WO2021099581A1

WO2021099581A1 - Process for the preparation of fibrin-based materials and fibrin-based materials obtained by said process

Info

Publication number: WO2021099581A1
Application number: PCT/EP2020/082911
Authority: WO
Inventors: Léa TRICHET; Clément RIEU; Thibaud CORADIN
Original assignee: Sorbonne Universite; Centre National De La Recherche Scientifique
Priority date: 2019-11-22
Filing date: 2020-11-20
Publication date: 2021-05-27

Abstract

The present invention relates to a method for the preparation of fibrin-based biomaterials, to the fibrin-based biomaterials obtained by such a method, and to said fibrin-based biomaterials for their medical use such as for their application and/or their implantation in damaged soft tissue and/or organ in need thereof.

Description

PROCESS FOR THE PREPARATION OF FIBRIN-BASED MATERIALS AND

FIBRIN-BASED MATERIALS OBTAINED BY SAID PROCESS

The present invention relates to the field of biocompatible materials, in particular to biomaterials that can be used in vertebrates for repairing damaged soft tissues (e.g. skin, muscles, nerves, blood vessels, ligaments, etc....) and/or organs (e.g. heart, liver, urinary bladder, etc....).

More particularly, the present invention relates to a method for the preparation of fibrin-based biomaterials, to the fibrin-based biomaterials obtained by such a method, and to said fibrin-based biomaterials for their medical use such as for their application and/or their implantation in damaged soft tissue and/or organ in need thereof.

A biomaterial is any substance that has been engineered to interact with biological systems for a medical purpose - either a therapeutic (treat, augment, repair or replace a tissue function of the body) or a diagnostic one. Biomaterials can be used as extracellular matrices ("scaffolds") for tissue growth and may include various therapeutic agents such as cells, growth and/or differentiation factors, active molecules such as hormones, antibiotics, chemotherapeutics, etc....

To achieve this purpose, biomaterials must meet some specific requirements. They are generally in the form of polymeric matrices or biopolymers, having high porosity and an adequate pore size to facilitate cell seeding and diffusion throughout the whole structure of both cells and nutrients. Biodegradability is also often an essential factor since scaffolds should preferably be absorbed by the surrounding tissues without the necessity of a surgical removal.

Among biopolymers, fibrin is a blood-borne protein responsible for hemostasis, which forms a clot together with platelets upon injury. During tissue and vascular injury, fibrinogen is converted enzymatically by thrombin to fibrin and subsequently to a fibrin-based blood clot (also called thrombus) which is the final product of the blood coagulation step in hemostasis. Fibrinogen is a 340 kDa protein made of three pairs of chains, called a, b and g that assemble in a specific symmetric shape, forming three aligned globules linked together by coiled coil connectors constituted by the three different chains. The central domain, called domain E, gathers the amino-terminal parts of the 6 chains, linked together by di-sulfide bonds. The distal domains, called domain D, are made of the carboxy-terminal ends of the b and g chains. The carboxy-terminal ends of a chains, called aCs, further extend in a random- coiled manner, with globular domains at their ends. Those globular domains interact together as well as with the E domain, and hence do not float freely. During clotting, thrombin cleaves two pairs of fibrinopeptides, FpA and FpB, from the central E domain, revealing two sites A and B which bind to sites a and b located on the g and b chains respectively in the D domain of an adjacent fibrin. Such interactions promote the polymerization of fibrin monomers in a half staggered manner, which results in a highly fibrillar network. A certain delay, called clotting time, exists before the formation of a macroscopic clot. Lateral assembly of protofibrils only occurs at the end of the clotting time, which can vary from seconds to hours depending on pH and ionic strength.

Fibrin has been used extensively as a biopolymer scaffold in tissue engineering. Fibrin is advantageous opposed to synthetic polymers, in particular when cost, inflammation, immune response, toxicity and cell adhesion are concerned and also to collagen gels, in particular when immune response is concerned. Fibrin satisfies many requirements of scaffold functions. Biomaterials made up of fibrin can attach many biological surfaces with high adhesion. Fibrin alone or in combination with other materials has been used as a biological scaffold for stem or primary cells to regenerate adipose tissue, bone, cardiac tissue, cartilage, liver, nervous tissue, ocular tissue, skin, tendons, and ligaments, and shows high cell adhesion and proliferation. Fibrin glue is also commercialized as a biological glue. Thus, fibrin is a versatile biopolymer, which shows a great potential in tissue regeneration and wound healing. An additional and important advantage is that fibrin can be extracted from patient own's blood and thus makes an autologous material. In contrast to other biomaterials such as type I collagen, fibrin shaping has so far faced major limitations related to the required addition of thrombin to fibrinogen to trigger fibrillogenesis. Indeed, its enzyme-induced self-assembly mechanism makes it difficult to shape, which limits the possibility to recreate 3D structures mimicking targeted tissues and organs.

Different methods have already been proposed to prepare fibrin biomaterials. More particularly, the international patent application WO 2007/109137 discloses a process for the preparation of fibrin microthreads comprising mixing a solution of fibrinogen with a solution of one or more molecules capable of forming fibrin, such as thrombin, co-extruding the mixture through an orifice into an aqueous buffered medium, incubating the extruded solution until filament formation is observed, and drying the resulting filaments. However, this process is limited to the production of microthreads, and does not lead to a 3D structure, and the mechanical properties of such filaments are not necessarily optimized to be used in various clinical applications such as tissue reconstruction. In addition, in said process, the use of two separate solutions, respectively of fibrinogen and of thrombin, is required to avoid premature spontaneous fibrillation of fibrin before the extrusion step and delay of clotting upon mixing fibrinogen and thrombin may be observed.

The aim of the present invention is to overcome the drawbacks of the techniques of the prior art by proposing a method that would make possible to easily prepare fibrin-based biomaterials; to control and ensure immediate clotting, to create 3D structures mimicking targeted tissues and organs; to widen the possibilities of fibrin-based biomaterials shaping; and/or to lead to biomaterials having appropriate mechanical and bioactivity properties so that they can be used in various clinical applications such as tissue reconstruction.

A first object of the present invention is a method for the preparation of a fibrin-based biomaterial in the form of a gel, wherein said method comprises at least the following steps: i) preparing an aqueous acidic solution comprising fibrinogen, said aqueous acidic solution having a pH value inferior to the isoelectric point of fibrinogen, ii) adding thrombin to the aqueous acidic solution comprising fibrinogen of step i), so as to form an aqueous acidic solution comprising fibrin, iii) incubating said aqueous acidic solution comprising fibrin at a temperature ranging from about 30 to about 70°C, and wherein: said step iii) is a step iii-1) performed during a time sufficient to form a viscous solution, and said method additionally comprises a step iv-1) of contacting said viscous solution with a buffer solution comprising at least one buffer having a pH value superior to the isoelectric point of fibrinogen, so as to form a gel; or said step iii) is a step iii-2) performed during a time sufficient to directly form a gel.

Surprisingly, the inventors have found that the implementation of an incubating step iii-1) of an acidic aqueous solution of fibrin at a temperature as defined above and a neutralization step iv-1), or of an incubating step iii-2) of an acidic aqueous solution of fibrin at a temperature as defined above lead to a fibrin-based gel which can be easily shaped. More particularly, this method is versatile and makes it possible to easily prepare fibrin-based biomaterials in the form of gels; to create 3D structures mimicking targeted tissues and organs; to widen the possibilities of fibrin-based biomaterials shaping; and/or to lead to biomaterials having appropriate mechanical and bioactivity properties so that they can be used in various clinical applications such as tissue reconstruction.

Step i)

In the present invention, the term "gel" means a visco-elastic solid structure composed of a three-dimensional ordered network swelled with a solvent such as water. In one preferred embodiment, the fibrinogen-based biomaterial of the invention is in the form of a hydrogel.

Fibrinogen used in step i) may be from any mammal source. As examples, fibrinogen may be obtained from any of a wide range of species, in particular from humans, non-human primates (e.g. monkeys), horses, cattle, pigs, sheep, rabbits, guinea pigs, hamsters, rats, mice, etc... Fibrinogen can also be obtained from the host's own blood or from an allogenic donor.

The concentration of fibrinogen in the aqueous acidic solution preferably ranges from about 10 to 100 mg/ml_, more preferably from about 15 to 70 mg/ml_, and even more preferably from about 20 to 60 mg/ml_. According to a particularly preferred embodiment of the present invention, the concentration of fibrinogen in the aqueous acidic solution is about 40 mg/ml_.

The isoelectric point of fibrinogen is about 5.5. Therefore, within the meaning of the present invention a pH value inferior to the isoelectric point of fibrin means a pH value inferior to 5.5, preferably a pH value superior to 2, more preferably a pH value ranging from about 2.5 to 4.0, and even more preferably of about 3.4 to 3.8. Thanks to these pH ranges, the fibrinogen in the aqueous acidic solution remains stable [e.g. does not form a gel during step i)] and/or does not precipitate, at room temperature (i.e. at 18-25°C) or in cold conditions (-80°C to 18°C).

The pH of the aqueous acidic solution of fibrinogen can be adjusted to the desired value with any organic or mineral acid. According to the invention, hydrochloride acid and acetic acid are preferred. Hydrochloride acid (HCI) is particularly preferred.

The concentration of organic or mineral acid will depend on the pH of the initial solution, the type of acid and/or other components such as buffer used to prepare the aqueous acidic solution of fibrinogen.

According to an embodiment of the present invention, the aqueous acidic solution of fibrinogen comprises HCI at a concentration ranging from about 10 to 160 mmol/L, preferably at a concentration ranging from about 30 to 90 mmol/L, and even more preferably around 60 mmol/L, so as to obtain a pH around 3.6.

To adjust the physical properties of the viscous solution or the gel formed during step iii), the aqueous acidic solution can comprise at least one monovalent or divalent metal cation selected from the group consisting of Na⁺, Ca²⁺, Mg²⁺ and Zn²⁺.

Among monovalent or divalent metal cations, Ca²⁺ is particularly preferred.

Said monovalent or divalent metal cation may be used in the aqueous acidic solution in the form of a metal salt selected from the group consisting of metal halides (e.g. metal chlorides).

Among metal salts, metal chlorides are particularly preferred such as

CaCI₂.

The concentration of the monovalent or divalent metal cation in the aqueous acidic solution may range from about 0.001 to 200 mmol/L, and preferably from about 0.1 to 100 mmol/L.

In a preferred embodiment, the aqueous acidic solution does not comprise other glycoprotein(s) than fibrinogen such as fibronectin.

Step i) is preferably performed at room temperature, for example at a temperature ranging from about 18 to 25°C.

The aqueous acidic solution obtained in step i) has preferably an ionic strength ranging from about 10 mM to about 200 mM, more preferably from about 50 mM to about 100 mM, and even more preferably from about 65 mM to about 85 mM.

Step iij

Thrombin is added to the aqueous acidic solution of fibrinogen during step ii).

Addition of thrombin to fibrinogen in acidic conditions effectively cleaves the Fps, FpA and FpB, transforming fibrinogen into fibrin. Thrombin is added to the aqueous acidic solution of fibrinogen such that the concentration of thrombin in the aqueous acidic solution of fibrinogen preferably ranges from about 0.01 U/mL to 200 U/mL, and even more preferably from about 5 to 40 U/mL. According to a particularly preferred embodiment of the present invention, the concentration of thrombin in the aqueous acidic solution of fibrinogen is about 20 U/mL.

The addition of thrombin may be performed directly, i.e. by adding neat thrombin to the aqueous acidic solution of fibrinogen; or indirectly, i.e. by preparing a solution comprising thrombin and adding said solution to the aqueous acidic solution of fibrinogen. Preparing a solution comprising thrombin and adding said solution to the aqueous acidic solution of fibrinogen is preferred.

When step ii) is performed by adding a solution comprising thrombin, the solution is preferably an aqueous neutral solution of thrombin, for example having a pH ranging from about 7 to 8.

Said solution of thrombin may comprise from about 1 to 1000 U/mL, and preferably from about 100 to 250 U/mL of thrombin, with respect to the total volume of said solution comprising thrombin.

After step ii), a fibrin-based solution is obtained. In other words, the addition of thrombin in the aqueous acidic solution of fibrinogen enables the formation of fibrin, preferably in such a way that the whole fibrinogen has been converted into fibrin.

Step ii) is preferably performed at room temperature, for example at a temperature ranging from about 18 to 25°C. Step iii)

As previously stated, the incubating step iii) is applied to the aqueous acidic solution comprising fibrin.

According to a preferred embodiment of the present invention, the incubating step iii) is carried out at a temperature ranging from about 35°C to 39°C, more preferably at a temperature ranging from about 33°C to 40°C, and even more preferably at a temperature of about 37°C.

The incubating step iii) may be performed for example by incubating the aqueous acidic solution comprising fibrin into a regulated water bath ("bain-marie") or into an oven.

During the incubation step iii), at least partial denaturation occurs and fibrin proteins gather to form aggregates.

In a preferred embodiment, the method does not comprise a pre incubating step at a neutral or basic pH.

The time during which incubating step iii) [either step iii-1) or step iii- 2)] is performed, generally depends on the pH and/or the ionic strength of the acidic medium, and/or the concentration of fibrin in the acidic medium.

The person skilled in the art knows how to adapt the time of step iii-1) or iii-2) to either form a viscous solution and/or a gel by at least following visually the appearance of the acidic medium.

Step iii-1) for obtaining material D

When step iii) is performed during a short time [also called step iii-1)], step iii-1) is preferably performed during a time of at most 25 min.

In this embodiment, a pH of at most 4.0, and preferably of at most 3.8, for the aqueous acidic solution of fibrinogen is particularly preferred. A pH of at least 2.0, and preferably of at least 2.5 can be used during step iii-1).

After step iii-1), a fibrin-based viscous solution (or visco-elastic solution) is obtained.

In a preferred embodiment, step iii-1) is performed during a time ranging from 1 min to 25min, advantageously from 5 min to 20 min, and more advantageously from 5 min to 15 min.

Said times for step iii-1) are generally applicable when the pH is about 3.6 and/or the ionic strength is about 75 mM and/or the concentration of fibrin is of 40 mg/mL, and may be different when the pH and/or the ionic strength and/or the concentration of fibrin of the acidic medium vary.

In a preferred embodiment, step iii-1) is performed during a time such that the loss tangent tan d of the acidic medium, defined as the ratio of the loss modulus G" over the storage modulus G', starts to decrease, and is higher than 0.5.

The loss tangent tan d, loss modulus G", and storage modulus G' can be measured using a cone-plate or plate-plate rheometer with an amplitude of y=0,l% and an angular frequency of w = 1 rad/s.

The viscosity of the solution at the end of step iii-1) can range from about 2 Pa.s to 200 Pa.s, said viscosity being dynamically measured with a rheometer.

Step iii-2) for obtaining material El or E2

When step iii) is performed during a large time [also called step iii-2)], step iii-2) is preferably performed during a time of at least 30 min.

In this embodiment, a pH of at most 4.0, and preferably of at most 3.8, for the aqueous acidic solution of fibrinogen is particularly preferred.

After step iii-2), a fibrin-based gel is directly obtained. Indeed, the time used during step iii-2) is sufficient to transform the aqueous acidic solution of step ii) into a viscous solution and then into a gel.

In a preferred embodiment, step iii-2) is performed during a time ranging from 30 min to 24h, advantageously from 45 min to 12h, and more advantageously from lh to 5h.

Said times for step iii-2) are generally applicable when the pH is about 3.6 and/or the ionic strength is about 75 mM and/or the concentration of fibrin is of 40 mg/mL, and may be different when the pH and/or the ionic strength and/or the concentration of fibrin of the acidic medium vary.

In a preferred embodiment, step ii-2) is performed during a time such that the loss tangent tan d of the acidic medium is lower than 0.5. Step iii-2) also triggers molecular assembly.

The incubating step iii-2) is preferably chosen among extrusion, 3D printing including extrusion 3D printing and ink-jet 3D printing, molding, coating, freeze-casting, electrochemical assembling, and electro-spraying. In this embodiment, the incubating step iii-2) is also a forming and/or shaping step.

In one preferred embodiment, the incubating step iii-2) is chosen among extrusion, molding, coating, and electro-spraying.

According to a particularly preferred embodiment of the present invention, the step iii-2) is molding or coating.

When step iii-2) is a molding step, the aqueous acidic solution comprising fibrin is preferably put into an appropriate mold, and the mold containing the aqueous acidic solution comprising fibrin is then incubated.

When step iii-2) is a coating step, the aqueous acidic solution comprising fibrin is preferably put in contact with an object heated to the desired temperature to induce gelation of the solution in contact, or with the object at room temperature that is subsequently incubated. In a preferred embodiment, solution of fibrin is poured on/in the object, excess solution is removed and the object is incubated. In a second preferred embodiment, the object heated to the desired temperature to induce gelation is dipped in the solution of fibrin and removed. This latter coating method is well-known under the expression "dip coating".

When step iii-2) is an extrusion step, the aqueous acidic solution comprising fibrin can be placed in an extrusion die heated at an incubating temperature as defined in the present invention.

Step iv-1) for obtaining material D

Step iv-1) triggers molecular assembly. Indeed, after the fibrin proteins interact upon incubation step iii), assembly is created during step iv- 1). The pH of the buffer solution of step iv-1) is preferably greater than the isoelectric point of fibrinogen, and more preferably ranges from about 6.5 to 9, more preferably from about 6.5 to 8, and even more preferably from about 7 to 7.8.

The buffer of step iv-1) is preferably selected from organic buffers commonly used in cell cultures such as for example 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid (HEPES), trishydroxymethylaminomethane (Tris buffer), 2-[l,3-dihydroxy-2-(hydroxymethyl) propan-2-yl]amino] ethanesulfonic acid (TES buffer) and physiological media such as for example Dubelcco's Modified Eagle's Medium (DMEM).

HEPES buffer is preferred.

The concentration of the HEPES buffer in the buffer solution preferably ranges from about 10 to 250 mmol/L, and more preferably from about 25 to 150 mmol/L. A particularly preferred concentration is about 100 mmol/L.

In order to increase the osmotic pressure, the buffer solution preferably comprises a thickening agent such as polyethyleneglycol (PEG), in particular a PEG having a molecular weight ranging from about 3000 and 10000 g/mol.

The amount of thickening agent may range from about 0.1 to 50 wt.%, preferably from about 2 to 10 wt.%, and more preferably from about 2 to 5 wt.%, with respect to the total weight of the buffer solution.

The contacting step iv-1) is preferably chosen among extrusion, 3D printing including extrusion 3D printing and ink-jet 3D printing, molding, freeze-casting, electrochemical assembling, coating and electro-spraying.

In this embodiment, the contacting step iv-1) is also a forming and/or shaping step.

According to a preferred embodiment of the present invention, the contacting step iv-1) is an extrusion step or a 3D printing step. For example, 3D printing can be carried out to produce a fibrin-based scaffold. More particularly, fibrin viscous solution obtained in step iii-1) can be used as an ink to be 3D printed into a fibrin-based scaffold.

Any apparatus known to those skilled in the art can be used for the extrusion step iv-1).

As examples, mention may be made of a syringe equipped with a cylindric needle, an extruder, and a co-extruder equipped with a Y blending connector.

A co-extruder equipped with a Y blending connector is particularly useful when the method implements biomaterials other than fibrin such as collagen, so as to produce composite biomaterials.

The rate of extrusion will depend on the type of extrusion apparatus that is used and on the viscosity of the mixture to be extruded. Typically, the rate of extrusion ranges from about 0.2 to 5 ply's, and preferably from about 0.5 to 1 ply's, for example with a 0.34 pm nozzle.

The extruded fibrin-based biomaterial may have different forms depending on the shape of the orifice or die used during the extrusion step.

In particular, the fibrin-based biomaterial can be extruded in the form of threads of various diameters (e.g. microthreads) or can be 3D-printed ("Extrusion-Based printing": EBB or "plotting") on a planar surface in a recipient comprising the buffer solution, to form an object having any desired printable 3D shape like square sheets, disks, etc...

Step iv-1) is preferably performed at room temperature, for example at a temperature ranging from about 18 to 25°C.

At the end of step iv-1), the fibrin-based biomaterial can be recovered from the buffer solution, and optionally dried.

Step iv-2j for obtaining material E2

The method according to the invention can optionally comprise after step iii-2), a step iv-2) of contacting said gel with a buffer solution comprising at least one buffer having a pH value superior to the isoelectric point of fibrinogen.

Step iv-2) induces a stiffening of the gel obtained after step iii-2).

In other terms, when the incubating step iii) is performed during a large time [also called step iii-2)], the method can further comprise a step iv- 2) consisting of contacting the fibrin-based gel with a buffer solution comprising at least one buffer having a pH value superior to the isoelectric point of fibrinogen, so as to stiffen the fibrin-based biomaterial produced according to step iii-2).

The pH of the buffer solution of step iv-2) preferably ranges from about 6.5 to 9, more preferably from about 6.5 to 8, and even more preferably from about 7 to 7.8.

The buffer of step iv-2) is preferably selected from organic buffers commonly used in cell cultures such as for example 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid (HEPES), trishydroxymethylaminomethane (Tris buffer), 2-[[l,3-dihydroxy-2-(hydroxymethyl) propan-2-yl]amino] ethanesulfonic acid (TES buffer) and physiological media such as for example Dubelcco's Modified Eagle's Medium (DMEM).

HEPES buffer is preferred.

The buffer solution may comprise a thickening agent such as polyethyleneglycol (PEG), in particular a PEG having a molecular weight ranging from about 3000 and 10000 g/mol. However, a thickening agent is not generally used.

If such a thickening agent is used, its amount may range from about 0.1 to 50 wt.%, preferably from about 2 to 10 wt.%, and more preferably from about 2 to 5 wt.%, with respect to the total weight of the buffer solution. Step iv-2) is preferably performed at room temperature, for example at a temperature ranging from about 18 to 25°C.

At the end of step iv-2), the fibrin-based biomaterial can be recovered from the buffer solution, and optionally dried.

Use of collagen - Step ii'j

The conditions of the method of the present invention also allow the combined use of other biomaterials and fibrinogen.

Other biomaterials such as collagen which are soluble at acidic pH and form fibers at neutral pH are particularly preferred.

For that matter, the method can further comprise a step ii') of preparing an aqueous acidic solution comprising at least collagen.

According to this particular embodiment of the invention, the concentration of collagen in the aqueous acidic solution comprising at least collagen may range from about 1 to 120 mg/ml_, and preferably this concentration is equal to about 60 mg/ml_.

The pH of the aqueous acidic solution comprising at least collagen preferably ranges from about 2 to 5, and even more preferably is about 3.6, for example so as to match the pH of the fibrin solution.

The pH of the aqueous acidic solution comprising at least collagen may be adjusted to the desired value with any organic or mineral acid, preferably with the same acids as those used to adjust the pH of the aqueous acidic solution comprising fibrinogen and thrombin, i.e. hydrochloride acid or acetic acid, hydrochloride acid being most preferred.

Collagen may be any form of fibrillar collagen (e.g., type I, type II, type III or type V). According to a preferred embodiment of the present invention, collagen is collagen I.

It has to be noted that said aqueous acidic solution of collagen is preferably not subjected to incubation. Indeed, the incubation of collagen at temperatures such as the one used in step iii) of the inventive method, can result in partial or complete transformation of collagen into gelatin. In the present invention, the use of collagen is particularly appropriate when the method comprises a step iii-1) of forming a viscous solution of fibrin.

Thus, in a particular embodiment, the incubating step iii-1) is followed by mixing the aqueous acidic solution comprising at least collagen with the viscous solution obtained after step iii-1), so as to obtain a composite mixture.

Then, the contacting step iv-1) can be carried out by contacting said composite mixture with the buffer solution.

After step iv-1), a fibrin-based composite material comprising fibrin and collagen is obtained.

A co-extruder equipped with a Y blending connector is particularly useful when two separate aqueous acidic solution, a first one comprising fibrin, and a second one comprising collagen are used, enabling continuous change of the respective amounts of fibrin aqueous acidic solution and of collagen aqueous acidic solution. In particular, when the aqueous acidic solution of collagen is co-extruded with the viscous solution of fibrin, the amount of said aqueous acidic solution of collagen may vary from 0.1 to 99.9 wt.%, with regard to the amount of the aqueous acidic solution of fibrin.

When the method comprises a step iii-2) of forming a gel of fibrin, the aqueous acidic solution comprising at least collagen can be mixed with the gel so as to obtain an impregnated gel, and then the obtained impregnated gel can be submitted to step iv-2).

Other steps

The method of the present invention can implement at least one therapeutic agent, such as cells, in particular stem cells, growth and/or differentiating factors, antibiotics, chemical therapeutics, etc ....

For that matter, the method of the present invention can further comprise the step of adding at least one therapeutic agent to the aqueous acidic solution of fibrin, the viscous solution of fibrin, the fibrinogen-based biomaterial in the form of a gel obtained after step iii-2), or to the fibrin-based biomaterial in the form of a gel obtained after step iv-1), or step iv-2). In the latter alternative, the addition may be made for example by impregnation of the fibrin-based biomaterial with a composition comprising said therapeutic agent(s).

The therapeutic agent may also be added into the aqueous acidic collagen solution if it exists.

The one skilled in the art will choose the most appropriate method to incorporate said therapeutic agent(s) into the biomaterial, in particular, he will verify that the pH conditions of these solutions will not alter the intrinsic properties of the added therapeutic agent(s).

In a preferred embodiment, the method of the present invention does not use any chemical and/or physical crosslinking.

A second object of the present invention is a fibrin-based biomaterial obtained according to the method defined in the first object of the present invention, wherein it is in the form of a gel and it comprises at least denatured fibrin proteins.

In the present invention, the presence of fibrin proteins in the fibrin- based biomaterial can be shown thanks to MALDI-TOF analysis.

During the method of the present invention, partial denaturation of the D domains leads to a fibrin-based biomaterial comprising at least denatured fibrin proteins. Indeed, fibrin is at least partially denatured.

In the present invention, denaturation of the thermolabile D domains in the fibrin-based biomaterial can be shown by differential scanning calorimetry (DSC).

More particularly, the thermolabile D domains of at least some fibrin proteins are destabilized or denatured, and subsequently interact by their extremities to form a gel.

It is noted that at least partial denaturation of fibrin proteins occurs during step iii) of incubation. By contrast, the E domains of the fibrin-based biomaterial are advantageously not denatured. Indeed, the E domains of the fibrin-based biomaterial remain intact (below 80°C).

In a preferred embodiment, the fibrin-based biomaterial comprises:

- non-denatured domains E, and

- partially denatured domains D.

Non-denatured domains E in the fibrin-based biomaterial can be shown by differential scanning calorimetry (DSC).

The fibrin-based biomaterial obtained according to the method defined in the first object of the present invention preferably does not comprise other glycoprotein(s) than fibrin such as fibronectin.

More particularly, the fibrin-based biomaterial do not comprise fibrinogen (the whole fibrinogen has been converted into fibrin thanks to thrombin during step ii)).

The fibrin-based biomaterial of the present invention is a massive or solid material.

The fibrin-based biomaterial of the present invention preferably comprises aggregates (as clearly shown in figure 1 as detailed in the example part). Such aggregates are not present in the fibrin-based materials of the prior art.

More particularly, these aggregates are formed by the denatured fibrin proteins. The aggregates are preferably non-fibrous aggregates.

In a preferred embodiment, the fibrin-based biomaterial of the present invention is not a cross-linked material.

According to a first variant, the fibrin-based material is obtained according to steps i), ii), i ii- 1) and iv-1) [material D and it is in the

form of an opaque gel.

The gel obtained according to the first variant preferably comprises denatured fibrin proteins and non-denatured fibrin proteins. The gel obtained according to the first variant is stable over time.

In the present invention, the expression "stable gel" means that the gel does not dissolve when agitated in a volume of at least 40 ml of non denaturing buffer such as HEPES.

The gel obtained according to the first variant can have a storage modulus ranging from about 1000 Pa to 4000 Pa, and more preferably from about 1500 Pa to about 3500 Pa.

In the present invention, the storage modulus is measured with an Anton Paar M302 Rheometer with a plate-plate geometry, preferably set with an amplitude of about 0,1%, and an angular frequency of about 1 rad/s.

The gel obtained according to the first variant can have a pH value ranging from about 6.8 to 7.8.

In a preferred embodiment, the gel obtained according to the first variant comprises fibers and/or fibrils.

According to a second variant, the fibrin-based material is obtained according to steps i), ii), and iii-2) fmaterial El and it is in the form of

a transparent or opaque gel, and preferably a transparent gel.

The gel obtained according to the second variant preferably comprises at least 80% by weight of denatured fibrin proteins, more preferably at least 90% by weight of denatured fibrin proteins, and even more preferably denatured fibrin proteins only, with respect to the total weight of fibrin proteins.

The gel obtained according to the second variant is stable over time as defined above for the gel of the first variant.

Additionally, the gel obtained according to the second variant can be stable to most conditions known to disperse protein-based hydrogels, such as acidic, neutral, basic buffers (pH 10.7), low and high ionic strength (MQ water and PBS10X => 1 = 1.6 M), temperature (about 100°C), dilution (MQ water), and salt-in solutions or chaotropes (KSCN, KI, CaCh). The gel obtained according to the second variant can have a storage modulus ranging from about 400 Pa to 10000 Pa, preferably from about 600 Pa to 3000 Pa, and more preferably from about 500Pa to about 1200 Pa.

The gel obtained according to the second variant can have a pH value ranging from about 2.5 to 4.

The gel obtained according to the second variant is preferably a non- fibrous material. In other terms, it does comprise fibers and/or fibrils.

According to a third variant, the fibrin-based material is obtained according to steps i), ii), iii-2), and iv-2) fmaterial E2 and it is in the

form of a transparent or opaque gel, and preferably a transparent gel.

The gel obtained according to the third variant preferably comprises at least 80% by weight of denatured fibrin proteins, more preferably at least 90% by weight of denatured fibrin proteins, and even more preferably denatured fibrin proteins only, with respect to the total weight of fibrin proteins.

The gel obtained according to the third variant is stable over time as defined above for the gel of the first variant.

Additionally, the gel obtained according to the third variant can be stable to most conditions known to disperse protein-based hydrogels, such as acidic, neutral, basic buffers (pH 10.7), low and high ionic strength (MQ water and PBS10X => 1 = 1.6 M), temperature (about 100°C), dilution (MQ water), and salt-in solutions chaotropes (KSCN, KI, CaCh).

The gel obtained according to the third variant can have a storage modulus ranging from about 500 Pa to 10000 Pa, and more preferably from about 1500 Pa to about 2500 Pa.

The gel obtained according to the third variant can have a pH value ranging from about 6.8 to 7.8.

The gel obtained according to the third variant is preferably a non- fibrous material. In other terms, it does comprise fibers and/or fibrils. The fibrin-based biomaterials defined in the second object of the invention or obtained according to the method defined in the first object of the present invention may be used to repair damaged soft tissues and/or organ in need thereof.

A third object of the present invention is therefore a fibrin-based biomaterial as defined in the second object of the present invention or obtained according to the method as defined in the first object of the present invention, for medical or surgical use, and more preferably for an application and/or an implantation in damaged soft tissues and/or organs in need thereof.

In particular, the biomaterial in the form of threads or microthreads has an application as suture material.

As another example, when said biomaterial takes the form of a 3D- printed material, it has an application as an implant to repair damaged tissue and/or organs in need thereof. The tissue repair includes tissue augmentation or the replacement of all or part of tissue, and the tissue repaired can be or can include skin, muscle, or connective tissue. The tissue repair can be necessitated by a traumatic injury, a congenital malformation, or tissue loss, malfunction, or malformation resulting from an infection or surgical procedure. The organ repair can include organ reconstruction, in particular of heart, liver, urinary bladder, etc...

The invention is further illustrated by the following examples.

EXAMPLES

EXAMPLE 1: Preparation of a fibrin-based biomaterial

1.1 Preparation of an aqueous acidic solution of fibrin

An aqueous acidic solution of fibrinogen was prepared by dissolving 1 g of human fibrinogen lyophilized from 20 mM citrate pH 7.4 solutions (Merck, >90% clottable proteins, CAS 9001-32-5) into 22 ml_ of distilled water. The final pH of the stock solution was of 6.6 to 6.8. The concentration of fibrinogen in the stock solution was measured with the extinction coefficient at 280 nm and was 40 ± 2 mg/mL. The stock solution of fibrinogen was aliquoted and kept at -80°C.

At the time of use, the pH of the stock solution of fibrinogen was decreased to 3.6 by addition of HCI 1.2M made from HCI 37% (VWR, AnalaR NORMAPUR, CAS No. 7647-01-0) to reach a final concentration of 60 mM HCI.

The final concentration of fibrinogen in the aqueous acidic solution was 38 mg/mL.

Lyophilized thrombin from bovine plasma (Sigma, 40-300 NIH U/mg, lkU) was dissolved in sterile PBS IX to reach a concentration of 200 U/mL. The resulting solution of thrombin was aliquoted and kept at -20°C.

At the time of use, the thrombin solution was added to the aqueous acidic solution of fibrinogen such that the concentration of thrombin in the aqueous acidic solution of fibrinogen was 20 U/mL.

Then, a solution of 100 mM of CaCh was added such that the resulting aqueous acidic solution comprises 10 mM of CaCh.

1.2 Incubation of the aqueous acidic solution of fibrin

The aqueous acidic solution of fibrin prepared at step 1.1 was then incubated at 37°C for 10 minutes in a controlled-temperature bath, so as to form a fibrin-based viscous solution.

1.3 Preparation of a fibrin-based ael bv extrusion

A syringe equipped with a blunt needle having an opening diameter of 370 pm was filled with the viscous solution of fibrin and then extruded in a 100 mM HEPES buffering solution (Sigma, >99,5%, CAS No. 7365-45-9) containing 2.5 wt.% of PEG (Fluka, Polyethylene glycol 3Ό00, CAS No. 25322- 68-3) at the speed of 0.5 pb s.

The pH of the buffering solution was 7.4.

A fibrin-based biomaterial in the form of thread having a diameter of around 250 pm was thus obtained. The figure 1 represents a TEM image of a fibrin-based gel obtained according to the method described in 1.1 to 1.3. The resulting gel exhibits a composite structure, with thin fibers connecting aggregate-like islands. Connections are represented by arrows.

It has a young modulus of 0.10 ± 0.02 MPa, a strength of 0.33 ±0.04 MPa, a strain at break of 251 ± 63 %in a 100 mMHepes2.5% PEG buffering solution.

Only parts of the fibrin proteins are denatured and form aggregates. Upon neutralization step iv-1), the strengthening of the network is hypothesized to rely on aCs interactions promoted by the proximity of proteins.

Fibrin threads were seeded with normal human dermal fibroblasts (PromoCell™, passage 17) at 420,000 cells/mL in low glucose DMEM with 10% in volume fetal bovine serum (GIBCO) and 2 mg/ml_ aminocaproic acid. After 72 hours culture, threads were rinsed with PBS IX and fixed with PFA 4 %in mass. Cells were permeabilized with Triton X-100. The nuclei and the actin filaments were fluorescently labeled (DAPI, Invitrogen and Alexa FluorR 488 phalloidin, Invitrogen). Observations of the samples were carried out under fluorescence microscope (Axio Imager D.l, Zeiss). Normal Human Dermal Fibroblasts demonstrated a high affinity for the fibrin threads, with high cellular densities obtained as it can be shown in annexed figure 2.

1.4 Preparation of a fibrin-based ael bv 3D printing

Printing of a fibrin-based construct was carried with the home-made 3D printer shown 13 on annexed figure 3. This device 1 was build using 3 stepper motors 2 to move the needle 3 in 3D space.

A syringe pump 4 made of a 200 step/turn stepper motor 5, aluminum tube 6, and M6 lead screws 7 is plugged to a Reprap ® 1.4 board 8 stacked on an Arduino® Mega 9. The syringe pump 4 was connected to the needle 3 using a 400 pm PTFE tubing 10. A printing platform 11 where the buffer solution 12 in a petri-dish 13 was placed can be leveled to ensure constant distance between the needle 3 and the dish 13 for horizontal movement. The software Repetier ® was used to slice 3D models and monitor the printer thanks to a computer 14. The syringe pump 4 was filled with the incubated acidic solution of fibrin 15 and then printed thanks to this home-made 3D printer 1 controlled by the Arduino® controller 9, through the needle 3, in a 25 mM HEPES buffer solution 12 (Sigma, >99,5%, CAS No. 7365-45-9) at the speed of 2 mm/s and a layer thickness of 0.3 mm.

EXAMPLE 2: Preparation of a fibrin-based biomaterial

2.1 Preparation of an aqueous acidic solution of fibrin

An aqueous acidic solution of fibrinogen was prepared by dissolving 1 g of human fibrinogen lyophilized from 20 mM citrate pH 7.4 solutions (Merck, >90% clottable proteins, CAS 9001-32-5) into 22 ml_ of distilled water. The final pH of the stock solution was of 6.6 to 6.8. The concentration of fibrinogen in the stock solution was measured with the extinction coefficient at 280 nm and was 40 ± 2 mg/ml_. The stock solution of fibrinogen was aliquoted and kept at -80°C.

The final concentration of fibrinogen in the aqueous acidic solution was 38 mg/ml_.

2.2 Incubation of the aqueous acidic solution of fibrin and preparation of a fibrin-based gel The aqueous acidic solution of fibrin prepared at step 2.1 was then incubated at 37°C for 1 hour in a controlled-temperature bath, so as to form a first gel which is transparent.

The figure 4 represents a TEM image of the fibrin-based biomaterial obtained after step 2.2.

It has a storage modulus of 890 Pa.

2.3 Stiffening of the fibrin-based ael

The gel obtained in step 2.2 was neutralized with 100 mM HEPES buffering solution.

A gel which is transparent is obtained.

It has a storage modulus of 2000 Pa.

COMPARATIVE EXAMPLE 3: Preparation of fibrin-based biomaterials which are not part of the invention

3.1 Preparation of an aqueous neutral solution of fibrin

An aqueous neutral solution of fibrinogen was prepared by dissolving 1 g of human fibrinogen lyophilized from 20 mM citrate pH 7.4 solutions (Merck, >90% clottable proteins, CAS 9001-32-5) into 22 ml_ of distilled water. The final pH of the stock solution was of 6.6 to 6.8. The concentration of fibrinogen in the stock solution was measured with the extinction coefficient at 280 nm and was 40 ± 2 mg/ml_. The stock solution of fibrinogen was aliquoted and kept at -80°C.

At the time of use, the thrombin solution was added to the aqueous neutral solution of fibrinogen such that the concentration of thrombin in the aqueous acidic solution of fibrinogen was 20 U/mL.

Then, a solution of 100 mM of CaCh was added such that the resulting aqueous neutral solution comprises 10 mM of CaCh. 3.2 Preparation of an aqueous acidic solution of fibrin

An aqueous solution of fibrinogen was prepared by dissolving 1 g of human fibrinogen lyophilized from 20 mM citrate pH 7.4 solutions (Merck, >90% clottable proteins, CAS 9001-32-5) into 22 ml_ of distilled water. The final pH of the stock solution was of 6.6 to 6.8. The concentration of fibrinogen in the stock solution was measured with the extinction coefficient at 280 nm and was 40 ± 2 mg/ml_. The stock solution of fibrinogen was aliquoted and kept at -80°C.

The final concentration of fibrinogen in the aqueous neutral solution was 38 mg/ml_.

3.3 Preparation of a fibrin-based ael

The aqueous neutral solution of fibrin prepared at step 3.1 was transformed into an opaque gel after 10 min at 25°C.

The aqueous acidic solution of fibrin prepared at step 3.2 was contacted at 25°C with a 100 mM HEPES buffering solution (Sigma, >99,5%, CAS No. 7365-45-9) containing 2.5 wt.% of PEG, so as to form an opaque gel.

It is noted that the processes used to provide the fibrin-based gels do not comprise an incubating step iii) as defined in the present invention. Thus, the fibrin-based materials obtained are not part of the invention. TEM images of the fibrin-based biomaterials respectively obtained from the aqueous neutral solution of fibrin, and from the aqueous acidic solution of fibrin have shown different fibrin-based materials in terms of structure.

In addition, the storage modulus of the fibrin-based biomaterial obtained from the aqueous neutral solution of fibrin could not be measured under the same conditions as the ones applied for the other fibrin-based materials.

The fibrin-based biomaterial obtained from the aqueous acidic solution of fibrin has a storage modulus of 530 Pa.

Claims

1. A method for the preparation of a fibrin-based biomaterial in the form of a gel, wherein said method comprises at least the following steps: i) preparing an aqueous acidic solution comprising fibrinogen, said aqueous acidic solution having a pH value inferior to the isoelectric point of fibrinogen, ii) adding thrombin to the aqueous acidic solution comprising fibrinogen of step i), so as to form an aqueous acidic solution comprising fibrin, iii) incubating said aqueous acidic solution comprising fibrin at a temperature ranging from 30°C to 70°C, and wherein: said step iii) is a step iii-1) performed during a time sufficient to form a viscous solution, and said method additionally comprises a step iv-1) of contacting said viscous solution with a buffer solution comprising at least one buffer having a pH value superior to the isoelectric point of fibrinogen, so as to form a gel; or said step iii) is a step iii-2) performed during a time sufficient to directly form a gel.

2. The method according to claim 1, wherein the concentration of fibrinogen in said aqueous acidic solution ranges from 10 to 100 mg/mL

3. The method according to claim 1 or 2, wherein the pH of said aqueous acidic solution of fibrinogen ranges from 2.5 to 4.0.

4. The method according to anyone of the preceding claims, wherein said aqueous acidic solution of fibrinogen comprises at least one monovalent or divalent metal cation selected from the group consisting of Na⁺, Ca²⁺, Mg²⁺ and Zn²⁺.

5. The method according to claim 4, wherein the concentration of said monovalent or divalent metal cation in said aqueous acidic solution ranges from 0.001 to 200 mmol/L.

6. The method according to anyone of the preceding claims, wherein said method comprises after step iii-2), a step iv-2) of contacting said gel with a buffer solution comprising at least one buffer having a pH value superior to the isoelectric point of fibrinogen.

7. The method according to anyone of the preceding claims, wherein said buffer is selected from 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, trishydroxymethylaminomethane, 2-[[l,3-dihydroxy-2-

(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid, and physiological media.

8. The method according to anyone of the preceding claims, wherein the buffer is 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid and the concentration of said buffer in the buffer solution ranges from 10 to 250 mmol/L.

9. The method according to anyone of the preceding claims, wherein the pH of the buffer solution ranges from 6.5 to 9.

10. The method according to anyone of the preceding claims, wherein the buffer solution comprises a thickening agent.

11. The method according to anyone of the preceding claims, wherein the incubating step iii) is an incubating step iii-1) performed during at most 25 min.

12. The method according to anyone of claims 1 to 10, wherein the incubating step iii) is an incubating step iii-2) performed during at least 30 min.

13. The method according to anyone of the preceding claims, wherein the contacting step iv-1) or the incubating step iii-2) is chosen among extrusion, 3D printing including extrusion 3D printing and ink-jet 3D printing, molding, freeze-casting, electrochemical assembling coating, and electro spraying.

14. The method according to anyone of the preceding claims, wherein said method further comprises a step ii') of preparing an aqueous acidic solution comprising at least collagen.

15. The method according to anyone of the preceding claims, wherein said method further comprises the step of adding at least one therapeutic agent to the aqueous acidic solution comprising fibrin, the viscous solution, the fibrinogen-based biomaterial in the form of a gel obtained after step iii-2), or to the fibrin-based biomaterial in the form of a gel obtained after step iv-1) or step iv-2).

16. A fibrin-based biomaterial obtained according to the method as defined in anyone of the preceding claims, wherein it is in the form of a gel and it comprises at least denatured fibrin proteins.

17. A fibrin-based biomaterial as defined in claim 16, for medical or surgical use.

18. The fibrin-based biomaterial according to claim 17, for an application and/or an implantation in damaged soft tissues and/or organs in need thereof.