consolidation step by maintaining the contact between the at least one surface of the ice-templated scaffold with a second buffer, at room temperature until the resulting collagen material presents a denaturation onset temperature of at least 45°C; wherein the second buffer has an ionic composition which is preferably derived from PBS buffer, which maintains a pH value above 7, and which osmolarity is above 0.1 Osm / L, preferably above 0.6 Osm / L; wherein the first buffer and the second buffer are the same or different.

2. The process according to claim 1, wherein the collagen is type I collagen.

3. The process according to claim 1 or claim 2, wherein the aqueous solution of collagen has a concentration ranging from 1 mg.mL1to 120 mg.mL1, preferably from 10 mg.mL1to 60 mg.mL1, more preferably 40 mg.mL1.

4. The process according to any one of claims 1 to 3, wherein the aqueous solution of collagen further comprises excipients, additives, active substances such as growth factors or interleukins, or mixtures thereof.

5. The process according to any one of claims 1 to 4, wherein in the first step, the ice-templating of the aqueous solution of collagen is performed by a method selected from: a) dipping a mold comprising the aqueous solution of collagen in a cryogenic liquid, at a dipping-speed ranging from 1 mm.min1to 100 mm.min1, preferably 10 mm.min1; b) placing a mold comprising the aqueous solution of collagen on a thermally conductive surface and cooling said mold at a rate ranging from - l0C.min-1to - 50oC.min-1, preferably - 50C.min-1, from room temperature to - 80°C; and c) placing a mold comprising the aqueous solution of collagen in a constant temperature field at - 80°C.

6. The process according to any one of claims 1 to 5, wherein in the consolidation step, the contact at room temperature between the at least one surface of the ice-templated scaffold and the second buffer is maintained for at least 5 days, preferably at least 8 days, more preferably at least 10 days.

7. The process according to any one of claims 1 to 6, wherein the obtained porous collagen material is stored in aqueous medium until use.

8. The process according to any one of claims 1 to 7, further comprising a step of topotactic fibrillogenesis by exposure to ammonia vapors, comprising exposing at least one surface of the ice-templated collagen scaffold to ammonia vapors for a period of time ranging from 24 hours to 72 hours, followed by elimination of ammonia vapors; said further step of topotactic fibrillogenesis by exposure to ammonia vapors being conducted: i) between the first step of ice-templating and the second step of topotactic fibrillogenesis in buffer; ii) concomitantly with the second step of topotactic fibrillogenesis in buffer; or iii) successively to the second step of topotactic fibrillogenesis in buffer.

9. The process according to claim 8, wherein the topotactic fibrillogenesis by exposure to ammonia vapors is performed concomitantly with the second step of topotactic fibrillogenesis in buffer, wherein one surface of the ice-templated collagen scaffold is exposed to ammonia vapors in the conditions of claim 8, while a different surface of the ice-templated collagen scaffold is contacted with the first buffer in the conditions of the pre-fibrillogenesis step.

10. The process according to any one of claims 1 to 9, wherein the ice-templated scaffold obtained in the first step has the shape of a tube, and the second step is performed on both the luminal and outer surfaces of the tube, leading to a buffer-topotactically-fibrillated tube; and further comprising: a third successive step comprising: deposing, at room temperature, an aqueous solution of collagen on one of the outer or luminal surfaces of the buffer-topotactically-fibrillated tube, to form a collagen-deposited surface; and contacting, at room temperature, the collagen-deposited surface with a third buffer, to form a collagen-dense surface; wherein the third buffer has an ionic composition which is preferably derived from PBS buffer, which maintains a pH value above 7, and which osmolarity is above 0.1 Osm / L, preferably above 0.6 Osm / L; and optionally a fourth successive step of turning inward the outer surface of the tube obtained in the third step, so that the luminal surface of the tube be the former outer surface.

11. A porous collagen material obtained by the process according to any one of claims 1 to 10.

12. The porous collagen material according to claim 11, whose shape is a planar shape or a three-dimensional shape; preferably whose shape is a tube, a cylinder, or a sheet.

13. The porous collagen material according to claim 11 or claim 12, seeded with living cells.

14. Use of a porous collagen material according to any one of claims 11 to 13, as scaffold for in vitro cell culture.

15. Use of a porous collagen material according to any one of claims 11 to 13, as scaffold for tissue engineering.

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