RU2813067C2 - Multi-dimensional hemostatic product and method of its manufacture - Google Patents

Abstract

FIELD: medical equipment.

SUBSTANCE: group of inventions relates to a hemostatic product obtained by three-dimensional additive printing, the use of hemostatic fluid material as printing ink for a three-dimensional additive printer, and a method of manufacturing a hemostatic product. The hemostatic product has three dimensions and consists of a stack of layers located one on top of the other, counting from the first outer layer to the second outer layer, in which adjacent layers of the stack of layers are connected to each other and in which a layer of the stack of layers includes a part obtained from a hemostatic fluid a material whose composition includes: non-cross-linked fibrillar type collagen, in which the content of fibrous collagen and/or fibrillar collagen is at least 70 wt.% of the total mass of collagen; monosaccharide; and glycosaminoglycan. The use of hemostatic fluid material as a printing ink for a three-dimensional additive printer, where the hemostatic fluid material is obtained from hemostatic powder mixed with saline solution. The method includes the step of a) providing a three-dimensional model for a three-dimensional additive printer, where the three-dimensional model corresponds to the structure of the hemostatic product being manufactured, and processing the three-dimensional model to determine a printable template including a plurality of layers designed to be stacked one on top of the other to form a stack of layers, corresponding three-dimensional model. The method includes step b) supplying a three-dimensional additive printer with at least one hemostatic fluid material that is used as a printing ink, where the fluid material is obtained from a hemostatic powder mixed with a saline solution. The method includes step c) printing a hemostatic product using a three-dimensional additive printer by applying printing ink to sequentially deposit one layer on top of another, where the formation includes applying a hemostatic fluid material using a three-dimensional additive printer, resulting in the formation of at least one part of at least one layer from a pack of layers.

EFFECT: provision of an easy-to-use hemostatic product, which, in particular, does not require complex preparation, can additionally be easily applied to a specific area to completely cover the target bleeding area, has high hemostatic efficiency, higher than existing hemostatic products, can be tailored to the specific needs of the patient and can be easily manufactured, can be used in cases of multiple bleedings and/or if the bleeding area has a complex shape, provides a convenient method of manufacturing a three-dimensional hemostatic product that allows to form a complex three-dimensional hemostatic product.

47 cl, 2 dwg

Description

TECHNICAL FIELD

The present invention relates to the field of technology of hemostatic (hemostatic) compositions, in particular, to printed hemostatic products, for example, a three-dimensional (3D) hemostatic product, and to a method for manufacturing a three-dimensional hemostatic product.

BACKGROUND ART

Wounds, both external and internal, caused by trauma or surgery, often lead to bleeding. Bleeding may be more or less significant. Bleeding is prevented and stopped as a result of a set of physiological phenomena called "hemostasis". Hemostasis promotes the fusion of damaged vessels and, in general, ensures the maintenance of the integrity of the vessel and tissue.

When a blood vessel is damaged, a natural multi-stage mechanism is triggered, which stops the bleeding. First, a constriction of the blood vessels occurs, which slows the bleeding and lasts for a period of 15 to 60 seconds, setting off a complex cascade of reactions. A fibrous network consisting of fibrin forms is formed around the platelet plaque: a ready-made blood clot is formed, which is protected from premature resorption by factor XIII, which stabilizes fibrin. Finally, the fibrin mesh thickens (retraction) and the edges of the wound are brought together: the wound becomes smaller. Within the stable, cross-linked fibrin, fibroblasts can then grow, organize within the thrombus into a connective matrix, and finally close the wound.

There is no solid fibrin in the circulatory system; if it were there, it would immediately clog the vital vessels. However, it contains a fibrin precursor, fibrinogen. Under the influence of thrombin, the synthesis of which is activated by blood clotting factors, fibrinogen is converted into insoluble fibrin.

Finally, within a few days or weeks after successful wound healing, the fibrin cluster is destroyed as a result of fibrinolysis.

Despite the considered biochemical phenomenon, especially in the case of extensive wounds or in the case of diffuse bleeding, there is often a need for “artificial” hemostasis.

To achieve hemostasis, there are “mechanical” solutions to the problem, such as pressure, ligation and electrocoagulation, which are used as first-line treatment. However, in some cases, such as diffuse capillary bleeding, bleeding of hypervascularized organs such as the spleen or liver, bleeding resulting in diffuse bleeding such as bone, and/or neurosurgical interventions, these solutions have little or no effectiveness.

In addition, there are “chemical” solutions, in particular those underlying some of the currently available hemostatic products. The components of such chemical solutions are typically "absorbent" or "active" type components.

Absorption-type hemostatic products, which are known to include polysaccharides such as regenerated oxidized cellulose or alginates, act primarily through mechanical action and simple absorption. Their disadvantage is often excessive swelling. While swelling results in rapid absorption of fluid, in particular blood, when used in a “closed” environment, such as in contact with the dura mater or in urological applications, it can also lead to unwanted pressure.

In addition, some products, which, it should be noted, include plant polysaccharides such as cellulose or alginates, when resorbed, may further cause inflammatory reactions, and/or the presence of these components may lead to the destruction of the product, which is not recognized by the host body. As a result, such products must be removed so that they do not remain in the body and thus do not lead to negative consequences.

Active hemostatic products, such as those containing thrombin or fibrin, are often blood-derived products. The disadvantage of such products is the risk of developing allergies and transmitting diseases, in particular if the disease vector cannot be inactivated by classical treatment. In addition, subsequent treatments are usually complex and/or expensive. Finally, they may typically require preparation before use, which can be limiting and even inconvenient in the event of an emergency.

In addition, the action of products containing fibrin and thrombin is based on the interaction between the two blood-derived products included in the product. Sometimes the reaction can occur without interaction with blood, in which case the products are said to float. In other words, the product is pushed out by blood, which continues to flow, possibly causing dilution or coagulation of the product, which forms a gel on top of the blood mass - and in this situation the blood flow is not blocked. Thus, hemostasis may not occur.

The hemostatic powder, the method of its preparation and the method of its use were discussed in international patent application WO 2012/146655, published on November 1, 2012, the contents of which are incorporated herein by reference in their entirety.

This hemostatic powder has satisfactory absorption capacity, good hemostatic (hemostatic) ability, almost no negative side effects, good ability to adhere to the edges of the wound and satisfactory penetration into the bloodstream at the site of application and/or limited swelling.

In addition to the good hemostatic properties mentioned, the advantage of this hemostatic powder is its very high fluidity, allowing it to be sprayed onto the bleeding site. The powder can be applied during most surgical procedures such as laparotomy, laparoscopy, celioscopy and robotic surgical techniques.

Hemostatic powder can be directly applied to the bleeding site without special preparation by the surgeon, which is also an advantage.

To facilitate the application of hemostatic powder to a specific bleeding site, in some cases it may be necessary to use special dosing devices (dispensers) for the powder.

The object of the present invention is to provide an easy-to-use hemostatic product, which in particular does not require complex preparation, which can additionally be easily applied to a specific area to completely cover the target bleeding site.

Another object of the present invention is to provide a hemostatic product having high hemostatic efficiency, superior to existing hemostatic products.

Another object of the present invention is to provide a hemostatic product that can be tailored to the specific needs of a patient and that can be easily manufactured.

Another object of the present invention is to provide a hemostatic product that can be used in cases of multiple bleedings and/or if the bleeding area has a complex shape.

It is also an object of the present invention to provide an easy-to-use method for manufacturing a three-dimensional hemostatic product that allows the formation of a complex three-dimensional hemostatic product.

SUMMARY OF THE INVENTION

To solve these problems, a multidimensional hemostatic product, a method for its manufacture and the use of a hemostatic fluid material disclosed in the accompanying claims are proposed.

In particular, a three-dimensional hemostatic product is proposed, consisting of a stack of layers located one on top of the other, counting from the first outer layer to the second outer layer, where adjacent layers are connected to each other; The hemostatic product is characterized in that at least one layer of the first and second outer layers of the stack of layers is obtained from a hemostatic fluid mixture consisting of:

- collagen of the fibrillar type, which is preferably not cross-linked and in which the content of fibrous collagen and/or fibrillar collagen is at least 70% by weight. from the total mass of collagen;

- at least one monosaccharide; And

- at least one glycosaminoglycan.

Preferred, but not limiting, aspects of the three-dimensional hemostatic device, which may be implemented individually or in combination, include the following:

- the first and second outer layers of the layer pack have the same composition.

- the peripheral part of each layer of the stack of layers is obtained from a fluid mixture identical to the fluid mixture of at least one layer selected from the first and second outer layers.

- all layers in a stack of layers have the same composition.

The invention also relates to the use of a hemostatic fluid material as a printing ink (printing material) for a three-dimensional printer, where the hemostatic fluid material is obtained from a hemostatic powder mixed with a saline solution, and the composition of the hemostatic powder includes:

- non-cross-linked fibrillar type collagen, in which the content of fibrous collagen and/or fibrillar collagen is at least 70% by weight. from the total mass of collagen;

- at least one monosaccharide; And

- at least one glycosaminoglycan.

The invention also relates to a method for manufacturing a three-dimensional hemostatic product using a three-dimensional additive printer, where the method includes the following steps:

- providing a three-dimensional model for a three-dimensional additive printer, where the three-dimensional model corresponds to the shape of the three-dimensional hemostatic product being manufactured, and processing the three-dimensional model to determine a template for printing, including a set of layers designed to be stacked one on top of the other to form a stack of layers corresponding to the three-dimensional model ;

- supplying a hemostatic fluid material to a three-dimensional additive printer for use as a printing ink, where the fluid material is obtained from a hemostatic powder mixed with a saline solution, and the composition of the hemostatic powder includes:

non-cross-linked fibrillar type collagen in which the content of fibrous collagen and/or fibrillar collagen is at least 70% by weight. from the total mass of collagen;

at least one monosaccharide; And

at least one glycosaminoglycan,

- deposition of hemostatic fluid material using a three-dimensional additive printer, which leads to the formation of at least one of the outer layers of the stack of layers.

In such a method for manufacturing a three-dimensional hemostatic product, a hemostatic fluid material is preferably used to form all layers of a stack of layers to form a three-dimensional hemostatic product (10).

Preferred, but non-limiting aspects of the hemostatic fluid material discussed above, which may be implemented individually or in combination, include the following:

- in the composition of hemostatic powder:

collagen is contained in an amount ranging from 80% to 90% of the mass. from the total weight of the hemostatic powder composition;

at least one monosaccharide is contained in an amount ranging from 1% to 12.5% by weight. from the total weight of the hemostatic powder composition; And

at least one glycosaminoglycan is contained in an amount ranging from 2% to 25% by weight. from the total weight of the hemostatic powder composition.

- in the composition of hemostatic powder:

collagen is contained in an amount ranging from 80% to 90% of the mass. from the total weight of the hemostatic powder composition;

at least one monosaccharide is contained in an amount ranging from 2.5% to 7.5% by weight. from the total weight of the hemostatic powder composition; And

at least one glycosaminoglycan is contained in an amount ranging from 5% to 12.5% by weight. from the total weight of the hemostatic powder composition.

- in the composition of hemostatic powder:

collagen is contained in an amount ranging from 84% to 88% of the mass. from the total weight of the hemostatic powder composition;

at least one monosaccharide is contained in an amount ranging from 4% to 6% by weight. from the total weight of the hemostatic powder composition; And

at least one glycosaminoglycan is contained in an amount ranging from 8% to 10% by weight. from the total weight of the hemostatic powder composition.

- in the hemostatic powder composition at least one monosaccharide is glucose and at least one glycosaminoglycan is chondroitin sulfate.

- in the hemostatic powder composition at least one glycosaminoglycan is selected from chondroitin sulfate, dermatan sulfate, hylauronic acid and mixtures thereof.

- the hemostatic powder composition additionally includes at least one blood coagulation factor in an amount of less than 0.1% by weight. from the total weight of the hemostatic powder composition.

- The blood clotting factor is thrombin.

- the saline solution includes or consists of distilled water and sodium chloride, where sodium chloride is contained in an amount ranging from 0.5% to 1.5% by weight. of the total weight of the saline solution, most preferably in an amount of 0.9% by weight. of the total mass of the saline solution.

- the weight of the saline solution is from 2 to 10 times the weight of the hemostatic powder, and preferably from 4 to 5 times the weight of the hemostatic powder.

- the hemostatic fluid material is prepared by mixing an amount of hemostatic powder of 1 g to 2 g with an amount of saline solution of 4 ml to 10 ml, preferably 5 ml to 10 ml of saline.

- hemostatic fluid material is prepared by mixing 1.65 g of hemostatic powder mixed with 7 ml of pure saline solution, or in any other similar ratio.

The invention also relates to a printed hemostatic product having at least three dimensions and consisting of a stack of layers located one on top of the other, counting from the first outer layer to the second outer layer, where adjacent layers of the stack of layers are connected to each other, and at least one layer of the stack of layers includes at least one portion derived from a hemostatic fluid material, the composition of which includes:

- non-cross-linked fibrillar type collagen, in which the content of fibrous collagen and/or fibrillar collagen is at least 70% by weight. from the total mass of collagen; And

- at least one monosaccharide.

Preferred, but not limiting, aspects of the printed hemostatic product, which may be implemented individually or in combination, include the following:

- at least one layer of the stack of layers includes several parts obtained from hemostatic fluid materials having different compositions.

- at least one layer of the stack of layers is made entirely of one hemostatic fluid material.

- all layers in a stack of layers have the same composition.

- the hemostatic product obtained by the printing method includes a set of adjacent layers, where each layer of the set of adjacent layers includes at least one part obtained from a hemostatic fluid material.

- at least one layer of the first and second outer layers of the stack of layers includes a portion derived from a hemostatic fluid material.

- at least one layer of the first and second outer layers is made entirely of the same hemostatic fluid material.

- the first and second outer layers of the layer pack have the same composition.

- each of the layers of the stack of layers has a peripheral part obtained from a fluid mixture identical to the fluid mixture of at least one layer selected from the first and second outer layers.

- at least one of the layers of the stack of layers includes a portion derived from a hemostatic fluid material having a swelling ability that is different from the swelling ability of other layers contained in the stack of layers.

- the collagen content in the hemostatic fluid material composition ranges from 70% to 99% by weight. of the total weight of the composition, preferably from 75% to 96% by weight, and more preferably from 80% to 90% by weight.

- the monosaccharide content in the composition of the hemostatic fluid material ranges from 1% to 12.5% by weight. of the total weight of the composition, preferably from 2% to 8% by weight, more preferably from 2.5% to 7.5% by weight. and more preferably from 4% to 6% by weight.

- the composition of the hemostatic fluid material includes at least one glycosaminoglycan.

- glycosaminoglycan content ranges from 1% to 30% by weight. of the total weight of the composition, preferably from 2% to 25% by weight, preferably from 4% to 15% by weight, more preferably from 5% to 12.5% by weight. and more preferably from 8% to 10% by weight.

- glycosaminoglycan selected from chondroitin sulfate, dermatan sulfate, hylauronic acid and mixtures thereof.

- the composition of the hemostatic fluid material includes at least one blood coagulation factor, in particular thrombin.

- the blood clotting factor is contained in an amount ranging from 0.01 IU/mg to 20 IU/mg of the composition, preferably ranging from 0.05 IU/mg to 10 IU/mg of the composition, preferably ranging from 0.1 IU/mg to 5 IU/mg of the composition and more preferably is from 0.2 IU/mg to 2 IU/mg of the composition.

- hemostatic fluid material is obtained from hemostatic powder, the composition of which includes:

collagen of the fibrillar type, in which the content of fibrous collagen and/or fibrillar collagen is at least 70% of the mass. of the total mass of collagen, and collagen is contained in the powder in an amount ranging from 75% to 96% of the mass. based on the total weight of the hemostatic powder composition, preferably from 80% to 90% by weight;

at least one monosaccharide in an amount ranging from 1% to 12.5% of the mass. based on the total weight of the hemostatic powder composition, preferably in an amount ranging from 2.5% to 7.5% by weight, and more preferably in an amount ranging from 4% to 6% by weight.

- the hemostatic powder additionally includes at least one glycosaminoglycan in an amount ranging from 1% to 30% by weight. of the total weight of the hemostatic powder composition, preferably from 2% to 25% by weight, preferably in an amount ranging from 4% to 15% by weight, more preferably in an amount ranging from 5% to 12.5% by weight, and more preferably in an amount ranging from 8% to 10% of the mass.

- the hemostatic powder additionally includes at least one blood coagulation factor in an amount of less than 0.5% by weight. from the total weight of the hemostatic powder composition, preferably in an amount of less than 0.1% of the mass.

The invention further relates to the use of a hemostatic fluid material as a printing ink for a three-dimensional printer, where the hemostatic fluid material is obtained from a hemostatic powder mixed with a saline solution, and the composition of the hemostatic powder includes:

- non-cross-linked fibrillar type collagen, in which the content of fibrous collagen and/or fibrillar collagen is at least 70% by weight. from the total mass of collagen;

- at least one monosaccharide.

Preferred, but not limiting, aspects of the use of the hemostatic fluid as a printing ink, which may be practiced alone or in combination, include the following:

- the collagen content in the hemostatic powder composition contained in the hemostatic fluid material ranges from 70% to 99% by weight. of the total weight of the composition, preferably from 75% to 96% of the mass. and more preferably from 80% to 90% of the mass.

- the monosaccharide content in the hemostatic powder composition contained in the hemostatic fluid material ranges from 1% to 12.5% by weight. of the total weight of the composition, preferably ranges from 2% to 8% by weight, more preferably from 2.5% to 7.5% by weight. and more preferably from 4% to 6% by weight.

- the composition of the hemostatic powder from which the hemostatic fluid material is formed includes at least one glycosaminoglycan.

- glycosaminoglycan content ranges from 1% to 30% by weight. of the total weight of the composition, preferably ranges from 2% to 25% by weight, preferably from 4% to 15% by weight, more preferably from 5% to 12.5% by weight. and more preferably from 8% to 10% by weight.

- glycosaminoglycan selected from chondroitin sulfate, dermatan sulfate, hylauronic acid and mixtures thereof.

- the composition of the hemostatic powder from which the hemostatic fluid material is formed includes at least one blood coagulation factor, in particular thrombin.

- the blood clotting factor is contained in an amount ranging from 0.01 IU/mg to 20 IU/mg of the composition, preferably ranging from 0.05 IU/mg to 10 IU/mg of the composition, preferably ranging from 0.1 IU/mg to 5 IU/mg of the composition and more preferably is from 0.2 IU/mg to 2 IU/mg of the composition.

- the saline solution includes at least one blood coagulation factor, in particular thrombin.

- the blood clotting factor is contained in an amount ranging from 10 IU/ml to 5000 IU/ml saline, preferably from 25 IU/ml to 2500 IU/ml saline, more preferably from 50 IU/ml to 1000 IU/ml saline and more preferably from 100 IU/ml to 500 IU/ml saline solution.

- the composition of the hemostatic powder from which the hemostatic fluid material is formed includes:

collagen of the fibrillar type, in which the content of fibrous collagen and/or fibrillar collagen is at least 70% of the mass. of the total mass of collagen, where collagen is contained in an amount ranging from 75% to 96% of the mass. of the total weight of the hemostatic powder composition, preferably ranges from 80% to 90% by weight;

at least one monosaccharide in an amount ranging from 1% to 12.5% of the mass. based on the total weight of the hemostatic powder composition, preferably in an amount ranging from 2.5% to 7.5% by weight, and more preferably in an amount ranging from 4% to 6% by weight.

- the composition of the hemostatic powder from which the hemostatic fluid material is formed additionally includes at least one glycosaminoglycan in an amount ranging from 1% to 30% of the mass. of the total weight of the hemostatic powder composition, preferably from 2% to 25% by weight, preferably in an amount ranging from 4% to 15% by weight, more preferably in an amount ranging from 5% to 12.5% by weight, and more preferably in an amount ranging from 8% to 10% of the mass.

- hemostatic powder from which the hemostatic fluid material is formed additionally includes at least one blood coagulation factor in an amount of less than 0.5 wt%. from the total weight of the hemostatic powder composition, preferably in an amount of less than 0.1% of the mass.

- the weight of the saline solution used to obtain the hemostatic fluid material is 2-10 times the weight of the hemostatic powder and preferably 4-5 times the weight of the hemostatic powder.

- hemostatic powder from which the hemostatic fluid material is formed includes at least 80% of the mass. particles whose size ranges from 20 microns to 300 microns.

- hemostatic powder from which the hemostatic fluid material is formed includes at least 90% of the mass. particles whose size is less than 350 microns.

Finally, the invention relates to a method for producing a hemostatic product having at least three dimensions using a three-dimensional additive printer, where the method includes the following steps:

a) providing a three-dimensional model for a three-dimensional additive printer, where the three-dimensional model corresponds to the structure of the hemostatic product being manufactured, and processing the three-dimensional model to define a printable template comprising a plurality of layers designed to be stacked one on top of the other to form a stack of layers corresponding to the three-dimensional model ;

b) supplying a three-dimensional additive printer with at least one hemostatic fluid material that is used as a printing ink, where the fluid material is obtained from a hemostatic powder mixed with a saline solution, and the composition of the hemostatic powder includes:

non-cross-linked fibrillar type collagen, in which the content of fibrous collagen and/or fibrillar collagen is at least 70% by weight. from the total mass of collagen; And

at least one monosaccharide,

c) printing the hemostatic product using a three-dimensional additive printer by applying printing ink to sequentially deposit one layer on top of another, where formation) includes applying a hemostatic fluid material using a three-dimensional additive printer, resulting in the formation of at least one portion of at least one layer from a pack of layers.

Preferred, but not limiting, aspects of the method of making a hemostatic device, which may be performed individually or in combination, include the following:

- at least one layer of the stack of layers is made entirely by deposition of a hemostatic fluid material.

- upon receipt of the hemostatic product (10), the hemostatic fluid material is used to form several layers contained in a stack of layers.

- upon receipt of the hemostatic product (10), the hemostatic fluid material is used to form all the layers contained in the stack of layers.

- in step b) a set of hemostatic fluid materials are supplied to the three-dimensional additive printer, which are used as printing inks, and these hemostatic fluid materials have different compositions.

- the composition of the hemostatic powder from which the hemostatic fluid material is formed includes:

collagen of the fibrillar type, in which the content of fibrous collagen and/or fibrillar collagen is at least 70% of the mass. of the total mass of collagen, where collagen is contained in an amount ranging from 75% to 96% of the mass. of the total weight of the hemostatic powder composition, preferably from 80% to 90% of the mass;

at least one monosaccharide in an amount ranging from 1% to 12.5% of the mass. based on the total weight of the hemostatic powder composition, preferably in an amount ranging from 2.5% to 7.5% by weight, and more preferably in an amount ranging from 4% to 6% by weight.

- the composition of the hemostatic powder from which the hemostatic fluid material is formed additionally includes at least one glycosaminoglycan in an amount ranging from 1% to 30% of the mass. based on the total weight of the hemostatic powder composition, preferably in an amount ranging from 2% to 25% by weight, preferably in an amount ranging from 4% to 15% by weight, more preferably in an amount ranging from 5% to 12.5% by weight. , and more preferably in an amount ranging from 8% to 10% of the mass.

- the composition of the hemostatic powder from which the hemostatic fluid material is formed additionally includes at least one blood coagulation factor in an amount of less than 0.5 wt%. from the total weight of the hemostatic powder composition, preferably in an amount of less than 0.1% of the mass.

- the saline solution used in preparing the hemostatic fluid material includes at least one coagulation factor, and the coagulation factor is contained in an amount ranging from 10 IU/ml to 5000 IU/ml saline solution, preferably from 25 IU/ml to 2500 IU/ml saline, more preferably 50 IU/ml to 1000 IU/ml saline and more preferably 100 IU/ml to 500 IU/ml saline.

- the weight of the saline solution used to obtain the hemostatic fluid material is 2 to 10 times the weight of the hemostatic powder, and preferably 4 to 5 times the weight of the hemostatic powder.

- printing step c) is carried out in a natural atmosphere, preferably at normal temperature.

- after printing step c) leading to the formation of the hemostatic product, coating step d) is performed, in which the outer surface of the hemostatic product is coated with a solution comprising a blood clotting factor, in particular thrombin.

- step d) of coating is performed by spraying a solution including a blood clotting factor onto the outer surface of the hemostatic product.

- after performing step c) of printing, leading to the formation of the hemostatic product, step e) soaking is performed, in which the hemostatic product is soaked in a solution including a blood clotting factor, in particular thrombin.

- the last step of manufacturing the hemostatic product consists of keeping the printed hemostatic product in a natural atmosphere for a predetermined holding period, where the predetermined holding period is preferably less than 10 minutes, preferably less than 5 minutes and more preferably less than 1 minute.

- the entire method is performed in a natural atmosphere without any stimulation of the hemostatic device, in particular without photostimulation or thermal stimulation.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Other features and advantages of the invention will become more apparent upon reading the following description, which is given for illustrative and non-limiting purposes, and which is accompanied by the following drawings, in which:

In FIG. 1 is a schematic representation of a process for manufacturing a three-dimensional (3D) hemostatic product using a 3D additive printer in which at least a hemostatic fluid material is used as the printing ink;

In FIG. Figure 2 shows an example of the electrophoresis results discussed in Example 10.

INFORMATION CONFIRMING THE POSSIBILITY OF IMPLEMENTING THE INVENTION

The inventors of the present invention have surprisingly found that a three-dimensional (3D) hemostatic product can be manufactured by using a hemostatic fluid material as a printing ink for a 3D printer, although a hemostatic powder composition obtained from collagen that is not cross-linked.

As shown in the description below, the hemostatic flowable material has a structure that allows the material to be easily applied using a 3D printer and provides high adhesive strength within a product formed from sequential layers deposited on top of each other, naturally bonded to each other.

In the description below, unless otherwise indicated, weight percentages are based on the total dry matter weight of the composition contained in a single article, for example, a hemostatic powder or hemostatic fluid or hemostatic product discussed herein.

In the context of the present invention, the term "total dry matter weight of the hemostatic powder composition" refers to the total weight of the hemostatic powder composition free of solvent, in particular water, and thus the total weight based on the weight of the anhydrous product.

Likewise, in the context of the present invention, the term “total dry matter weight of the hemostatic fluid composition” refers to the total weight of the hemostatic fluid composition free of solvent, particularly water, and thus the total weight based on the weight of the anhydrous product.

Likewise, in the context of the present invention, the term “total dry matter weight of a hemostatic product composition” refers to the total weight of the hemostatic product composition free of solvent, particularly water, and thus the total weight based on the weight of the anhydrous product.

In addition, the weights of the components and the resulting percentages may correspond to the weight of the anhydrous material in those components, in other words, the weight of the component excluding the weight of water that it may contain. This may also refer to percentages (shares) received.

The collagen content in the hemostatic powder composition used to obtain the hemostatic fluid material may be greater than or equal to 70% by weight. of the total weight of the hemostatic powder composition, in particular, greater than or equal to 75% by weight, in particular, greater than or equal to 77% by weight, preferably greater than or equal to 80% by weight.

In addition, the collagen content in the hemostatic powder composition may be less than or equal to 99% by weight. of the total weight of the hemostatic powder composition, in particular, less than or equal to 96% by weight, in particular, less than or equal to 93% by weight, preferably less than or equal to 90% by weight.

Thus, the collagen content in the hemostatic powder composition can range from 70% to 99% by weight. of the total weight of the hemostatic powder composition, in particular from 75% to 96% by weight, in particular from 77% to 93% by weight, preferably from 80% to 90% by weight. Preferably, the collagen content is approximately 86% by weight. from the total weight of the hemostatic powder composition.

Collagen is the main structural protein in the mammalian body. Collagen is composed of tropocollagen (TC) molecules that are approximately 280 to 300 nm in length and approximately 1.5 nm in diameter.

The term "fibrous collagen" refers to collagen in the form of a fiber corresponding to an aggregate of fibrils. The diameter of the fibers is usually from 1 µm to 10 µm. The term "fibrillar collagen" refers to collagen in the form of fibrils. In particular, the fibril diameter is typically between 10 nm and 1 µm. Thus, fibrils are formed from unevenly spaced arrays of tropocollagen molecules, and such fibrils can form collagen fibers. Fibrous and/or fibrillar collagen is usually insoluble, while non-fibrillar collagen is highly soluble.

In particular, the definitions given by Markus Buehler in the publication “Nature designs tough collagen: explaining the nanostructure of collagen fibrils”, PNAS, August 15, can be accepted as definitions of fibrous collagen and fibrillar collagen. 2006, vol. 103, no. 33, pp. 12285-12290.

Currently, more than 28 different types of collagen are known, which are divided into 3 main categories: fibrillar type collagens, non-fibrillar type collagens and FACIT collagens (Fibril Associated Collagens with Interrupted Triple Helices - fibril-associated collagens with discontinuous triple helices).

Fibrillar-type collagens are collagens that primarily include fibrillar and/or fibrous collagens and substantially no non-fibrillar collagens (eg, type I collagen). Likewise, non-fibrillar type collagens are collagens that mainly include non-fibrillar collagens. Some nonfibrillar type collagens may consist only of nonfibrillar collagens (eg, type IV or V collagen).

Industrial extraction and purification of collagen typically consists of the destruction of source tissues to 1) remove all or most contaminating proteins and 2) to achieve the level of structurization required for subsequent use of the finished product. Collagen extraction is usually carried out under acidic or alkaline conditions, which promote the solubilization of non-fibrillar monomolecular soluble collagen. The resulting collagen inevitably contains a mixture of fibrillar/fibrous collagen and non-fibrillar collagen. The ratio of fibrillar/fibrous collagen to non-fibrillar collagen depends on the type of tissue selected for extraction and extraction.

The resulting product differs from collagen obtained by artificially mixing only fibrillar collagen and only non-fibrillar collagen. In an article entitled "Extraction of collagen from connective tissue by neutral salt solutions" (Proceedings of the National Academy of Sciences, Volume 41 Number I, January 15, 1955, Jerome Gross, John H. Highberger, Francis O. Schmitt), shows the differences between fibrillar and non-fibrillar collagen obtained after special extraction, which, as discussed above, leads to the formation of a mixture of these two types of collagen.

The hemostatic powder from which the hemostatic fluid material is obtained contains collagen of the fibrillar type, which includes fibrous and/or fibrillar collagen in an amount of at least 60% by weight, in particular at least 70% by weight, in particular at least at least 75% by weight, preferably at least 80% by weight. of the total mass of collagen.

In particular, collagen includes at least 85%, in particular at least 90%, in particular at least 95%, preferably at least 98% of the mass. fibrous and/or fibrillar collagen from the total mass of collagen in the hemostatic powder composition.

Preferably, the content of fibrous and/or fibrillar collagen in the composition is from 85% to 95% by weight. of the total mass of collagen in the composition, and most preferably from 85% to 90% of the mass.

This means that, therefore, in a preferred embodiment, the content of non-fibrillar collagen in the hemostatic powder composition is from 5% to 15% by weight. of the total mass of collagen in the composition, and most preferably from 10% to 15% of the mass.

Very preferred, in particular for use as a hemostatic powder preparation, is a composition with the specified ratios of fibrous and/or fibrillar collagen to non-fibrillar collagen. Indeed, fibrous and/or fibrillar collagen must be present in sufficient quantity to achieve hemostasis, and non-fibrillar collagen must also be present in sufficient quantity to ensure adhesive strength in the article, but not in too much quantity to avoid excessive swelling.

The collagen may be selected from type I collagens or type I and III collagens. Collagen can be extracted from various tissues, in particular skin (hides) and/or tendons, of any organism, in particular various species of pigs, cows or horses.

Collagen may largely consist of porcine-derived fibrous collagen extracted from skin (hide) and/or tendon. In the case of collagen extracted from tendons, the extraction can be carried out as discussed in international patent application WO 2010/125086.

The above collagen, in particular fibrous and/or fibrillar collagen, can be extracted by acid or alkaline extraction. According to one specific embodiment, collagen is obtained by alkaline extraction. According to one specific embodiment, the collagen may be the collagen disclosed in patent application FR 2944706.

Preferably, the collagen is obtained by alkaline extraction, which allows for maximum fibrous and/or fibrillar collagen content in the extracted collagen. Additionally, the alkaline extraction can be optimized to control the ratio of fibrillar/fibrous collagen to non-fibrillar collagen in the extracted collagen. Unlike acid extraction, alkaline extraction involves hydrolysis of proteoglycans. It leads to tissue destruction and separation of fibers without modifying their shape. Under acidic conditions during the method, swelling of internal collagen molecules in the fibers leads to their partial destruction with the release of large quantities of non-fibrillar soluble collagen.

Collagen can be used in the form in which it is obtained as a result of extraction, i.e. without further processing, or it can be cross-linked, in particular by classical cross-linking methods such as thermal dehydration, the use of cross-linking agents, for example formaldehyde and/or glutaraldehyde, oxidized polysaccharides, for example according to the method discussed in international patent application WO 2010/125086, and/or the use of oxidized amylopectins or glycogen. However, cross-linking of collagen is undesirable because it complicates the manufacturing method but does not necessarily improve the efficiency of hemostasis.

Thus, the collagen added to the composition is preferably not further processed and, in particular, not cross-linked. A clear advantage of using non-cross-linked collagen is the simplification of the manufacturing method.

Including non-cross-linked collagen in a hemostatic fluid that can be used to formulate a hemostatic product also has many advantages as it limits the leaching of chemicals during product degradation, which can increase the toxicity of the product. This also speeds up the resorption of the finished product and eliminates the formation of toxic intermediate products during resorption.

The hemostatic powder composition used to obtain the hemostatic fluid material further includes at least one monosaccharide, alone or in mixture with other monosaccharides. Monosaccharides may be selected from ribose, sucrose, fructose, glucose and mixtures thereof. The monosaccharide present in the hemostatic composition, either alone or in mixture with monosaccharides, is in particular glucose.

The monosaccharide content in the hemostatic powder composition can range from 1% to 12.5% by weight. of the total weight of the composition, in particular from 1.5% to 10% by weight, in particular from 2% to 8% by weight. and especially preferably from 2.5% to 7.5% by weight. Most preferably, the monosaccharide content is approximately 5% by weight. from the total mass of the composition.

The collagen/monosaccharide weight ratio in the hemostatic powder composition may be from 5 to 100, in particular from 7 to 65, in particular from 10 to 50 and more preferably from 11 to 40. Most preferably, the collagen/monosaccharide weight ratio in the composition is approximately 19.

Monosaccharide, in particular ribose, sucrose, fructose, glucose and mixtures thereof, and in particular glucose, contributes to the production of particles, mainly comprising fibrous and/or fibrillar collagen and monosaccharides with the desired characteristics, in particular with the desired size and density . The addition of a monosaccharide to the collagen mixture further reduces the electrical charges in the composition, resulting in a powder suitable for placement in containers such as tubes, pneumatic delivery devices, dispensing devices for spraying or application. This also facilitates the formation of a hemostatic fluid when mixing the hemostatic powder with a saline solution, and the resulting hemostatic fluid has properties particularly suitable for its use as a printing ink for a 3D additive printer.

Thus, the presence of a monosaccharide makes it easier and/or cheaper to obtain particles of the required density and/or size, in particular, with the aim of improving the hemostatic properties of the powder composition.

Grinding collagen fibers in the absence of additives reduces the size of the fibers and reduces the density of the powder. In addition, the finished product has a significant electrical charge, which prevents operations with the finished product. Adding a monosaccharide before grinding the collagen causes the preparation to harden into a mixture that can be quickly ground (limiting denaturation), resulting in a powder with a reduced electrical charge (suitable for placing the powder in containers such as dispensing devices) and a final density suitable for application and restoration of the composition. As noted above, this also makes it possible to obtain from the hemostatic powder hemostatic fluid materials having certain properties, preferably suitable for additive printing, in particular, suitable in particle size and density, as well as the rheology of the hemostatic fluid material.

Contrary to what might be expected, this addition of a monosaccharide does not affect the activity of the finished product. In particular, the addition of a monosaccharide does not change the bioactivity of the finished product. The monosaccharide does not have a hemostatic effect.

Moreover, the added monosaccharide does not act as a blowing agent, as shown in WO 01/97873. According to WO 01/97873, heating the dilute solution results in the formation of gelatin. High concentrations of gelatin can be added to produce a very concentrated solution, but in this case the finished product contains gelatin rather than collagen. It is known that gelatin has less hemostatic activity than collagen, since platelet aggregation requires the presence of collagen fibrils and the structure of natural collagen, which is absent in gelatin.

According to one embodiment, the composition comprises, preferably consists of particles comprising, preferably consisting of collagen and a monosaccharide, in particular selected from ribose, sucrose, fructose, glucose and mixtures thereof, in particular glucose.

The composition may include at least one blood clotting factor. Blood clotting factors are well known to those skilled in the art. Preferably, one of the coagulation factors is thrombin. More preferably, the hemostatic powder composition includes only thrombin as a blood coagulation factor.

Clotting factors, particularly thrombin, can be obtained from animal sources (isolated from animal tissues and body fluids) or from recombinant sources (produced by cultures of genetically modified cells). The coagulation factor may, for example, be thrombin isolated from human tissues and body fluids.

If a blood coagulation factor, in particular thrombin, is present, its content is preferably less than 0.5% by weight. from the total weight of the hemostatic powder composition, preferably less than 0.1% of the mass.

In the case of thrombin, international units (IU) are usually used. Thus, the thrombin content of the composition may be from 0.01 IU/mg to 20 IU/mg of the composition, in particular from 0.05 IU/mg to 10 IU/mg, in particular from 0.1 IU/mg to 5 IU/mg, preferably 0.2 IU/mg to 2 IU/mg. Most preferably, the thrombin content - if present - is approximately 0.83 IU/mg of the composition.

In addition to the mixture of collagen and monosaccharide, the composition may include at least one other carbohydrate compound, which may be a glycosaminoglycan. The carbohydrate compound may be part of a composition with or without a clotting factor such as thrombin.

The glycosaminoglycan may be selected from chondroitin sulfates, dermatan sulfate, hyaluronic acid and mixtures thereof, in particular chondroitin sulfates.

Glycosaminoglycans may help increase the rate of blood absorption of the hemostatic composition. In particular, glycosaminoglycans can accelerate the contact between blood and hemostatic materials, in particular collagen and thrombin.

The glycosaminoglycan content in the composition can range from 1% to 30% by weight. of the total weight of the composition, in particular from 2% to 25% by weight, in particular from 3% to 20% by weight, in particular from 4% to 15% by weight, preferably from 5% to 12.5% by weight. Most preferably, the glycosaminoglycan content - if present - is approximately 9% by weight. from the total mass of the composition.

The collagen/glycosaminoglycan weight ratio in the composition can be from 2.5 to 50, in particular from 3.5 to 35, in particular from 5 to 25 and preferably from 6.5 to 20.

According to one embodiment, the composition comprises at least one, in particular one, monosaccharide and at least one, in particular one, glycosaminoglycan, in particular as defined above, and in particular in the amounts indicated higher.

The carbohydrate compounds are preferably monosaccharides and glycosaminoglycans.

The carbohydrate content in the composition can range from 2% to 25% by weight. of the total weight of the composition, in particular from 5% to 23% by weight, in particular from 7% to 21% by weight, preferably from 10% to 18% by weight.

The weight ratio of collagen/carbohydrate compound in the composition can be from 2 to 40, in particular from 2.5 to 30, in particular from 3 to 20 and preferably from 3.5 to 15.

The expression "total mass of carbohydrate compounds" refers to the total mass of the above-mentioned monosaccharides and the mass of other carbohydrate compounds mentioned above.

According to one embodiment, the composition includes, preferably consists of:

- collagen, mainly including fibrous and/or fibrillar collagen, in which the content of fibrous and/or fibrillar collagen is at least 50% by weight. of the total mass of collagen, and

- at least one, in particular one monosaccharide.

Preferably the composition includes, preferably consists of:

- collagen, in particular in an amount ranging from 70% to 99% of the mass. of the total weight of the composition, in particular in an amount ranging from 75% to 96% by weight, in particular in an amount ranging from 77% to 93% by weight, preferably in an amount ranging from 80% to 90% by weight. , where the content of fibrous and/or fibrillar collagen in the collagen is at least 50% of the mass. of the total mass of collagen, and

- at least one monosaccharide, in particular glucose, in an amount ranging from 1% to 12.5% by weight. of the total weight of the composition, in particular from 1.5% to 10% by weight, in particular from 2% to 8% by weight. and preferably from 2.5% to 7.5% by weight.

According to another embodiment, the composition includes, preferably consists of:

- collagen, mainly including fibrous and/or fibrillar collagen, where the content of fibrous and/or fibrillar collagen in the collagen is at least 50% by weight. from the total mass of collagen;

- at least one, in particular one monosaccharide;

- at least one, in particular one blood coagulation factor.

Preferably, the composition includes, preferably consists of:

- collagen, in particular in an amount ranging from 70% to 99% of the mass. of the total weight of the composition, in particular in an amount ranging from 75% to 96% by weight, in particular in an amount ranging from 77% to 93% by weight, preferably in an amount ranging from 80% to 90% by weight. , where the content of fibrous and/or fibrillar collagen in the collagen is at least 50% of the mass. from the total mass of collagen;

- at least one monosaccharide, in particular glucose, in an amount ranging from 1% to 12.5% by weight. of the total weight of the composition, in particular in an amount of from 1.5% to 10% by weight, in particular in an amount of from 2% to 8% by weight, and preferably in an amount of from 2.5% up to 7.5% wt., and

- at least one, in particular one blood coagulation factor, in particular thrombin, in an amount ranging from 0.01 IU/mg to 20 IU/mg of the composition, in particular from 0.05 IU/mg to 10 IU /mg, in particular from 0.1 IU/mg to 5 IU/mg, preferably from 0.2 IU/mg to 2 IU/mg.

According to another embodiment, the composition includes, preferably consists of:

- collagen, mainly including fibrous and/or fibrillar collagen, where the content of fibrous and/or fibrillar collagen in the collagen is at least 50% by weight. from the total mass of collagen;

- at least one, in particular one monosaccharide, and

- at least one, in particular one glycosaminoglycan.

Preferably, the composition includes, preferably consists of:

- collagen, in particular in an amount ranging from 70% to 99% of the mass. of the total weight of the composition, in particular in an amount of from 75% to 96% by weight, in particular in an amount of from 77% to 93% by weight, preferably from 80% to 90% by weight, where the content fibrous and/or fibrillar collagen in collagen is at least 50% of the mass. from the total mass of collagen;

- at least one monosaccharide, in particular glucose, in an amount ranging from 1% to 10% by weight. of the total weight of the composition, in particular in an amount of from 1% to 12.5% by weight, in particular in an amount of from 1.5% to 10% by weight, in particular in an amount of from 2 % to 8% by weight, and preferably in an amount ranging from 2.5% to 7.5% by weight, and

- at least one glycosaminoglycan, in particular chondroitin sulfate, in an amount ranging from 1% to 30% of the mass. of the total weight of the composition, in particular in an amount of from 2% to 25% by weight, in particular in an amount of from 3% to 20% by weight, in particular in an amount of from 4% to 15% wt., preferably in an amount ranging from 5% to 12.5% wt.

According to another embodiment, the composition includes, preferably consists of:

- collagen, in which the content of fibrous and/or fibrillar collagen is at least 50% by weight. from the total mass of collagen;

- at least one, in particular one monosaccharide;

- at least one, in particular one blood clotting factor, and

- at least one, in particular one glycosaminoglycan.

Preferably, the composition includes, preferably consists of:

- collagen, in particular in an amount ranging from 70% to 99% by weight, in particular in an amount ranging from 75% to 96% by weight, in particular in an amount ranging from 77% to 93% by weight. , preferably in an amount ranging from 80% to 90% of the mass. from the total weight of the composition, in particular, from the weight of dry matter in the composition, where the content of fibrous and/or fibrillar collagen in the collagen is at least 50% of the mass. from the total mass of collagen;

- at least one monosaccharide, in particular glucose, in an amount ranging from 1% to 10% by weight. of the total weight of the composition, in particular in an amount of from 1% to 12.5% by weight, in particular in an amount of from 1.5% to 10% by weight, in particular in an amount of from 2 % to 8% by weight, and preferably in an amount ranging from 2.5% to 7.5% by weight;

- at least one blood coagulation factor, in particular thrombin, in an amount ranging from 0.01 IU/mg to 20 IU/mg of the composition, in particular from 0.05 IU/mg to 10 IU/mg, in particular , from 0.1 IU/mg to 5 IU/mg, preferably from 0.2 IU/mg to 2 IU/mg, and

- at least one glycosaminoglycan, in particular chondroitin sulfate, in an amount ranging from 1% to 30% of the mass. of the total weight of the composition, in particular in an amount of from 2% to 25% by weight, in particular in an amount of from 3% to 20% by weight, in particular in an amount of from 4% to 15% wt., preferably in an amount ranging from 5% to 12.5% wt.

According to one preferred embodiment, the composition includes, preferably consists of:

- fibrillar type collagen, mainly including fibrous and/or fibrillar collagen, where fibrillar type collagen is obtained, for example, by extraction in an alkaline medium and is contained in an amount of approximately 85% of the mass. from the total weight of the composition;

- glucose in an amount of approximately 4.9% by weight. from the total weight of the composition;

- thrombin in an amount ranging from 0.2 IU/mg to 2 IU/mg of the composition; And

- chondroitin sulfate in an amount of approximately 10% of the mass. from the total mass of the composition.

According to another specific embodiment, the composition includes, preferably consists of:

- fibrillar type collagen, mainly including fibrous and/or fibrillar collagen, where fibrillar type collagen is obtained, for example, by extraction in an alkaline medium and is contained in an amount of approximately 85% of the mass. from the total weight of the composition;

- glucose in an amount of 5% by weight. from the total weight of the composition, and

- chondroitin sulfate in an amount of 10% of the mass. from the total mass of the composition.

In the context of the present description, the expression "an amount of approximately X%" refers to a value that differs from the quoted value by 20%, either less (minus) or more (plus); in other words, an amount of approximately 10% means 8% to 12%, in particular the difference is plus or minus 10%, preferably plus or minus 5%.

When adding a powdered coagulation factor, in particular thrombin, the powdered coagulation factor is preferably mixed with the already prepared powdered homogeneous molecular mixture of collagen/monosaccharide.

If both a glycosaminoglycan (eg chondroitin sulfate) and a clotting factor (eg thrombin) are added, they are preferably mixed together first and the resulting mixture is added to the pre-prepared collagen/monosaccharide mixture (already ground into powder).

Thrombin is not stabilized by either carbohydrate or collagen. Thrombin is never in contact with the monosaccharide solution (unlike described in WO 98/57678), which prevents protein denaturation and rehydration of the powder, resulting in the powder not being able to completely re-dry.

The composition in powder form may in particular include or consist of:

- particles comprising or consisting of collagen of the fibrillar type and at least one monosaccharide, in particular glucose, wherein, in particular, said particles have the size, particle size distribution and/or density specified in the present description, and

- optionally particles comprising or consisting of at least one saminoglycan, in particular chondroitin sulfates, and/or at least one coagulation factor, in particular thrombin, where in particular said particles have a size, particle size distribution and/or density, specified in this description.

As stated above, the advantage of the hemostatic powder according to the invention is the possibility of grinding adapted to the use of the hemostatic powder, in particular grinding to form particles of small sizes, in particular less than 500 μm, more preferably less than 350 μm.

For example, the hemostatic powder composition may preferably include at least 50% by weight. particles whose size ranges from 200 microns to 400 microns.

The average particle size distribution of the particles constituting the hemostatic powder is preferably from 10 μm to 500 μm, in particular from 50 μm to 400 μm.

Preferably at least 90% by weight, in particular 100% by weight. The particles making up the hemostatic powder can pass through a sieve with a mesh size of 500 microns, in particular 400 microns.

At least 90% of the mass. and, in particular, at least 95% of the mass. The particles constituting the hemostatic powder can be retained by a sieve with a mesh size of 10 μm, in particular 20 μm, preferably 30 μm, preferably 50 μm. For example, at least 90% of the mass. and preferably 90% wt. The particles that make up the hemostatic powder have a size exceeding 20 microns and can be retained by a sieve with a mesh size of 20 microns.

According to one specific example, a hemostatic powder composition is prepared, which includes at least 50% of the mass. particles whose size is less than 200 microns, 90% of the mass. particles whose size is less than 350 microns, and 98% of the mass. particles whose size is less than 400 microns. In this case, the particles constituting the hemostatic powder preferably have an average particle size distribution in the range of 20 μm to 300 μm, preferably from 25 μm to 270 μm. For example, 80% wt. The particles that make up the hemostatic powder have a granulometric composition in the range from 20 microns to 300 microns. This particular example of a hemostatic powder is particularly preferred for preparing a hemostatic fluid material that is used in a 3D additive printer, discussed in more detail below.

Re-separation of the powder is preferably chosen so that the powder can be hydrated. If the particle size of the powder is too small, then the powder will not form a hydrated matrix that satisfies the required properties and aspects.

The hemostatic composition in powder form, in particular, includes:

- particles including collagen and monosaccharide, and

- optionally at least a glycosaminoglycan and/or a blood clotting factor such as thrombin.

The hemostatic powder composition may include:

- particles comprising collagen, a monosaccharide and optionally at least one glycosaminoglycan and/or coagulation factor;

- particles comprising collagen, a monosaccharide, optionally a blood clotting factor and optionally glycosaminoglycan particles;

- particles including collagen and a monosaccharide, and particles including at least one glycosaminoglycan and/or coagulation factor.

In the context of the present invention, the expression "dry powder" means that the composition includes a limited content of solvent, in particular water. Such limited content may be less than 5% by weight, in particular less than 3% by weight. and preferably less than 1 wt%. from the total mass of the composition.

Such a dry form can be obtained by simple evaporation of the solvent used, dehydration under the influence of organic solvents.

As stated above, the hemostatic powder discussed is prepared from non-cross-linked collagen because its use simplifies the manufacturing process and has been shown to have high hemostatic efficacy despite the fact that the collagen contained in the powder is not cross-linked.

The authors of the present invention unexpectedly discovered that, despite the fact that the collagen was not cross-linked, mixing the hemostatic powder discussed above with saline solution allows to obtain a hemostatic product, the rheological properties of which, in particular viscosity, allow it to be used for the formation of complex 3D hemostatic product, using, for example, a hemostatic fluid material as a starting material, sequentially deposited in the form of layers using a 3D additive printer.

This finding was unexpected because, on the contrary, it was known that cross-linked collagen was required to obtain a working and stable hemostatic collagen paste from a mixture of collagen powder and saline solution. In particular, this was stated in the US patent published on January 2, 1990 under the number US 4891359. Indeed, it is known that the formation of cross-links provides stability to molecules due to additional chemical bonds in the molecular structure, and such additional chemical bonds are usually required for translation molecules into an aqueous solution (transition to a hydrated form).

It was also not expected that the hemostatic fluid material could be directly introduced into a 3D printer to produce a 3D hemostatic product without the need to adapt the printing method of traditional 3D additive printers, in particular extrusion 3D printers.

Accordingly, according to one preferred embodiment, the dry hemostatic powder discussed above, in which the collagen is not cross-linked, is thus subjected to hydration, for example by adding saline or water, resulting in the formation of a hemostatic fluid that is used as a printed material. inks for 3D printer.

As used herein, the term "flowable material" refers to compositions whose consistency, in the absence of deforming force, allows the composition to maintain some shape, but under the influence of a deforming force, such as pressure, the composition is deformed. The minimum shear stress required to initiate flow of the product corresponds to the lower yield strength.

The flowable material is neither a liquid, nor a sponge, nor a powder, but rather is some kind of paste, gel or matrix having a certain viscosity, which depends on the voltage applied to the flowable material.

Preferably, the viscosity of the flowable material is from 20 Pa⋅s to 10,000 Pa⋅s (corresponding to a yield range of 0.0001 (Pa⋅s) ^-1 to 0.05 (Pa⋅s) ^-1 ) at no or low applied stress. voltage (for example, less than 10 Pa). However, at higher voltages, the flowable material may have a higher viscosity, for example, from 1000 Pa.s to 1,000,000 Pa.s.

The term "flowable" refers to a composition that, for example, can be passed through a syringe and/or tube. Preferably, it can also be passed through the nozzle of the 3D printer head, as discussed below.

As used herein, the terms “hemostatic fluid,” “fluid hemostat,” and “hemostatic matrix” are used interchangeably and refer to the same specific composition.

Thus, to obtain the hemostatic fluid material discussed above, the hemostatic powder and saline solution are mixed, during which the hemostatic powder is suspended in water or saline solution.

The hemostatic powder used to make the hemostatic fluid material can, for example, be produced by a process that includes at least the following steps:

a) forming an aqueous suspension comprising, preferably consisting of fibrillar type collagen, generally including fibrous and/or fibrillar collagen, and a monosaccharide such as glucose;

b) recovering the product in the form of a sediment, paste or gel, in particular by centrifugation or decantation;

c) drying the product, for example by evaporation;

d) grinding the product to the required particle size, in particular in a hammer mill; And

e) optionally adding thrombin and/or chondroitin sulfates, in particular in solid form, in particular in powder form.

The formation in step a) of an aqueous suspension comprising fibrous/fibrillar collagen and a monosaccharide results in a homogeneous distribution of the monosaccharide around the collagen molecules. In addition, the close contact between the molecular particles of collagen and the monosaccharide leads, after dehydration, to the formation of a solid precipitate suitable for obtaining a powder with the required high density after grinding. In contrast, mixing collagen powder and glucose powder does not result in a homogeneous and sprayable powder, particularly due to density and electrical charge limitations.

In step a) collagen may be present in a concentration ranging from 30 g/l to 150 g/l.

The monosaccharide may be added to the suspension or homogeneous collagen paste in the amount specified in the description, in particular ranging from about 2% to 5% by weight. from the mass of collagen.

In step a) the monosaccharide may be present in a concentration ranging from 0.3 g/L to 10 g/L.

The aqueous collagen suspension of step a) may be acidic and in particular include an acid such as hydrochloric acid. The acid may be present in a concentration of from 0.01 M to 0.5 M, and in particular from 0.02 M to 0.1 M, preferably about 0.05 M. The suspension may be a homogeneous paste.

Step b) may involve pouring the slurry into the mold.

Step c) is carried out in such a way as to obtain the densest possible precipitate (preferably with the best possible final particle size) with a very high density and the minimum possible number of air bubbles (less than 5%) within the precipitate.

After step d) a powder sieving step can be carried out, in particular to obtain particles of the required size.

According to one of the preferred embodiments, step a) consists of the formation of a mixture comprising 95% of the mass. non-cross-linked fibrillar type collagen and 5% wt. monosaccharide (for example, glucose).

As mentioned above, a glycosaminoglycan (eg, chondroitin sulfate) can be added to the mixture formed by non-cross-linked fibrillar type collagen and a monosaccharide. Preferably, the glycosaminoglycan is added to the mixture in a mixture/glycosaminoglycan weight ratio of 70/30 to 100/0, preferably in a weight ratio of 80/20 or 85/15 or 90/10 or 95/5. The indicated mass ratios can be applied to mixtures of any type formed by non-cross-linked fibrillar type collagen and a monosaccharide.

For example, with a mass ratio of 95/5, the hemostatic powder composition may include 95 wt%. a mixture obtained from 95% collagen and 5% glucose, and 5% wt. chondroitin sulfate. Thus, the hemostatic powder includes 90.25% of the mass. collagen and 4.75% wt. glucose and 5% wt. chondroitin sulfate.

Alternatively, with a weight ratio of 85/15, the hemostatic powder composition may include 85% wt. a mixture obtained from 95% collagen and 5% glucose, and 15% wt. chondroitin sulfate. Thus, the hemostatic powder includes 80.75% of the mass. collagen, 4.25% wt. glucose, and 15% wt. chondroitin sulfate.

Alternatively, at a weight ratio of 80/20, the hemostatic powder composition may include 80% wt. a mixture obtained from 95% collagen and 5% glucose, and 20% wt. chondroitin sulfate. Thus, the hemostatic powder includes 76% of the mass. collagen, 4% wt. glucose, and 20% wt. chondroitin sulfate.

Alternatively, at a weight ratio of 90/10, the hemostatic powder composition may include 90 wt%. a mixture obtained from 95% collagen and 5% glucose, and 10% wt. chondroitin sulfate. Thus, the hemostatic powder includes 85.5% of the mass. collagen, 4.5% wt. glucose, and 10% wt. chondroitin sulfate.

According to one specific example, the hemostatic powder composition used to obtain the hemostatic fluid material includes chondroitin sulfate, the content of which is 10 wt%. from the total mass of the mixture, and the finished composition includes:

- collagen: 86.36% wt. from the total weight of the composition;

- glucose: 4.54% wt. from the total weight of the composition;

- chondroitin sulfate: 9.09% wt. from the total mass of the composition.

If thrombin is also added, then its content in the finished composition is less than 0.01% by weight. from the total mass of the composition. In the above mixture, thrombin may be present in an amount of 0.083 IU/mg of the composition.

Greater or smaller amounts may be added to all of the above powder products during the grinding process to produce a powder containing various particle sizes appropriate to the type of grinding and duration thereof.

As stated, the hemostatic powder is then suspended in saline. The saline solution is preferably standard sterile saline solution used in operating rooms.

The brine may contain chloride salts of monovalent or divalent cations, such as calcium chloride or sodium chloride, at a concentration of 0 to 300 mM, preferably at a concentration of 100 to 200 mM, in particular at a concentration of about 150 mM.

Preferably the solution consists of distilled water containing sodium chloride in an amount of from 0.5% to 1.5% and preferably about 0.9%.

The saline solution is preferably pure, that is, it consists of a mixture of sodium chloride and distilled water without the addition of any other components.

Alternatively, the saline solution may include a clotting factor such as thrombin. In this case, the hemostatic powder mixed with the saline solution preferably does not include a blood clotting factor.

For example, the clotting factor is contained in an amount of 10 IU/ml to 5000 IU/ml saline, preferably in an amount of 25 IU/ml to 2500 IU/ml saline, more preferably in an amount of 50 IU/ml to 1000 IU /ml saline and more preferably in an amount of from 100 IU/ml to 500 IU/ml saline.

The saline solution can be stored in various forms, such as one volume in a large container or in a special container of a certain volume.

Preferably, the saline solution is part of a kit for preparing the hemostatic fluid material, and such kit also includes a certain amount of hemostatic powder in the container.

The hemostatic fluid composition obtained from the hemostatic powder may include dried non-cross-linked fibrillar collagen, the content of which ranges from 60% to 99% by weight. of the total weight of the composition, in particular from 75% to 90% of the mass. and preferably from 83% to 88% of the mass.

The hemostatic fluid composition obtained from the hemostatic powder may include a monosaccharide, in particular glucose, the content of which is from 1% to 10% by weight. of the total weight of the composition, in particular from 3% to 7% of the mass. and preferably from 4% to 5% by weight.

The hemostatic fluid composition obtained from the hemostatic powder may include glycosaminoglycan (lyophilized particles), in particular chondroitin sulfate, the content of which ranges from 1% to 30% by weight. of the total weight of the composition, in particular from 5% to 12% of the mass. and preferably from 8% to 10% by weight.

The hemostatic fluid composition obtained from the hemostatic powder may include a blood clotting factor, preferably thrombin, the content of which is less than 1% by weight. from the total mass of the composition. Preferably, if a blood clotting factor is present, its content is preferably less than 0.5% by weight. based on the total weight of the composition, preferably less than 0.1 wt%.

Optionally, the hemostatic fluid composition derived from the hemostatic powder may include a cross-linking initiator, particularly oxidized glycogen or retinol, or a photoinitiator. Such initiators are not necessary for the manufacture of a hemostatic product; on the contrary, they are used at later stages for the functionalization of the hemostatic device. The cross-linking initiator or photoinitiator is selected to allow the reaction in the hemostatic device to occur only when subjected to a specific stimulation. In particular, the cross-linking initiator does not cause spontaneous cross-linking - the cross-linking initiator must be activated.

Hemostatic fluid material obtained from hemostatic powder mixed with a certain amount of saline solution can be used as a hemostatic agent.

The hemostatic fluid material can also be used as a pharmaceutical composition, in particular a hemostatic drug.

As already noted, the hemostatic fluid material is suitable for the manufacture of a 3D hemostatic product and can preferably be directly fed into a 3D printer.

Preferably, the hemostatic fluid material is suitable for use as a raw material for the production of printing ink for any type of 3D printer, in particular a so-called bioprinter, for example 3D printers operating on the principle of extrusion of feed material.

In the case where the 3D printer includes multiple print heads, typically multiple printing inks of different compositions are used, and at least one of them is the hemostatic fluid material of the invention. In one particular case, several hemostatic fluids of different compositions are sent to a 3D printer to be used as printing inks.

Some information about bioprinters can be found in the article by Ibrahim T. Ozbolat and Monika Hospodiuk, entitled “Current advances and future perspectives in extrusion-based bioprinting,” which was published in the 2015 issue of Biomaterials (see Biomaterials 76 (2016) 321-343), the contents of which are incorporated herein by reference in their entirety.

Prior to preparing the hemostatic fluid for use in a 3D printer, all active components may be contained together in powder form in a specific container. This is very beneficial in several ways. First, it makes the product easier to store, since it only requires careful handling of the container containing the hemostatic powder and not the saline solution, which is a commonly available product.

It is also very important for the production process that only the hemostatic powder needs to be sterilized before storage, which, for example, is not the case if some components are first mixed with saline (eg thrombin) and then mixed with the hemostatic powder.

It should be noted that if a clotting factor such as thrombin is not added to the hemostatic powder or saline solution, then the clotting factor should be added after the hemostatic product is completed.

To do this, the hemostatic product can, for example, be soaked in a solution containing a blood clotting factor.

The solution including the coagulation factor can also be used to coat the outer surface of the hemostatic device. Such a coating can, for example, be obtained by spraying a solution onto the outer surface of a hemostatic device.

Optionally, as will be discussed below, the hemostatic product produced by 3D printing can also be sterilized upon completion of the manufacturing process, and thus the hemostatic powder does not need to be sterilized.

The container may be in the form of a cartridge, which is directly suitable for use in a 3D printer.

Preferably, when producing a hemostatic fluid material from which a 3D hemostatic product is manufactured using a 3D additive printer, the weight of the saline solution used to hydrate the hemostatic composition is 2 to 10 times the weight of the hemostatic powder, preferably 4 to 5 times the weight of the hemostatic powder .

The hemostatic fluid material of the invention has the advantage of having rheological properties that make it suitable for bioprinting.

In particular, the hemostatic fluid particles are small enough to pass through the print head of the printer without clogging the printer nozzle (typically less than 400 microns in size).

Preferably, the lower yield limit of the hemostatic fluid material is from 500 Pa to 20000 Pa, preferably less than 15000 Pa, more preferably from 1000 Pa to 3000 Pa.

The amount of hemostatic powder in the printing ink cartridge can be from 1 g to 2 g.

The amount of saline solution in the printing ink cartridge is 4 ml to 10 ml, more preferably 5 ml to 10 ml.

According to one preferred example, the ink in the ink cartridge is a hemostatic fluid prepared from 1.65 g of hemostatic powder mixed with 7 ml of pure saline solution.

Printing ink cartridges having a larger volume may be considered (particularly if the 3D printed hemostatic product has a large volume), but the ratio between the amount of hemostatic powder and the amount of saline forming the hemostatic fluid should preferably remain as specified higher. For example, an ink cartridge includes a hemostatic fluid made from 16.5 g of hemostatic powder mixed with 70 ml of pure saline solution.

When adding saline solution to a container containing hemostatic powder, the container is preferably rotated, for example, about its axis to facilitate the introduction of saline solution into the hemostatic powder. If the user administers the saline solution manually, rotation of the container can also be done manually. If necessary, however, the method can be automated.

Tapping and/or gently shaking the container while adding the saline solution is also helpful in introducing the saline solution into the hemostatic powder.

After adding the saline solution to the container, the container is turbulent and shaken to mix the hemostatic powder with the saline solution. Turbulation can be accomplished by mixing the components with a spatula or by shaking.

Turbulization is preferably carried out for a period of from 5 seconds to 60 seconds, more preferably for at least 30 seconds, for example 20 seconds. However, a shaking duration of 10 seconds to 30 seconds, for example 20 seconds, is sufficient to hydrate the hemostatic powder.

Shaking is preferably done manually, but it can also be automated.

Stirring or shaking by hand may involve moving the container up and down several times. For example, the dispensing device may be moved up and down at least 10 to 30 times, preferably 20 times. To improve mixing efficiency, the dosing device can also be inverted and then moved up and down several times. In this second manual mixing phase, the dosing device 1 can also be moved up and down at least 10 to 30 times, preferably 20 times.

After shaking, the container containing the resulting hemostatic fluid is preferably left static for at least 30 seconds, preferably at least 60 seconds, and more preferably at least 90 seconds.

The most likely duration of holding in a static state is from 30 seconds to 120 seconds, preferably about 90 seconds.

The period of holding in a static state allows hydration of the hemostatic powder to occur and its initial swelling, which leads to the formation of a hydrated hemostatic fluid material.

The hemostatic fluid material can be prepared at room temperature and allowed to stand for a period of time ranging from one minute to several hours. Maximum swelling can be achieved in less than 5 minutes.

The above production of hemostatic fluid material can be performed directly in the cartridge that is installed in the bioprinter, or in an intermediate container, before transferring the hemostatic fluid material to the printing cartridge.

The advantage of the hemostatic fluid material thus obtained is homogeneity. In particular, in the container, the hemostatic fluid material has a substantially uniform fluidity. This is particularly advantageous because the result is that the hemostatic fluid material will be deposited from the container with the same characteristics both at the beginning of the operation, when there is a lot of material, and at the end, when there is little material left.

After obtaining the hemostatic fluid material by hydrating the hemostatic powder under the action of a saline solution, the hemostatic fluid material is suitable for use for several hours, for example at least 8 hours, without loss of properties or technical characteristics.

In FIG. 1 schematically shows the production of a 3D hemostatic product using a 3D additive printer 20, the design of which only shows the deposition nozzle 21 and the container body 22 with printing ink.

Printing is preferably carried out in a natural atmosphere, at room temperature.

The advantage of this printing method is that the 3D hemostatic product can be applied, for example, directly in the operating room. Of course, printing can also be carried out in any other room, in particular outside the operating room, and independently of other surgical operations.

Alternatively, the 3D hemostatic product can be directly printed onto the surface of the body of the patient being treated, for example, directly onto a bleeding area or bleeding wound of the patient undergoing surgery.

However, the hemostatic product does not necessarily need to be printed directly on the surface of the body of the patient being treated. The printing can be performed in the absence of the patient, not on the patient's body, in particular, outside the operated area of the patient's body.

The nozzle 21 of the 3D printer 20 serves to deposit the hemostatic fluid material 23 from the ink container 22 onto the supporting surface 24 and form subsequent layers 11 one on top of the other to form a 3D hemostatic product 10.

The 3D printer is adjusted to spatially structure the starting material, including and preferably consisting of a hemostatic fluid material, which is formed by computerized layer deposition, resulting in the formation of a 3D hemostatic product 10.

Preferably, the 3D hemostatic product to be manufactured has a shape that can be determined by a 3D model that is provided as input to the 3D additive printer 20.

The 3D additive printer 20 typically includes a processing device configured to process the 3D model to determine a printable template including a plurality of layers designed to be stacked on top of each other to form a stack of layers corresponding to the 3D model. Such a printing template also depends on the type of source material used as the printing ink and the nature of the 3D hemostatic product being manufactured.

The simplest 3D hemostatic product is preferably produced from a single material, i.e. a hemostatic fluid material arranged in a stack of layers that are naturally connected to each other (layers are said to add to one another). The phrase "naturally connected to each other" implies that there is no need for additional active processing to connect two adjacent layers to each other, in particular, there is no need for cross-linking.

The layers, printed one on top of the other, are connected to each other through self-assembly. There are no cross-links or covalent bonds between the layers.

Preferably, the hemostatic fluid material of the invention used to print the hemostatic product has a high concentration of polymers (due to the concentration of collagen) that promote its self-assembly.

In addition, the hemostatic fluid material according to the invention mainly consists of non-cross-linked fibrillar type collagen, which further promotes self-assembly.

Finally, the hemostatic fluid material according to the invention has thixotropic properties that promote self-assembly, that is, it is particularly promising for the manufacture of products by printing.

When the bioprinter applies a force greater than the lower yield strength of the material to the hemostatic fluid material of the invention, the viscosity of the hemostatic fluid material is reduced, allowing the printing process to occur. This phenomenon is called shear liquefaction.

After shear stress is applied by the printer, the thixotropic properties of the hemostatic fluid material of the invention allow it to return to its original, uncompressed, more viscous and stable state after a certain time, long enough for the printing method to apply another layer to the first layer and at the same time allowing the materials from which it is produced these layers can be mixed, but at the same time short enough for quick adhesion of the layers and stabilization of the product obtained by the printing method.

Preferably, after settling, the hemostatic fluid material of the invention returns to its original undeformed state within a time of less than 10 minutes, preferably less than 5 minutes, more preferably less than 1 minute or less than 30 seconds.

The invention also includes a 3D hemostatic product in which only one of the outer layers of the stack of layers or both outer layers (top layer and bottom layer) are obtained by deposition of hemostatic fluid material, while some or all of the intermediate layers contained between the outer layers are obtained from another material, which is also adapted for application using a 3D printer.

For example, the invention may include 3D printing an artificial body part (e.g., part of a tendon, cartilage tissue, meniscus, intervertebral disc, etc.), which is coated with a hemostatic fluid material to facilitate implantation of the product into the patient's body and corresponding healing process.

To ensure that not only the top and bottom layers of the 3D product have hemostatic properties (which happens in cases where only these outer layers are made of a hemostatic flowable material), printing can be carried out in such a way that each layer is formed from several printing inks. In this way, it is possible to produce intermediate layers, the peripheral part of which is made of a hemostatic fluid material. This method of additive printing makes it possible to obtain a 3D product that has a shell (outer walls) with hemostatic properties, while the central part of the 3D product can be made from a different material.

According to the invention, 3D hemostatic products can also be obtained from various printing inks formed from hemostatic fluid materials having different compositions. This would be useful, for example, in the production of multilayer products comprising layers with different hemostatic properties.

Preferably, the 3D additive printer 20 may be an extrusion printer equipped with a plurality of dispensing nozzles, with each dispensing nozzle dispensing a specific printing ink. This makes it possible to conveniently 3D print a hemostatic product consisting of different layers, each of which can be composed of specific compositions contained in different printing cartridges. In particular, one layer may include several parts, each of which is printed with a certain printing ink, in particular, a certain hemostatic flowable material.

In addition, each layer of the 3D hemostatic product can be produced according to a different pattern, and the overlapping layers can be printed in different orientations and patterns.

The 3D hemostatic product 10 can be obtained by superimposing two-dimensional (2D) layers on each other.

As stated above, the 2D layer can be made from hemostatic fluid materials of different compositions, from which different parts of the layer are obtained.

Suitably, by forming multiple pieces of multiple hemostatic fluid materials having different hemostatic abilities, a 2D layer characterized by a hemostatic gradient can be formed.

As stated above, 2D layers can be printed in various patterns, such as curved lines, circles, polygons, dots, waves.

Between overlapping layers or within a layer, the patterns can have different orientations, for example, the patterns can be oriented perpendicularly or diagonally at different angles. In addition, the distance between patterns within a layer can also vary.

In addition, the layers superimposed on each other can have different thicknesses, shapes and sizes.

The 2D layer may be solid or alternatively may have voids.

As a result, hemostatic products with complex shapes can be printed. Printed hemostatic products may also include areas having specific functions and a collection of areas with different functions.

The printing method can also produce a hemostatic product having a shape that can change over time, for example, with hydration. In particular, the printed hemostatic product is a 3D hemostatic product, i.e. a product that has three dimensions in space and a fourth dimension corresponding to the transformation of the product over time. Such 3D printed products having a fourth dimension are also called 4D products.

For more detailed information, please refer to the article by A. Sydney Gladman et al., entitled: "Biomimetic 4D printing" and published in Nature Materials in 2016 (see Nature Materials, vol. 15, p. 413 -418 (2016)), the contents of which are incorporated herein by reference in their entirety.

This makes it possible to obtain, for example, a 3D hemostatic product in which each layer or at least two different layers can have different hemostatic properties and/or different swelling abilities, for example upon hydration.

The composition of the finished printed hemostatic product may include fibrillar collagen, preferably partially fibrillar collagen, the content of which is from 60% to 99% by weight. of the total weight of the composition, in particular from 75% to 90% of the mass. and preferably from 83% to 88% of the mass.

The composition of the finished hemostatic product obtained by printing may include a monosaccharide, the content of which ranges from 1% to 10% by weight. of the total weight of the composition, in particular from 3% to 7% of the mass. and preferably from 4% to 5% by weight.

The composition of the finished hemostatic product obtained by printing may include glycosaminoglycan, the content of which ranges from 1% to 30% by weight. of the total weight of the composition, in particular from 5% to 12% of the mass. and preferably from 8% to 10% by weight.

The composition of the finished printed hemostatic product may include a blood clotting factor, preferably thrombin, the content of which is less than 1% by weight. from the total mass of the composition. Preferably, if a blood clotting factor is present, the content is preferably less than 0.5 wt%. based on the total weight of the composition, preferably less than 0.1 wt%.

The presence of collagen in the hemostatic fluid material used to produce a 3D hemostatic product is necessary to ensure the adhesive strength of the product, in particular the adhesive strength between the different layers of the product. Particularly preferred is the use of non-cross-linked fibrillar collagen. In addition, the use of fibrillar collagen promotes hemostasis.

As stated above, the hemostatic fluid material contains a high concentration of polymers (corresponding to the concentration of collagen) that facilitate the printing process and allow the production of hemostatic products by a self-assembly manufacturing method. In particular, this allows the printing process to produce self-supporting and durable cohesive hemostatic products without an additional reinforcement step, for example by natural cross-linking or cross-linking initiated by, for example, photostimulation or thermal stimulation.

As discussed above, when preparing a hemostatic powder, monosaccharides and, in particular, glucose are preferably used, since this leads to a decrease in electrical charges in the composition.

In addition, the use of a monosaccharide may also be due to its hydrophilicity, as well as the fact that monosaccharides usually carry polarized charges and can form hydrogen bonds. This gives monosaccharide molecules solubility not only in water, but also in other polar solvents. The monosaccharide may also contribute to the preservation of a possible coagulation factor.

Glycosaminoglycans and, in particular, chondroitin sulfates are also highly hydrophilic. Controlling the relative content of glycosaminoglycans in the hemostatic fluid composition used to make the printed hemostatic article allows the swelling ability of the printed article to be varied. In particular, it affects the swelling ability of the template obtained from a particular hemostatic fluid material.

It may be particularly advantageous to print a hemostatic product consisting of a superposition of layers with different concentrations of chondroitin sulfates, since chondroitin sulfates promote deformation of the product through swelling upon hydration.

For example, layers enriched in chondroitin sulfates may swell to a greater extent than layers primarily derived from collagen and containing smaller amounts of chondroitin sulfates. Differences in the degree of hydration and swelling throughout the printed hemostatic product can be used to deform and/or expand the hemostatic product upon hydration, for example, to better cover a bleeding wound.

The addition of glycosaminoglycans and in particular chondroitin sulfates to the hemostatic fluid also has the advantage of facilitating the printing process. In particular, they appear to, like a lubricant, facilitate the flow of hemostatic fluid material through the nozzle of the bioprinter print head.

A blood clotting factor, such as thrombin, which can be added at any stage of the process for making a hemostatic product, is used to improve blood clotting. By using various hemostatic fluid materials to print different layers of a 3D hemostatic article, the hemostatic fluid material containing the clotting factor can be deposited during printing only in the target areas, for example, in those areas where blood clotting is required.

The 3D hemostatic product produced by the 3D printing method according to the invention can be used within a short time after being produced on a 3D printer, for example within a few minutes. Because the bioprinting method is convenient and automated, the 3D hemostatic product can be printed for immediate use according to predefined patterns, such as the type of wound being healed or the bleeding site.

Preferably, the hemostatic product can be dried and applied dry to the bleeding site. If the 3D hemostatic product needs to be used at a later time, for example much later than the optimal recommended time of use indicated above, then the 3D hemostatic product can be partially dried, for example, in a natural atmosphere and/or in an autoclave. In this case, the hemostatic product may be rehydrated before application to the wound. It is preferable to rehydrate the 3D hemostatic device prior to use, although this is not absolutely necessary since the product may be rehydrated by blood surrounding the application site.

The 3D printed hemostatic product can be dried at room temperature or freeze-dried to better preserve the triple helical structure of collagen.

As stated above, an advantage of the hemostatic fluid material of the invention is that the material does not require cross-linking before or during additive printing to produce a stack of interconnected layers. However, the invention may include cross-linking during printing to more precisely control the adhesion between layers and optionally to print according to a more complex layout. Cross-linking can be carried out, for example, under the influence of UV radiation or using any adapted chemical cross-linking agent.

Additionally, the 3D printed hemostatic product can be cross-linked before or after drying. Cross braces can be formed locally to create a local reinforced area or to locally secure a printed form. This is particularly advantageous when obtaining a product with high resistance to manipulation and a product used in severe bleeding. However, it should be noted that the cross-linking step is not absolutely necessary for the printing process to produce dense, self-supporting and durable 3D products.

Alternatively, to reinforce the hemostatic product and, in particular, to slow down or limit its swelling and deformation, the cross-links can span the entire hemostatic product.

Local or global cross-linking can be induced by heat, light or any suitable initiator.

As discussed above, if a certain amount of cross-linking is required, the cross-linking initiator is preferably present in the hemostatic fluid composition used to print the hemostatic article.

If the hemostatic product does not contain a blood clotting factor, then a blood clotting factor such as thrombin solution can be introduced into the product by impregnation, immersion, spraying before or after drying. Alternatively, the clotting factor may be introduced into the article by absorption or precipitation.

The components of the hemostatic fluid material may be provided sterile for manufacture, but are not required to do so. In the latter case, the printed hemostatic product can be sterilized before use.

The dried, sterile hemostatic product obtained by printing can be applied to a bleeding wound.

Alternatively, the dried, sterile printed hemostatic product may be soaked in saline and/or clotting factor solution prior to application to the bleeding wound.

The properties and appropriate use of the printed hemostatic product depend on the properties of each of the layers constituting the printed hemostatic product.

For example, the composition and structure of the printed hemostatic product may be selected to allow the product to swell and deform to conform to a bleeding wound.

A possibly applied template enriched with coagulation factor may accelerate local blood clotting.

An optionally applied "pro-adhesive" template may improve local adhesive strength within the article or its adhesion to a bleeding wound.

Optionally, an outer layer enriched with water may be used to successfully prevent adhesion of the article to surrounding tissues.

The advantage of using a hemostatic product obtained by printing using the hemostatic fluid material discussed above is that it increases the contact surface with the wound. In particular, contact with the bleeding area becomes more dense. In particular, this becomes important when the area of bleeding is in soft tissues and parenchymal organs.

A 3D hemostatic product manufactured using a 3D additive printer can have any type of three-dimensional structure, from a simple one, having a closed volume, to a very complex one, including many lattices and angles.

The 3D hemostatic product may, for example, have the shape of a rod, a hollow cylinder, a ball, etc. The 3D hemostatic product may also be a bandage having a cellular structure, for example a membrane-shaped bandage at least several millimeters thick (eg, 5 mm to 50 mm).

Preferably, the hemostatic article may undergo resorption, for example, within a period of several weeks to several months after application, depending on the size, thickness of the article, or possible cross-linking.

As stated above, the invention also relates to a method of hemostasis comprising applying a printed hemostatic product, such as the one described above, to a bleeding part of an animal, including a human. In particular, the hemostatic product produced by the printing process can be used in surgical procedures, in particular laparotomy, laparoscopy, celioscopy and robotic surgical techniques.

The hemostatic product obtained by printing, discussed above, can also be used as a healing agent for internal and external wounds. The expression "healing agent" refers to an article that promotes clinically satisfactory healing of tissues in contact with the agent.

DESCRIPTION OF EXAMPLES OF IMPLEMENTATION OF THE INVENTION

Example 1

Protocol for determining hemostatic capacity in vitro

During the entire determination, citrated (approximately 0.1 M) human blood is kept at 37°C in a water bath. The test article (10 mg) is placed in a 5 ml polypropylene tube with a snap cap, and then citrate-treated fresh blood (2 ml) is added. Then add such an amount of CaCl ₂ that the final concentration of CaCl ₂ in the blood is 15 mM, and then the tube is closed. The contents of the test tube are vigorously mixed in a circular motion (10 times), and then the test tube is immersed in a water bath; the tube is returned to the vertical position every 10 seconds. The time elapsed before clot formation is noted, which corresponds to hemostatic capacity.

Example 2

Protocol for particle size determination

A known amount of product, in particular powder, is sifted through sieves with mesh sizes of 50 μm, 100 μm, 200 μm, 300 μm and 400 μm for 2 minutes (per sieve). Weigh the fractions obtained in each of the sieves. The proportion of particles in each size range is determined.

Example 3

Protocol for assessing the swelling of a composition

Weigh a 15 ml flask ( _m0 in mg) and then add X mg of dry composition powder ( _m0+x in mg). 0.15 Μ NaCl aqueous solution (2 ml) was added and the composition was allowed to swell for 20 minutes; then the flask is centrifuged at 1000 rpm.

Excess NaCl is removed with a Pasteur pipette, drops are removed by inverting the flask onto filter paper; then weigh the flask with the wet powder (m ₁ in mg).

The degree of swelling is calculated as follows: ((m ₁ -m ₀ )/(m _0+x -m ₀ )).

Example 4

Preparation of fibrillar type collagen by alkaline extraction

Pieces of pig skin (30 kg) degreased with acetone are left to swell for 3 hours in 100 kg of 0.05 M NaOH solution. The leather pieces are then finely ground in a knife mill and the resulting paste is diluted with 50 liters of 0.05 Μ NaOH. The resulting mixture is sieved under pressure through a sieve with a mesh size of 1 mm. The resulting paste is adjusted to pH 6-7.5 by adding HCl, and the resulting precipitate is collected by centrifugation or filtration through a 1 mm sieve.

The retentate is dehydrated with acetone by methods known to those skilled in the art. The retentate thus dehydrated consists of fibrillar type collagen containing a large amount of fibrillar/fibrous collagen compared to non-fibrillar collagen. Typically, the extracted collagen comprises from 85% to 95% wt. fibrillar/fibrous collagen from the total mass of collagen and from 5% to 15% of the mass. non-fibrillar collagen from the total mass of collagen.

Example 5

Obtaining hemostatic powder #1

30 g of fibrillar type collagen obtained according to Example 4 was added to 1 L of 0.02 Μ HCl aqueous solution, and the mixture was stirred for 5 hours. Then powdered fructose is added to the resulting homogeneous paste in an amount of 2% (0.6 g) by weight. from the mass of collagen.

The mixture is homogenized for 1 hour, then poured out and dehydrated. After drying, the dry product is ground at a rate of 25 g/min using a Fitzpatrick hammer mill at 7000 rpm. with controlled heating. The product is then screened using mechanical sieves to remove particles larger than 400 microns.

Then dermatan sulfate is added to the powder in an amount of 2% by weight. by weight of dry powder (0.612 g).

The mixture is then homogenized using a ball mill, lyophilized thrombin is added to the mixture in an amount of 15 IU/mg powder, and finally the mixture is homogenized using a ball mill.

Example 6

Obtaining hemostatic powder #2

7.5 kg of fibrillar type collagen obtained according to Example 4 was added to 50 L of 0.05 Μ HCl aqueous solution, and the mixture was stirred for 16 hours. Then powdered fructose is added to the resulting homogeneous paste in an amount of 5% (375 g) by weight. from the mass of collagen.

The mixture is homogenized for 3 hours and then distributed onto dewatering trays. After drying, the dry product is ground into fractions with a productivity of 5 g/min using a hammer crusher at a speed of 12,000 rpm. with controlled heating. The product is then screened using mechanical sieves to remove particles larger than 400 microns and particles smaller than 50 microns.

The particle size distribution is determined by determining that the size distribution is such that 60% of the sample mass has a particle size distribution in which the particle size exceeds 200 microns.

Then purified chondroitin sulfates are added to the powder in an amount of 20% by weight. by weight of dry powder (1.575 kg). The mixture is homogenized using a ball mill.

Finally, lyophilized thrombin is added to the mixture in an amount of 10 IU/mg powder. As in previous examples, the mixture is homogenized using a ball mill.

Example 7

Obtaining hemostatic powder #3

1000 g of fibrillar type collagen obtained according to Example 4 was added to 60 ml of 0.02 Μ HCl aqueous solution, and the mixture was stirred for 5 hours. Then powdered glucose is added to the resulting homogeneous paste in an amount of 5% (50 g) by weight. from the mass of collagen.

The mixture is homogenized for 1 hour, then poured out and dehydrated. After drying, the dry product is ground at a rate of 25 g/min using a Fitzpatrick hammer mill at 7000 rpm. with controlled heating. The product is then screened using mechanical sieves to remove particles larger than 400 microns and particles smaller than 50 microns.

Then chondroitin sulfate is added to the powder in an amount of 10% by weight. based on the mass of dry powder (105 g). The mixture is then homogenized using a ball mill.

This powder composition has a bulk density of approximately 0.408 g/ml.

Example 8

Obtaining hemostatic powder #4

500 g of fibrillar type collagen obtained according to Example 4 was added to 30 ml of 0.02 Μ HCl aqueous solution, and the mixture was stirred for 5 hours. Then powdered glucose is added to the resulting homogeneous paste in an amount of 5% (25 g) by weight. from the mass of collagen.

The mixture is homogenized for 1 hour, then poured out and dehydrated. After drying, the dry product is ground at a rate of 25 g/min using a Fitzpatrick hammer mill at 7000 rpm. with controlled heating. The product is then screened using mechanical sieves to remove particles larger than 400 microns and particles smaller than 50 microns.

Then chondroitin sulfate mixed with powdered thrombin is added to the powder in an amount of 10% by weight. by weight of dry powder (52.5 g). Such an amount of thrombin is added to the mixture so that its final amount is 0.85 IU/mg. The mixture is then homogenized using a ball mill.

This powder composition has a bulk density of approximately 0.425 g/ml.

Example 9

Obtaining hemostatic powder #5

750 g of fibrillar type collagen obtained according to Example 4 are mixed with 6675 ml of high-purity water. The mixture is stirred at a first stirring speed of 20 rpm for 10 minutes and then at a second stirring speed of 40 rpm for 15 minutes.

The mixture obtained as above is then stirred again at a first stirring speed of 20 rpm by introducing a glucose solution (37.5 g glucose in 300 ml water) into the mixture. The amount of added glucose corresponds to 5% of the mass. on the mass of collagen contained in the mixture. The resulting mixture was stirred at a second stirring speed of 40 rpm for 10 minutes. The resulting preparation is kept for 16 hours.

Then, 87.5 ml of 1 Μ aqueous HCl solution is added to the preparation while stirring at a stirring speed of 30 rpm. The resulting mixture is stirred at the first stirring speed of 35 rpm for 1 minute, then at the second stirring speed of 40 rpm for 1 minute, after which several mixing stages are carried out, each of 5 minutes, at the same mixing speed of 40 rpm, with short pauses between mixing stages.

The dense paste obtained in the previous operation is then divided into several samples having a similar shape and mass. The resulting paste samples are placed for 24 hours in a hermetically sealed container filled with an atmosphere saturated with ammonia. After this neutralization step, the paste samples are dried at 20°C for 96 hours, and the dry products are then ground at a capacity of 1 kg/hour in a low-temperature (cryogenic) Forplex mill at a speed of 8500 rpm. The powdered product is then sieved using mechanical sieves to remove particles larger than 200 µm and particles smaller than 50 µm to obtain a collagen-glucose mixture powder.

Then chondroitin sulfate (CS) powder, consisting of particles whose size ranges from 50 μm to 200 μm, is added to the powdered mixture of collagen and glucose in an amount of 10% by weight. of the dry matter mass in the powdered mixture of collagen and glucose. For example, 30 g of chondroitin sulfate powder is mixed with 300 g of collagen and glucose powder mixture. Lyophilized thrombin is also added to this Hemostatic Powder #5 at 1000 IU/g. The mixture is then homogenized in a V-mixer. The bulk density of the finished hemostatic powder is approximately 0.4 g/ml.

Example 10

Characteristics of collagen: presence of soluble collagen in collagen, determination of the relationship between fibrillar/fibrous collagen and non-fibrillar collagen

The objective of the experiment was to determine the ratio of fibrillar/fibrous collagen to non-fibrillar collagen in collagen (extracted collagen or collagen ground into powder). This ratio can be determined from the ratio of insoluble (corresponding to fibrillar/fibrous collagen) and soluble collagen (corresponding to non-fibrillar collagen) in collagen.

The experiment consists of solubilizing approximately 2.5 g of test collagen in 166 ml of water at pH 13 for 16 hours. Then the solution is centrifuged (10,000 rpm for 10 minutes). The liquid above the precipitate (corresponding to non-fibrillar collagen) is separated from the residue (corresponding to fibrous/fibrillar collagen). The residue is directly dried, successively treated in acetone baths and in a controlled air flow. The pΗ value of the liquid above the sediment is adjusted to pH 3 by adding acetic acid and hydrochloric acid with a concentration of 6 M. Solid collagen is obtained from the liquid above the sediment by adding 0.6 M NaCl and centrifugation. It is then dried by successive treatments in acetone baths and controlled air flow.

The masses of collagen in the residue (M _residue ) and in the liquid above the sediment (M _{liquid_above_sediment} ) are calculated, and using the formula M _residue /(M _residues + M _{liquid_above_sediment} )×100 the percentage of fibrous collagen in the total amount of collagen is calculated.

According to the invention, the ratio M _residue /(M _residue + M _{liquid_above_sediment} ) must exceed 80% both in the collagen used to obtain the powder and in the finished powdered collagen. Preferably this ratio is greater than 85%.

For example, in the experiment described above, in which three batches of collagen prepared as described in Example 4 were tested, very similar ratios were found to be 92.67%, 94.60% and 91.51%, respectively. After grinding the collagen contained in these three batches, the ratios also remained very close, being 91.63%, 88.02% and 88.69%, respectively.

Another way to detect the presence of fibrous/fibrillar collagen and soluble collagen is SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis).

In FIG. 2 shows the results of electrophoresis of sample S1 corresponding to the sediment of the first batch of material (obtained from collagen extracted as described in Example 4), sample S2 corresponding to the residue of this first batch, sample S3 corresponding to the sediment of the second batch of material (also obtained from collagen extracted as described in Example 4) and sample S4 corresponding to the remainder of this second batch.

The results show that in the case of residue-derived collagen, more fibers are unable to migrate through the acrylamide gel and they form a colored area near the top of the gel. In the preparation from this sample, each chain of collagen is not separated. Thus, alpha chains are present in very small quantities. Collagen obtained from the liquid above the sediment can migrate completely in the gel, there are no fibers blocked at the top of the gel, and the collagen chains are easily cleaved off during electrophoresis.

Literature cited

- WO 2012/146655

- "Nature designs tough collagen: explaining the nanostructure of collagen fibrils," by Markus Buehler (PNAS, August 15, 2006, vol. 103, no. 33, pp. 12285-12290)

- "Extraction of collagen from connective tissue by neutral salt solutions" by Gross, John H. Highberger and Francis O. Schmitt (Proceedings of the NATIONAL ACADEMY OF SCIENCES Volume 41 Number I January 15, 1955)

- WO 2010/125086

- FR 2944706

- WO 01/97873

- US 4891359

- "Current advances and future perspectives in extrusion-based bioprinting" by Ibrahim T. Ozbolat and Monika Hospodiuk (Biomaterials 76 (2016) 321-343)

- "Biomimetic 4D printing" by A. Sydney Gladman et al. entitled (Nature Materials volume 15, pages 413-418 (2016))

Claims (68)

1. A hemostatic product obtained by three-dimensional additive printing, having at least three dimensions and consisting of a stack of layers located one on top of the other, counting from the first outer layer to the second outer layer, in which adjacent layers of the stack of layers are connected to each other and in which at least one layer of the stack of layers includes at least one portion derived from a hemostatic fluid material, the composition of which includes: - non-cross-linked collagen of the fibrillar type, in which the content of fibrous collagen and/or fibrillar collagen is at least 70 wt.% of the total mass of collagen; - at least one monosaccharide; And - at least one glycosaminoglycan. 2. The printed hemostatic product according to claim 1, wherein at least one layer of the stack of layers includes various parts made from hemostatic fluid materials having different compositions. 3. A hemostatic product obtained by printing according to claim 1, in which at least one layer of the stack of layers is made entirely of one hemostatic fluid material. 4. A hemostatic product obtained by printing according to claim 3, in which all layers in a stack of layers have the same composition. 5. A hemostatic product obtained by printing according to any one of claims. 1-3, wherein at least one of the layers of the stack of layers includes a portion derived from a hemostatic fluid material having a swelling ability that is different from the swelling ability of other layers contained in the stack of layers. 6. A hemostatic product obtained by printing according to any one of claims. 1-5, including a plurality of adjacent layers, where each layer of the plurality of adjacent layers includes at least one portion derived from a hemostatic fluid material. 7. A hemostatic product obtained by printing according to any one of claims. 1-6, wherein at least one layer of the first and second outer layers of the stack of layers includes a portion derived from a hemostatic fluid material. 8. The printed hemostatic product according to claim 7, wherein at least one layer of the first and second outer layers is made entirely of a single hemostatic fluid material. 9. A hemostatic product obtained by printing according to any one of claims. 7, 8, in which the first and second outer layers of the stack of layers have the same composition. 10. The printed hemostatic product according to claim 9, wherein each of the layers of the stack of layers has a peripheral portion made from a fluid mixture identical to the fluid mixture of at least one layer selected from the first and second outer layers. 11. A hemostatic product obtained by printing according to any one of claims. 1-10, wherein the collagen content of the hemostatic fluid composition is from 70 to 99% by weight based on the total weight of the composition, preferably from 75 to 96% by weight, and more preferably from 80 to 90% by weight. 12. A hemostatic product obtained by printing according to any one of paragraphs. 1-11, wherein the monosaccharide content of the hemostatic fluid composition is from 1 to 12.5 wt.% of the total weight of the composition, preferably from 2 to 8 wt.%, more preferably from 2.5 to 7.5 wt.% and more preferably 4 to 6 wt.%. 13. A hemostatic product obtained by printing according to any one of claims. 1-12, in which the glycosaminoglycan content is from 1 to 30 wt.% of the total weight of the composition, preferably from 2 to 25 wt.%, preferably from 4 to 15 wt.%, more preferably from 5 to 12.5 wt.% and more preferably from 8 to 10 wt.%. 14. A hemostatic product obtained by printing according to any one of claims. 1-13, in which the glycosaminoglycan is chondroitin sulfate. 15. A hemostatic product obtained by printing according to any one of claims. 1-14, wherein the hemostatic fluid composition includes at least one blood coagulation factor, in particular thrombin. 16. The hemostatic product obtained by the printing method according to claim 15, in which the blood coagulation factor is contained in an amount of from 0.01 to 20 IU/mg of the composition, preferably from 0.05 to 10 IU/mg of the composition, preferably from 0.1 up to 5 IU/mg of the composition, and more preferably from 0.2 to 2 IU/mg of the composition. 17. A hemostatic product obtained by printing according to any one of paragraphs. 1-10, in which the hemostatic fluid material is obtained from a hemostatic powder, the composition of which includes: - collagen of the fibrillar type, in which the content of fibrous collagen and/or fibrillar collagen is at least 70 wt.% of the total mass of collagen, where collagen is contained in the powder in an amount ranging from 75 to 96 wt.% of the total mass of the hemostatic powder composition , preferably ranges from 80 to 90% by weight; - at least one monosaccharide in an amount of from 1 to 12.5 wt.% of the total weight of the hemostatic powder composition, preferably in an amount of from 2.5 to 7.5 wt.%, and more preferably in an amount of from 4 to 6 wt.%; And - at least one glycosaminoglycan in an amount of from 1 to 30% by weight of the total weight of the hemostatic powder composition, preferably from 2 to 25% by weight, preferably in an amount of from 4 to 15% by weight, more preferably in an amount, ranging from 5 to 12.5% by weight, and more preferably in an amount ranging from 8 to 10% by weight. 18. The hemostatic product obtained by the printing method according to claim 17, in which the hemostatic powder additionally includes at least one blood clotting factor in an amount of less than 0.5 wt.% of the total weight of the hemostatic powder composition, preferably in an amount of less than 0.1 wt. %. 19. Use of a hemostatic fluid material as a printing ink for a three-dimensional additive printer, where the hemostatic fluid material is obtained from a hemostatic powder mixed with a saline solution, and the composition of the hemostatic powder includes: - non-cross-linked collagen of the fibrillar type, in which the content of fibrous collagen and/or fibrillar collagen is at least 70 wt.% of the total mass of collagen; - at least one monosaccharide; And - at least one glycosaminoglycan. 20. Use according to claim 19, wherein the collagen content of the hemostatic powder composition from which the hemostatic fluid is formed is from 70 to 99 wt.% of the total weight of the composition, preferably from 75 to 96 wt.% and more preferably from 80 up to 90 wt.%. 21. Application according to any one of paragraphs. 19 and 20, wherein the monosaccharide content of the hemostatic powder composition from which the hemostatic fluid is formed is from 1 to 12.5 wt% of the total weight of the composition, preferably from 2 to 8 wt%, more preferably from 2.5 to 7.5 wt.% and more preferably from 4 to 6 wt.%. 22. Application according to any one of paragraphs. 19-21, in which the glycosaminoglycan content is from 1 to 30 wt.% of the total weight of the composition, preferably from 2 to 25 wt.%, preferably from 4 to 15 wt.%, more preferably from 5 to 12.5 wt.% and more preferably from 8 to 10 wt.%. 23. Application according to any one of paragraphs. 19-22, in which the glycosaminoglycan is chondroitin sulfate. 24. Application according to any one of paragraphs. 19-23, in which the hemostatic powder composition from which the hemostatic fluid is formed includes at least one blood coagulation factor, in particular thrombin. 25. Use according to claim 24, wherein the blood coagulation factor is contained in an amount ranging from 0.01 to 20 IU/mg of the composition, preferably from 0.05 to 10 IU/mg of the composition, more preferably from 0.1 to 5 IU/ mg of the composition and more preferably from 0.2 to 2 IU/mg of the composition. 26. Application according to any one of paragraphs. 19-23, in which the saline solution includes at least one blood coagulation factor, in particular thrombin. 27. Use according to claim 26, wherein the coagulation factor is contained in an amount of 10 to 5000 IU/ml saline, preferably 25 to 2500 IU/ml saline, more preferably 50 to 1000 IU/ml saline and more preferably 100 to 500 IU/ml saline solution. 28. Use according to claim 19, wherein the composition of the hemostatic powder from which the hemostatic fluid material is formed includes: - collagen of fibrillar type, in which the content of fibrous collagen and/or fibrillar collagen is at least 70 wt.% of the total mass of collagen, where collagen is contained in an amount ranging from 75 to 96 wt.% of the total mass of the hemostatic powder composition, preferably ranges from 80 to 90 wt.%; - at least one monosaccharide in an amount of from 1 to 12.5 wt.% of the total weight of the hemostatic powder composition, preferably in an amount of from 2.5 to 7.5 wt.%, and more preferably in an amount of from 4 to 6 wt.%; And - at least one glycosaminoglycan in an amount of from 1 to 30% by weight of the total weight of the hemostatic powder composition, preferably from 2 to 25% by weight, preferably in an amount of from 4 to 15% by weight, more preferably in an amount, ranging from 5 to 12.5% by weight, and more preferably in an amount ranging from 8 to 10% by weight. 29. Use according to claim 28, wherein the hemostatic powder from which the hemostatic fluid is formed further includes at least one blood coagulation factor in an amount of less than 0.5 wt.% of the total weight of the hemostatic powder composition, preferably in an amount of less than 0 .1 wt.%. 30. Application according to any one of paragraphs. 19-29, wherein the weight of the saline solution used to produce the hemostatic fluid is 2 to 10 times the weight of the hemostatic powder, and preferably 4 to 5 times the weight of the hemostatic powder. 31. Application according to any one of paragraphs. 19-30, in which the hemostatic powder from which the hemostatic fluid is formed includes at least 80 wt.% particles whose size ranges from 20 to 300 microns. 32. Application according to any one of paragraphs. 19-31, wherein the hemostatic powder from which the hemostatic fluid is formed includes at least 90 wt.% particles whose size is less than 350 microns. 33. A method for manufacturing a hemostatic product having at least three dimensions using a three-dimensional additive printer, where the method includes the following steps: a) providing a three-dimensional model for a three-dimensional additive printer, where the three-dimensional model corresponds to the structure of the hemostatic product being manufactured, and processing the three-dimensional model to define a printable template comprising a plurality of layers designed to be stacked one on top of the other to form a stack of layers corresponding to the three-dimensional model ; b) supplying a three-dimensional additive printer with at least one hemostatic fluid material that is used as a printing ink, where the fluid material is obtained from a hemostatic powder mixed with a saline solution, and the composition of the hemostatic powder includes: - non-cross-linked collagen of the fibrillar type, in which the content of fibrous collagen and/or fibrillar collagen is at least 70 wt.% of the total mass of collagen; - at least one monosaccharide; And - at least one glycosaminoglycan; c) printing the hemostatic product using a three-dimensional additive printer by applying printing ink to sequentially deposit one layer on top of another, where the formation includes applying a hemostatic fluid material using a three-dimensional additive printer, resulting in the formation of at least one portion of at least one layer of packs of layers. 34. The method according to claim 33, wherein at least one layer of the stack of layers is made entirely by deposition of a hemostatic fluid material. 35. Method according to any one of paragraphs. 33, 34, in which, when preparing a hemostatic product (10), a hemostatic fluid material is used to form a part from layers contained in a stack of layers. 36. Method according to any one of paragraphs. 33, 34, in which, upon receipt of the hemostatic product (10), the hemostatic fluid material is used to form all the layers contained in the stack of layers. 37. Method according to any one of paragraphs. 33-35, in which, in step b), a plurality of hemostatic fluid materials that are used as printing inks are supplied to the three-dimensional additive printer, and these hemostatic fluid materials have different compositions. 38. Method according to any one of paragraphs. 33-37, in which the composition of the hemostatic powder from which the hemostatic fluid material is formed includes: - collagen of fibrillar type, in which the content of fibrous collagen and/or fibrillar collagen is at least 70 wt.% of the total mass of collagen, where collagen is contained in an amount ranging from 75 to 96 wt.% of the total mass of the hemostatic powder composition, and preferably in an amount of 80 to 90% by weight; - at least one monosaccharide in an amount of from 1 to 12.5 wt.% of the total weight of the hemostatic powder composition, preferably in an amount of from 2.5 to 7.5 wt.%, and more preferably in an amount of from 4 to 6 wt.%; And - at least one glycosaminoglycan in an amount of from 1 to 30% by weight of the total weight of the hemostatic powder composition, preferably from 2 to 25% by weight, preferably in an amount of from 4 to 15% by weight, more preferably in an amount, ranging from 5 to 12.5% by weight, and more preferably in an amount ranging from 8 to 10% by weight. 39. The method of claim 38, wherein the hemostatic powder composition from which the hemostatic fluid material is formed further includes at least one blood clotting factor in an amount of less than 0.5 wt.% of the total weight of the hemostatic powder composition, preferably in an amount of less than 0.1 wt.%. 40. The method according to claim 38, wherein the saline solution used in preparing the hemostatic fluid material includes at least one blood clotting factor and the blood clotting factor is contained in an amount ranging from 10 to 5000 IU/ml saline solution, preferably from 25 up to 2500 IU/ml saline, more preferably 50 to 1000 IU/ml saline, and more preferably 100 to 500 IU/ml saline. 41. Method according to any one of paragraphs. 33-40, in which the weight of the saline solution used to obtain the hemostatic fluid material is 2 to 10 times the weight of the hemostatic powder and preferably 4 to 5 times the weight of the hemostatic powder. 42. Method according to any one of paragraphs. 33-41, in which printing step c) is carried out under ambient conditions. 43. Method according to any one of paragraphs. 33-42, in which, after printing step c) leading to the formation of the hemostatic product, coating step d) is performed, in which the outer surface of the hemostatic product is coated with a solution comprising a blood clotting factor, in particular thrombin. 44. The method according to claim 43, wherein step d) of coating is performed by spraying a solution including a blood clotting factor onto the outer surface of the hemostatic device. 45. Method according to any one of paragraphs. 33-44, in which, after printing step c) leading to the formation of the hemostatic product, a soaking step e) is performed, in which the hemostatic product is soaked in a solution including a blood clotting factor, in particular thrombin. 46. Method according to any one of paragraphs. 33-45, wherein the final step of manufacturing the hemostatic product is to expose the printed hemostatic product to ambient conditions for a predetermined holding period, wherein the predetermined holding period is preferably less than 10 minutes, more preferably less than 5 minutes, and more preferably less than 1 minute. 47. Method according to any one of paragraphs. 33-46, in which the entire method is carried out under ambient conditions without stimulation of the hemostatic device, in particular without photostimulation or thermal stimulation.