EP3801652A1

EP3801652A1 - Cellularised dressing and method for producing same

Info

Publication number: EP3801652A1
Application number: EP19740640.8A
Authority: EP
Inventors: Delphine FAYOL; Fabien Guillemot; Catherine VAN DER MEE; Christelle Laurensou
Original assignee: Urgo Recherche Innovation et Developpement; Poietis SAS
Current assignee: Urgo Recherche Innovation et Developpement; Poietis SAS
Priority date: 2018-06-07
Filing date: 2019-06-06
Publication date: 2021-04-14
Also published as: WO2019234365A1; FR3082123A1; FR3082123B1; US20210220510A1

Abstract

The invention relates to a cellularised dressing and to the method for producing such a dressing, said method preferably comprising a step of bioprinting cells.

Description

CELLULARIZED DRESSING AND METHOD OF MANUFACTURE

The present invention relates to a cellularized dressing and method of manufacturing such a dressing, which method preferably comprises a step of bio-printing cells.

The cellularized dressings or cutaneous substitutes are known and marketed for a long time. The advantage of the cellularized dressings lies in the fact that the exogenous supply of living cells contributes to the healing of the wound. The cells provided by the dressing participate directly or indirectly (through the secretion of factors) in the healing process.

Such dressings are generally in the form of at least one resorbable material (the at least one material naturally present in the cellular environment). By way of example, mention may be made of GRAFIX® products marketed by OSIRIS. These products are composed of a placental membrane containing a collagen-rich extracellular matrix (ECM), growth factors, fibroblasts, mesenchymal stem cells and epithelial cells. There may also be mentioned the product APLIGRAF®, marketed by Organogenesis, composed of keratinocytes, fibroblasts and bovine collagen. The product DERMAGRAFT®, marketed by the company Advanced Biohealing, contains dermal derivatives and human fibroblasts.

Such cellularized dressings, although having a proven effectiveness on healing, however, have risks of transmitting viruses such as, for example, prions (in particular for cellularized dressings containing compounds of animal origin).

Moreover, in a cellularized dressing, the cell density, the cell localization zone or the homogeneous distribution of the cells are poorly controlled parameters.

There is therefore a need for new cellularized dressings that do not have the drawbacks of the prior art.

The printing of cells, also called bio-printing, directly on bio-resorbable materials in order to reconstitute a dermis or epidermis is known and described in the patent application WO2016 / 115034 of Wake Forest University or in the application patent number WO0160 / 073782 from Organovo. These patent applications relate to the printing of extracellular cells and components (such as, for example, collagen, hyaluronic acid, etc.) to make skin ex vivo. The materials or substrates on which the cells are printed are materials conventionally used in cell cultures (hydrogels containing collagen, hyaluronan, polyethylene glycol, etc.). The patent application WO2016 / 115034 also describes a bio-mask comprising a hydrogel layer containing cells inside this hydrogel, said hydrogel being then bio-printed on a polyurethane structure. This patent application therefore does not describe the bio-printing of cells on the polyurethane structure. Indeed, there is no direct contact between the bio-printed cells and a non-absorbable material. Moreover, in the patent application WO2016 / 115034, the polyurethane structure corresponds to a bio-printed polyurethane gel: this polyurethane structure therefore does not allow the absorption of exudates.

Biomaterials and tissue engineering are also known to replace part or function of an organ or tissue.

Biomaterials are materials, either synthetic or living, that can be used for medical purposes to replace a part or function of an organ or tissue. The said biomaterials must respect several obligations:

be well tolerated by the recipient, ie do not cause infection, inflammation, allergy or rejection if it is living material;

- do not contain any toxic substances, such as endocrine disruptors or carcinogens;

respond to mechanical stresses to adapt to environmental pressures (blood pressure for vascular prostheses, millions of openings and closures for a heart valve, body weight for hip or knee prostheses, ...);

- Be able to be shaped, implantable or injectable, degradable (absorbable) or not depending on the case, possibly porous if they are to be colonized once implanted, ....

Tissue engineering consists in the manufacture of a tissue by multiplication of cells around a matrix or scaffolding ("scaffold" type). Concrete realization, however, faces various problems. For example, in an artificial environment cells tend to lose their ability to differentiate. Of the more the cells sometimes express atypical proteins which, after implantation, can cause inflammations or rejection reactions.

The therapeutic use of non-absorbable materials comprising cells is therefore described in the prior art (for example in the case of the manufacture of a tissue using a scaffold matrix). Nevertheless, when this matrix is not resorbable it is intended to be kept in place within the body for at least a long time, it is not intended to be withdrawn. It is the same for the use of non-absorbable biomaterials.

On the contrary, the present invention relates to the therapeutic use of non-absorbable materials (preferably synthetic materials), but said materials are not intended to replace a part or function of an organ or tissue. They are not intended to be held in place within the body, they are intended to be removed after regeneration of the organ or tissue on which they were applied. According to the invention, the materials thus have a transitional dressing role.

The bio-printing of cells as described in applications WO2016 / 115034 and WO20160 / 073782 is carried out on bio-resorbable materials. Bioprinting methods are also described in WO2011 / 107599, WO2016 / 097619 and WO2016 / 097620. These requests notably describe that bio-printing can be used to produce tissues (for example implantable tissues for regenerative medicine).

The bio-printing of cells on non-absorbable materials intended to be used in a transient manner is therefore not described in the prior art.

In the context of the present invention, cells are thus printed on nonabsorbable materials. Said materials are used as dressings. Such materials are not naturally present in the cellular environment and are not usually used in cell culture. Surprisingly the inventors have found that bio-printed cells on such materials were not only viable, able to proliferate, but were also capable of migration. The advantage of such printing or bio-printing is that it is possible to customize or adapt to each patient and each wound the dressing, allowing a tailor-made treatment to optimize the healing of wounds. Thus, depending on the healing phase in which the wound is located, it is possible to integrate cells of the dermis and cells of the epidermis or only one of these two cell types. It is also possible, within the same dressing, to vary the cell density from one place to another in order to optimize the treatment according to the morphology of the wound. In addition, the cellularized dressings according to the present invention make it possible to avoid the risks of virus transmission, in particular because they do not contain compounds of animal origin. Bio-printing also makes it possible to precisely locate on the dressing an area on which the cells will be present at a controlled concentration. For dressings that have grid fibers, the cells can be printed specifically on the grid, or outside the grid. The precision of this technique is of the order of ten pm. The cell density, the cell localization zone and / or the homogeneous distribution of the cells are thus better controlled in the dressings according to the invention compared with the cellularized dressings of the prior art.

In a first aspect, the invention thus relates to a cellularized dressing intended to be applied transiently to a wound, said dressing comprising cells on a non-resorbable material.

For purposes of the present invention, "cellularized dressing" means that the dressing comprises cells.

According to the invention, the term "intended to be applied transiently to a wound" means that the dressings are intended to be removed from the wound. This expression also means that the dressings according to the invention are in a form suitable for transient application to a wound. The dressings according to the invention have indeed a protective role and are intended to be removed once the organ or tissue of the wound has regenerated. The dressings according to the invention do not resorb, and they are not intended to be held in place for a long time (several days or weeks). Advantageously, the dressing covers all or part of the wound, preferably the entire wound.

According to the invention, the expression "said dressing comprising cells on a non-resorbable material" means that the cells are in direct contact with the non-resorbable material. Advantageously, the cells are not mixed with a hydrogel or incorporated inside a hydrogel. According to one embodiment of the invention, said dressing is devoid of hydrogel.

For the purposes of the present invention, the term "non-absorbable material" means that the material does not progressively dissolve within the wound, unlike the materials resorbable which they break down naturally. The removal / degradation of a non-absorbable material therefore requires a physical / mechanical action, in contrast to the degradation of a resorbable material.

Said non-resorbable material advantageously has the following properties: (1) it allows the absorption of the exudates, and / or (2) it can undergo a dimensional change (by gelling or deformation related to the absorption), and / or (3) it does not adhere to the tissues, and / or (4) it is preferably partially hydrophilic in the hydrated state, and / or (5) it exhibits hydrated slip, and / or (6) it is not cytotoxic. According to the invention, the "slipperiness in the hydrated state" means that the material has a surface state which does not allow the cells to adhere to it but which nevertheless keeps them alive.

Advantageously, said non-absorbable material is chosen from:

- an interface dressing,

an absorbent dressing, or

a hydrophilic polyurethane foam.

Advantageously, said non-absorbable material is a material comprising fibers, in particular the interface dressing or the absorbent dressing.

Advantageously, the non-absorbable material allows the absorption of exudates. Preferably, the non-resorbable material according to the invention is a hydrophilic polyurethane foam for the absorption of exudates.

By way of example, an interface dressing is as described in patent application EP2793773, that is to say an adherent interface dressing comprising: (i) a non-adherent, cohesive gel formed of a hydrophobic elastomeric matrix consisting of a triblock elastomer of styrene - (ethylene - butylene) - styrene or styrene (ethylene - propylene) - styrene type optionally combined with a diblock copolymer of the styrene - (ethylene - butylene) or styrene - (ethylene - propylene) type, said elastomer being strongly plasticized by means of a mineral oil, and dispersively containing a small amount of hydrophilic particles of a hydrocolloid, and (ii) an open-meshed flexible fabric, said fabric comprising yarns which are coated with the non-cohesive gel adherent so as to leave the stitches essentially unsealed, characterized in that the fabric is a thermofixed knit with screened yarns, said yarns being continuous yarns s with non-elastic filaments, which has in the transverse direction an extensibility measured according to EN 13726-4 of between 0.01 and 0.5 N / cm. According to a preferred embodiment, said nonadherent cohesive gel is formed of a hydrophobic elastomeric matrix comprising, per 100 parts by weight of elastomer chosen from a triblock elastomer of the styrene - (ethylene - butylene) - styrene or styrene (ethylene) type. - propylene) - styrene optionally combined with a diblock copolymer of styrene - (ethylene - butylene) or styrene - (ethylene - propylene) type, 1000 to 2000 parts by weight of a paraffin oil, and containing in dispersion from 2 to 20% by weight, based on the total weight of the elastomeric matrix, of hydrophilic particles of a hydrocolloid.

By way of example, an absorbent dressing is as described in the patent application EP2696828, that is to say an adhesive absorbent dressing comprising an absorbent nonwoven (6) and a fluid-impermeable and permeable protective support. the water vapor (4), characterized in that: (i) the support is constituted by the assembly of a continuous film (4a) and a perforated reinforcement coated, on at least one of its faces, with adhesive silicone gel (4b), without closing the openings of the frame, said frame covering the entire surface of the film, (ii) in that said dressing further comprises a non-absorbent veil (5) and a non-absorbent complementary woven fabric (7) which are fixed to each other on their periphery by wrapping said absorbent nonwoven, preferably without a point of attachment with the latter, and (iii) in that said non-absorbent web (5) adheres adhesive silicone gel (4b) coated on said frame.

According to one embodiment of the invention, the cells present in the dressing are cells adhering to a substrate (for example polystyrene in a box or a culture flask). They are chosen in particular from the cells of the dermis or epidermis. They are especially chosen from fibroblast-type cells and / or epithelial-type cells. Advantageously, the cells are chosen from fibroblasts and / or keratinocytes, in particular primary fibroblasts and / or primary keratinocytes. Even more advantageously, the cells are chosen from primary dermal fibroblasts and / or primary epidermal keratinocytes. The term "fibroblasts" refers to spindle-shaped, irregularly shaped cells that are responsible for fiber formation. In cell cultures, many other cell types can not be morphologically distinguished from fibroblasts. In organ and tissue cultures in which cell-to-cell ratios are conserved, it is possible to identify fibroblasts using accepted histological criteria. The term "epithelial cells" refers to cells opposite to each other that form a mosaic-like continuous tissue with very few intercellular substances as can be seen in in vitro cultures, tissues or tissues. organs. The term "fibroblast-like cells" refers to cells that are attached to a substrate and that appear elongated and bipolar. In cell cultures, various cell types have similar morphologies. Cells that take irregular shapes or Spindle shapes are often referred to as fibroblasts. The term "epithelial-like cells" refers to cells that are attached to a substrate and appear flat and polygonal in shape. In cell cultures, epithelial cells can take a variety of forms but tend to form into tight polygonal cell tissue.

According to one embodiment of the invention, in said dressing, the cells are (or have been previously) bio-printed on said non-absorbable material. In this embodiment the cells are also directly bio-printed on said non-absorbable material, said dressing therefore does not include a bio-printing of hydrogel. Some dressings have the property of not adhering to wounds, and the cells do not adhere to the materials generally used in dressings. It is therefore complicated to support cells on the surface of this type of dressing since the cells will not be able to adhere to it. One of the advantages of bio-printing is that it makes it possible to print the cells on the surface of this type of dressing, and to hold them there until the dressing is transferred to the wound. By way of example, the applications WO2016 / 115034, WO20160 / 073782 WO2011 / 107599, WO2016 / 097619 and WO2016 / 097620 describe bio-printing processes that can be used to bio-print a dressing according to the invention.

According to one embodiment of the invention, in said dressing, the cells are present (or have been bio-printed) at the level of the fibers of said material (ie on the fibers themselves) or within a or patterns defined by the fibers. Advantageously, according to the invention, the cells can thus be present (or be bio-printed) at the level of the fibers of a concentric pattern, a radial pattern, a geometric pattern or a non-geometric random pattern ( that is, not representing a geometric shape), or within at least one of these patterns. According to an even more preferred embodiment, in said dressing, the cells are present (or have been bio-printed) at the level of the fibers of said material, at the intersection of the fibers of said material and / or in the center of each quadrangle of said material. .

According to one embodiment of the invention, said dressing is saturated with liquid up to 90% of its absorption capacity. Preferably, said dressing is saturated with liquid at a content of between at least 50% of its absorption capacity, preferably at least 80%, and up to 90% of its absorption capacity. According to the invention, "between at least 50% and up to 90%" means all values between 50% and 90%, and in particular 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% and 90%. The dressing according to the invention must also ensure the absorption or gelling functions of the exudates. When adding the cells to the non-resorbable material or when bio-printing the cells, only a few picoliters of cellular ink are deposited or printed. The cells must be in an environment saturated with moisture or liquid to be able to survive and grow. It is therefore necessary to maintain a certain cell viability, while allowing the dressing to perform these functions. It is therefore important to find a balance between the absorption or gelling of the exudates by the dressing, and the cell survival. Preferably, the dressing should be sufficiently hydrated (but not saturated) so that the cells on its surface survive, and thus facilitate healing. The inventors have found that the dressing according to the present invention responds particularly to this equilibrium when the dressing is saturated with liquid at 90% of its absorption capacity. The absorption capacity of the dressing is measured according to standard NF EN 13726-1.

According to one embodiment of the invention, said dressing comprises a cell concentration of between 50 and 30,000 cells / cm ² , preferably between 200 and 20,000 cells per cm ² .

According to one embodiment of the invention, said dressing further comprises an active agent, preferably an active agent having a favorable role in the treatment of wounds. Advantageously, said active agent is chosen from an antiseptic, an antibacterial agent, an antibiotic, a painkiller, an anti-inflammatory, anesthetic or a compound that promotes the healing of the wound. By way of example, the antibacterials / antibiotics may be silver derivatives such as silver salts or other metals (for example silver sulfate, chloride or nitrate and silver sulfadiazine), complexes of silver or other metals (for example silver zeolites such as alphasan, or ceramics), metodinazole, neomycin, Polymyxin B, penicillins (Amoxycillin), clavulanic acid, tetracyclines, Minocycline, chlorotetracycline, aminoglycosides, Amikacin, Gentamicin or probiotics. The antiseptics may be chlorhexidine, triclosan, biguanide, hexamidine, thymol, Lugol, Povidone iodine, Benzalkonium Chloride and Benzethonium. The painkillers may be Paracetamol, Codeine, Dextropropoxyphene, Tramadol, Morphine and its derivatives, Corticosteroids and derivatives. Anti-inflammatory drugs may be Glucocorticoids, Nonsteroidal Anti-Inflammatory Drugs, Aspirin, Ibuprofen, Ketoprofen, Flurbiprofen, Diclofenac, Aceclofenac, Ketorolac, Meloxicam, Piroxicam, Tenoxicam, Naproxen, Indomethacin, Naproxcinod, Nimesulide, Celecoxib, Etoricoxib, Parecoxib, Rofecoxib, Valdecoxib, Phenylbutazone, Niflumic acid, Mefenamic acid. Other active ingredients promoting healing can also be used, for example retinol, vitamin A, vitamin E, N-acetyl-hydroxyproline, extracts of Centella Asiatica, papain, essential oils of thyme, niaouli, rosemary and sage, hyaluronic acid polysulfated oligosaccharides and their salts (in particular synthetic sulphated oligosaccharides having 1 to 4 unsaturated units such as the potassium salt of octasulfated sucrose or the silver salt of octasulfated sucrose), sucralfate, Allantoin, urea , metformin, enzymes (for example proteolitics such as streptokinase, trypsin or collagenase), peptides or protease inhibitors. Anesthetics such as benzocaine, lidocaine, dibucaine, pramoxine hydrochloride, bupivacaine, mepivacaine, prilocaine, or etidocaine may also be used.

According to one embodiment, the invention also relates to a kit comprising (a) a dressing according to the invention and (b) an asset as mentioned above.

The dressing according to the invention may also comprise any other material conventionally used by those skilled in the field of dressings, for example at least one protective bag or a culture box or any system making it easier to handle and / or his transfer.

In a second aspect, the invention also relates to the method of manufacturing a dressing as defined above. Example 1 illustrates a method for manufacturing a dressing according to the invention.

In one embodiment, the invention thus relates to a method of manufacturing a cellularized dressing as defined above, comprising a step of placing in contact, advantageously a direct contact, cells with a non-absorbable material. This contacting step may consist of a direct application of the cells with the non-absorbable material, an impregnation step or a printing step.

According to a preferred embodiment, the contacting step is a step of bioprinting cells on said non-absorbable material. In particular, the method of manufacture relates to the printing of two cell types (primary fibroblasts and primary keratinocytes) on three materials: an interface dressing, an absorbent dressing, a hydrophilic polyurethane foam (HPU). The bio-printing step is carried out using a bio-ink comprising the cells to be printed.

Advantageously, the method of manufacturing a dressing according to the invention does not include a step of bio-printing a hydrogel. Even more advantageously, the method of manufacturing a dressing according to the invention does not include a step of bio- printing a hydrogel having been mixed with cells or a hydrogel into which cells have been incorporated.

According to a preferred embodiment, the bio-printing of cells is carried out from a bio-ink in which the cells are in suspension or in the form of aggregates. Advantageously, said bio-ink consists of a culture medium comprising a concentration of suspended cells of about 0.1.10 ⁶ to 100.10 ^6, preferably from 1.10 ⁶ to 80.10 ^6. The bio-still may be prepared according to the following protocol: after a cell culture step (for example under standard conditions known to those skilled in the art), the cells intended to be bio-printed are recovered and then centrifuged (for example at 400g for 5 minutes). The cell pellet is then recovered and then suspended in a culture medium at a cell density of 70 × 10 ⁶ cells / ml. The bio-ink may also be in the form of cellular aggregates (or micro-aggregates). In this embodiment, the cell concentration is greater than 100 × 10 ⁶ cells / ml. These aggregates can be, for example, in the form of those described in the patent application WO2016 / 089825.

Advantageously, during the bio-printing step, said non-absorbable material is wet or dry, preferably moist. Even more advantageously, said material is wet or dry when the bio-printing step is performed on the interface dressing. Alternatively, said material is wet when the bio-printing step is performed on the absorbent dressing or on the hydrophilic polyurethane foam.

Before the bio-printing step the dressings can be prepared, in particular cut under sterile conditions if necessary.

Optionally, before the bio-printing step, said dressings may be wetted with a culture medium (for example with 1-2 ml of culture medium for a dressing of about 1.5 cm x 1.5 cm ) and then, if necessary, the excess culture medium can be absorbed. Advantageously, when the interface dressing is moistened, it is preferable to absorb the excess culture medium before the bio-printing step.

According to a preferred embodiment, the method according to the invention comprises the following steps:

optionally, a step of cell culture cells intended to be bio-printed,

optionally, a step of preparing a bio-ink comprising the cells intended to be bio-printed, optionally, a moistening step of nonabsorbable material, using a culture medium,

a step of bio-printing of cells on said non-absorbable material.

According to a preferred embodiment, said non-resorbable material is bio-printed at the level of the fibers of said material or within one or more units defined by the fibers. More particularly, according to the invention, the pattern may typically be a concentric pattern, a radial pattern, a geometric pattern, or a non-geometric random pattern (i.e., not representing a geometric shape), or the interior of at least one of these patterns.

According to a preferred embodiment, said non-resorbable material is bio-printed at the level of the fibers of said material, at the intersection of the fibers of said material and / or in the center of each quadrangle of said material.

In a third aspect, the invention also relates to the use of a dressing as defined above.

The invention thus relates to a method of treating a wound in a patient comprising:

topical administration, on the wound to be treated, of the cellularized dressing as defined above,

optionally, removal of said dressing.

According to one embodiment, said method may also comprise the following steps:

topical administration, on the wound to be treated, of a first cellularized dressing as defined above,

- removal of the first dressing,

topical administration, on the wound to be treated, of a second cellularized dressing as defined above,

- optionally, removal of the second dressing.

In a fourth aspect the invention also relates to a kit for obtaining a cellularized dressing according to the present invention, said kit comprising:

(a) a non-absorbable material, and

(b) cells intended to be contacted with (or bioprinted onto) the non-resorbable material, said cells being in a medium suitable for cell survival. According to the invention, a "suitable medium for cell survival" means, for example, a suitable culture medium. Such media are known to those skilled in the art.

In this aspect the dressing according to the invention may more particularly be adapted to the wound of the patient to be treated, and prepared shortly before its administration.

Said kit intended to obtain a cellularized dressing according to the present invention may also comprise an asset as mentioned above or any other material conventionally used by those skilled in the field of dressings, for example at least one protective bag or a culture box or any system facilitating its handling and / or transfer.

The invention will be better illustrated by the following examples and figures. The following examples are intended to clarify the object of the invention and to illustrate advantageous embodiments. These examples are not intended to restrict the scope of the invention.

FIGURES

Figure 1 shows the first print pattern on the Urgotul® interface dressing: the print spots are positioned at the intersection of the fibers.

Figure 2 shows the second print pattern on the Urgotul® interface dressing: print spots are added on each of the fibers.

3 shows the printing spots of the pattern on the absorbent dressing: the printing spots are positioned at the intersection of the fibers, on the fibers themselves, and in the center of each quadrille.

Figure 4 shows the results of primary fibroblast prints and seeding on the interface, the absorbent dressing and the HPU foam, when the dressings are wet. *** means that p <0.00l.

Figure 5 shows the results of control fibroblast priming impressions and seeding on the interface, the absorbent dressing and the HPU foam, when the dressings are dry. *** means that p <0.00l.

Figure 6 shows the standardized results of Figure 4, where a corresponds to the results obtained with the Urgotul® interface dressing, the absorbent Urgotul Absorb® dressing and the HPU foam. *** means that p <0.00l. Figure 7 shows the standardized results of Figure 5, where a corresponds to the results obtained with the Urgotul® interface dressing, the absorbent Urgotul Absorb® dressing and the HPU foam. *** means that p <0.00l.

Figure 8 shows the results of the primary keratinocyte control prints and sownings on the wet Urgotul® interface and the wet HPU foam. * means that p <0.05.

Figure 9 shows the normalized results of keratinocyte viability in which a represents the results obtained with the Urgotul® interface and b with the HPU foam. * means that p <0.05.

Figure 10 shows the immunolabeling results of collagen I fibroblasts printed on the interface (c, d) and foam HPU (e, f) moistened and control fibroblasts at the bottom of the culture wells (a, b).

FIG. 11 represents the immunolabeling results of the fibronectin synthesized by the fibroblasts printed on the humidified interface (c, d) and the foam HPU (e, f) and by the control fibroblasts at the bottom of the culture wells (a, b).

FIG. 12 represents the immunolabeling results of collagen III synthesized by the fibroblasts printed on the humidified interface (c, d) and foam HPU (e, f) and by the control fibroblasts at the bottom of the culture wells (a, b).

FIG. 13 represents the immunolabeling results of the Ki67 antigen present in the nucleus of the proliferative fibroblasts printed on the humidified interface (c, d) and the HPU foam (e, f) and by the control proliferative fibroblasts (a, b).

Figure 14 represents the percentage of Ki67 labeled cells.

Figure 15 shows the results of prints and control seedings of primary keratinocytes on the interface and the HPU foam. * means that p <0.05.

Figure 16 represents the number of days it took the keratinocytes to migrate from the dressings (the interface and the HPU foam) on which they were printed or witnessed.

FIG. 17 represents the immunolabeling results of the Ki67 antigen present in the nucleus of the proliferative keratinocytes printed on the humidified interface (c, d) and the HPU foam (e, f) and by the control proliferative keratinocytes (a, b).

Figure 18 shows the 100% confluent keratinocyte mat only below the dressing, obtained after 8 days of migration from samples of the HPU foam. EXAMPLES

Example 1 Method of Manufacturing a Dressing According to the Present Invention

The two cell types used are primary dermal fibroblasts and primary epidermal keratinocytes extracted from operative samples (mammoplasty and foreskin).

1. Cell culture

The DMEM fibroblast culture medium is composed of 10% fetal calf serum, 1% antibiotics: penicillin, streptomycin, amphotericin.

The culture medium for keratinocytes is the CNT-PR medium marketed by CellnTec.

The culture media of these two cell types are changed every 2 to 3 days.

2. Preparation of the bio-ink

Before use, the fibroblasts and keratinocytes are detached from the culture flask with 0.25% trypsin / EDTA and fetal calf serum is added after detachment of the cells to stop the enzymatic reaction. Trypan blue counting is performed to enumerate the population and determine cell viability. The cells are then centrifuged at 400 g for 5 minutes. The printing ink is prepared by suspending the cell pellet in culture medium at a density of 70x10 ⁶ cells / ml.

3. Fluorescence labeling of cells

The cells are optionally labeled with a fluorescent cell tracer, the Orange CMRA Dye CellTracker ™ (ThermoFischer Scientific, reference C34551) to visualize the cells after printing. In this case, the cell pellet obtained after trypsination is suspended in the CMRA cell tracer and the cells are put in the incubator for 15 minutes at 37 ° C. and then centrifuged again.

4. Dressing preparation

The three dressings (the Urgotul® interface dressing, the Urgotul Absorb® absorbent dressing and the HPU foam) are cut under sterile conditions, using a scalpel (about 1.5cm x 1.5cm) and positioned in wells of 12-well culture plates. In the case where the dressings are moistened, 1 ml of culture medium is deposited on the Urgotul® interface, and 2 ml is deposited on the absorbent dressing Urgotul Absorb® and the foam HPU which are thicker. After 20 minutes, the culture medium is removed from each of the culture wells containing the dressings to be able to position the culture plate during the printing step. In the case of the Urgotul® interface, it is preferable (and in some cases necessary) to deposit the dressing before printing on a sterile compress so that the excess of culture medium between the grid fibers is absorbed. Indeed, if the medium is still present between the grid of the interface, the imaging system hardly detects the dressing.

5. Cell seeding

The cell bioprinting performed in this example utilizes the laser-assisted bioprotection modality of the printer as described in patent applications WO2011 / 107599, WO2016 / 097619 and WO2016 / 097620. This bioprinting process requires the prior creation of a print file containing all the instructions to be executed by the machine. The pattern (geometry and spacing of points) is part of the information contained in this file. The bio-ink is first deposited on a cartridge consisting of a glass slide covered with a very thin layer of gold. When printing, the laser beam passes through this cartridge and reaches the area of the bio-ink. A cavity is formed and propagated to finally generate a jet that causes the formation of a drop of liquid and its deposit on the recipient. By moving on the donor blade, the laser beam generates drops that are deposited on the recipient in a predefined cell pattern. This laser-assisted bio-printing process is based on the physical phenomenon of material laser interaction and involves many parameters. Some are fixed during the design of the machine (as for example the wavelength of the laser), others can be adjusted by the operator according to the printing conditions (as for example the energy of the laser) .

In this example, the adjustable parameters in Table 1 below have been kept fixed. The reason was thus the only variable parameter during bioprinting. This pattern was created from the image of the receiving substrate and customized according to the geometric characteristics of the support. It was thus possible to print specifically on the grid of the interface and the absorbent dressing.

Table 1: The fixed print parameters during the bio-printing step performed in the examples

Imation system and a software tool

An imaging system and a software tool have been developed to automate the creation of custom print patterns based on observable gridlines on dressing materials. This tool makes it possible to match the observable grids on the dressings (Urgotul® interface and absorbent Urgotul Absorb® dressing) with the printing areas. It is also possible to vary the fiber density by modulating the spacing between the printing spots.

Two patterns were chosen for the Urgotul® interface. On the first, the printing spots are positioned at the intersection of the fibers (Figure 1). The second pattern is created by adding spots on each of the fibers (Figure 2). For the absorbent dressing, the spots of the pattern are located at the intersection of the fibers, on the fibers themselves, and in the center of each quadrille (Figure 3).

The dressing is deposited at the bottom of a well of a culture plate (12-well plate). Then, the imaging system performs image acquisition and reconstruction so as to restore the entire surface of the material (12 photos in total). In the second step, the software binarizes the image obtained in the first step by detecting the dark areas and the light areas. After binarization, the hollows (= centers of the grids) appear in black and the solid ones (= fibers), in white. The coordinates of the centers of the dark areas are then calculated. These points are on a fiber. By iterating in a similar way with the calculated points, it is possible to create patterns with a dot density per variable fiber. The pattern produced by the algorithm can then be loaded into the printer software for use as a template for printing the cells.

The imaging software must fulfill two objectives to be validated.

At first, the calculation must generate a good positioning of the points on the dressing, to reproduce the desired pattern. This objective was fulfilled during the software development. Imaging of the dressing interface can generate images with greater contrast than the absorbent dressing. Inadequate contrast is a source of errors in calculating the positioning of the points, which is the case with the absorbent dressing. An intermediate solution has been found: a pattern correction function has been added to the software. It allows to delete or add points manually and thus to correct errors in the calculation of the reason on a case-by-case basis.

In a second step, it is necessary to validate the correct positioning of the bio-printed drops on the dressing support. To be certain of correctly visualizing the result of the printing, it is primary fibroblasts and primary keratinocytes labeled with fluorescent tracer in the orange that have been printed in place of the initially planned hydrogel. The patterns of keratinocytes printed on the interface using the software makes it possible to specifically position the cell spots on the fibers of the interface. Regardless of the type of cell printed, the software leaves the choice as to the pattern to use. For example, the cell spots can be positioned automatically at the intersection of each fiber of the dressing, or the user can position himself the cell spots at a predefined distance (300-500-800pm ...). In this example, the pattern printed on the HPU foam is a square of 1 cm ² with a spacing between the cell spots (keratinocytes or fibroblasts) of 200 μm.

Example 2: Cell Viability Test

Before printing, the dressings are either dry or moistened. During the inoculation of the controls, 30,000 cells are deposited on each of the dressings in 7.5 μL of culture medium. Immediately after printing or pipetting (for the controls) the cells, the dressings are immersed in 2 ml of culture medium and are "flushed" (in order to recover the maximum of cells on the materials, successive "flush" are made using a pipette). The cells are then labeled and counted on Malassez cell. The marking is performed on the cells directly after printing. The cells are left in culture (post-impression) at least 24 hours before the cell viability test. The keratinocytes or fibroblasts in solution are then inoculated in a new culture well and are placed in an incubator at 37 ° C. and 5% CO ₂ . After 24 hours, the culture medium is removed and the cells are labeled with the solution of calcein and ethidium. The percentage of cell viability is calculated after counting the number of live cells and dead cells in 6 zones per culture well.

Firstly, the "live dead" technique is performed on the primary fibroblasts printed or control on the Urgotul® interface, the absorbent Urgotul Absorb® dressing and the dry and wet HPU foam. The "live dead" technique makes it possible to distinguish live cells from dead cells within the same culture. The ubiquitous intracellular esterase activity and the presence of an intact plasma membrane are the characteristics of living cells. These cells transform the non-fluorescent dye acetoxymethyl calcein (AM) into fluorescent calcein (green). Dead cells are characterized by a loss of the integrity of their plasma membrane. The ethidium homodimer-1 (EthD-1) penetrates these cells and binds to the nucleic acids, which results in the presence of red fluorescence.

In a second step, the cell viability was studied on the printed or control primary keratinocytes on the Urgotul® interface and on the wet HPU foam.

The statistical test used to analyze the cell viability count results is a Student's test whose a value is 0.05.

A. Result of viability of fibroblasts

The results of the primary fibroblast prints and seeding on the interface, the absorbent dressing, and the HPU foam are shown in Figure 4 (with wet dressings) and Figure 5 (with dry dressings). Standardized results are shown in Figures 6 and 7 where a corresponds to the results obtained with the Urgotul® interface dressing, the Urgotul Absorb® absorbent dressing and the HPU foam.

When the fibroblasts are printed on the interface, the absorbent dressing and the HPU foam are moistened, the viability of the fibroblasts is greater than 94%, and also very close to that of the control cells. The printed and control fibroblasts on these 3 wet dressings remain viable. The low value of the standard deviations proves that these results are reproducible.

On the other hand, the viability results of dry dressing impressions are highly variable except for the Urgotul® interface. The viability of the printed or control cells on the dry interface is close to the results on the wet interface. The cells therefore remain viable after printing on the wet or dry Urgotul® interface. The control cells on the absorbent Urgotul Absorb® dry dressing and the dry HPU foam give results of viability comparable to the results on these dressings but wet. The viability results of the fibroblasts printed on the absorbent dressing Urgotul Absorb® are of great variability. The result is 57% ± 46%. Finally, the cells printed on the dry HPU foam have a cell viability of 36%. A little more than half of the cells die after printing on this dry dressing compared to this same wet dressing. Typically, cells do not support environments and dry print media, which may explain this difference in cell viability.

B. Result of the viability of keratinocytes

The results of the primary keratinocyte control prints and seedings on the wet Urgotul® interface and the wet HPU foam are shown in Figure 8. Figure 9 shows the normalized results of keratinocyte viability in which a represents the results obtained with the Urgotul® interface and b with the HPU foam.

The cells printed on the wet Urgotul® interface have viability close to that of the control cells on the interface, with approximately 70% ± 7% viability. The viabilities between the control cells and the printed cells being close, printing on this medium is therefore not the cause of the 30% of dead cells.

The percentage viability of primary keratinocyte impressions on the wet HPU foam is 81% ± 6%. The control cells on this same dressing have a viability percentage of 90% ± 7%. The difference between these two values is significant.

The standardization of the results shows that the viability of the keratinocytes printed on the Urgotul® interface and on the HPU foam are very close to the viability of the controls.

Example 3: Cell Migration Test

Before printing the dressings are either dry or moistened. The control cells are inoculated onto the dressings, with 30,000 cells in 7.5 μl of culture medium. After the step of printing and pipetting (controls) of the cells, the dressings are kept either 30 minutes or 3 hours in an incubator at 37 ° C and 5% CO ₂ . This period is called the shelf life. Each dressing is subsequently inverted (printed side against the culture well) and immersed in 2 ml of culture medium. A stainless steel ring is deposited on each dressing so that it does not float. The culture medium is changed every 2 to 3 days. The dressings are kept in culture for 4 days for primary fibroblasts and 8 days for primary keratinocytes (migration time needed to reach 50% confluence), in order to subsequently label and immunostain the cells that migrated from the dressings onto the plastic surface of the culture wells. The conditions tested with wet dressings are the same as with dry dressings.

A. Results on fibroblast migration

No migration of the printed fibroblasts is observed from the wet Urgotul® interface while the control fibroblasts migrate after only one day.

Printed fibroblasts take 4 to 9 days to migrate from wet Urgotul Absorb® absorbent dressings after 30 minutes or 3 hours post-print. The control cells on this wet dressing put a comparable time to migrate: 4 days and 5 days with a waiting time respectively 30 minutes and 3 hours post-printing.

It only takes one day for the printed and control fibroblasts to migrate from the moist HPU foam with a storage time of 30 minutes. Finally, when this storage time is extended to 3 hours, the control fibroblasts take 5 days to migrate and no migration is observed from the dressings on which the fibroblasts were printed.

Control fibroblasts generally take longer to migrate from the Urgotul® interface, absorbent Urgotul Absorb® dressing and wet HPU foam if the shelf life is 3 hours. This result appears similar on the absorbent Urgotul Absorb® dressing and the HPU foam when the fibroblasts were printed. They take almost twice as long to migrate from the absorbent dressing and they do not migrate from the HPU foam. The storage time of 30 minutes seems more suitable for cells printed on wet dressings.

No migration was observed from the Urgotul® interface, Urgotul Absorb® absorbent dressing and dry HPU foam when the retention time was 3 hours, while the printed and control fibroblasts migrated from all dry dressings when conservation is 30 minutes. This storage time can be increased (beyond 30 minutes) using a larger seeding volume (7.5 μL <V <1 mL), while remaining below the saturation hydration volume for the cellularized dressing. can still play its role of absorbing wound exudates.

The migration time of the bio-printed fibroblasts was studied from the Urgotul® interface, and the wet HPU foam, after a storage time of 30 minutes. of the Additional results were acquired on 30 samples per condition. For each sample, one observes over a period of 4 days if the bio-printed cells migrate out of the dressing.

Under these conditions, for all the HPU foam samples on which the fibroblasts were printed, the migration was observed from 2 days.

The fibroblast migration results from the Urgotul® interface are more variable. The migration is observed after 2 days after printing for a good part of the interface samples (19 out of 30 samples). After 4 days the fibroblasts started to migrate from 4 samples, and no migration was observed since 7 samples.

B. Results on keratinocyte migration

For 50% of the Urgotul® interface dressings on which the keratinocytes were printed and pipetted, no migration was observed. From the remaining 50% of the samples, keratinocyte migration was observed between 2 and 4 days after manual cell printing or seeding. The migration time from the Urgotul® interface is relatively short but this migration is only observed from half of the Urgotul® interface samples.

Since 19 samples of HPU foam on which the keratinocytes were printed or controls, the migration was observed between 2 and 4 days. The migration of the printed keratinocytes was not observed on a sample of HPU foam which is negligible. The migration time of the printed and control keratinocytes from the HPU foam is short and concerns almost all the samples.

The keratinocytes printed on the Urgotul® interface and the HPU foam, just like the control keratinocytes, express for the most part the Ki67 antigen.

After 8 days of migration from samples of the HPU foam, surprisingly, it was observed (for 2 out of 4 wells) a keratinocyte carpet at 100% confluence only below the dressing. On the surface of this carpet, some keratinocytes begin to proliferate and stratify. In this case, the keratinocytes proliferated a lot. By contact inhibition, some of these cells do not express the Ki67 antigen and return to the quiescent phase. Other cells remained proliferative, and continued to divide on the surface of the confluent mat.

The percentage of proliferation (cells that express the Ki67 antigen) is calculated to be able to quantify the expression of the Ki67 antigen and compare the printed cells. with the control cells. The control keratinocytes have a proliferation percentage of 68% ± 18%. This great variability can be explained by a keratinocyte seeding density that is too low (2000 cells / cm ² ).

The percentage of proliferation of keratinocytes printed on the interface is 92%.

The percentage of proliferation of keratinocytes printed on the HPU foam is 80% ± 18%. This result is comparable to the percentage of proliferation of control keratinocytes. The keratinocytes printed on the HPU foam therefore do not undergo any change in their ability to proliferate.

Example 4 Markings and immunostaining on cells

After printing and pipetting fibroblasts or keratinocytes on the Urgotul® interface and the moistened HPU foam with a storage time of 30 minutes, the dressings are turned over (print side against the bottom of the culture well) during 4 days for fibroblasts and 8 days for keratinocytes. The cells are then fixed, then, in order to verify that the cellular metabolism is not affected by the contact of the dressing after printing, immunostaining is performed on the cells.

The markings made are:

-Actin, collagen I, collagen III, fibronectin and Ki67 on fibroblasts,

KI67 on keratinocytes.

The observation of the actin filaments is done using a phalloidin labeling. Phalloidin coupled with a red fluorescent marker (texas red) will bind to the actin filaments and prevent their depolymerization. The actin filaments then appear fluorescent in the red.

The cells are fixed with 4% formaldehyde. The cell membranes are permeabilized with a Triton solution, then treatment with BSA (bovine serum albumin) reduces nonspecific binding. The cells are then labeled with phalloidin and then observed under a fluorescence microscope.

A. Immunolabeling of Collagen I, Collagen III and Fibronectin

Collagen I and III are fibrillar polypeptides synthesized and secreted by primary dermal fibroblasts. Their role is to participate in the elasticity and resistance of the extracellular matrix of the dermis. Fibronectin is a glycoprotein also synthesized and secreted by primary dermal fibroblasts. It participates in adhesion and cell migration in the extracellular matrix.

The three labeled proteins are located in the cell cytoplasm. If no labeling is observed it is because the cells do not express and synthesize the targeted protein.

The cells are fixed and the cell membranes are permeabilized with methanol. The aspecific binding sites are saturated with a solution of BSA, then the cells are labeled first with the primary antibody, then in a second step with the secondary antibody (which binds to the primary antibody to fluoresce) and the Dapi (which marks the cell nuclei in blue). The cells are then observed under a fluorescence microscope.

B. Immunolabeling of Ki67 antigen

Ki67 is the antigen of a nuclear protein present in the proliferating cells in the Gl, S, G2 and M phase. The cells in the quiescent phase GO do not express this nuclear protein. This marking is located in the nucleus of the cells. If some cells do not express this antigen, it is because the cells are not proliferative. In order to quantify the results, the percentage of the number of cells in proliferative phase is calculated.

C. Results

Figure 10 shows the immunolabeling results of collagen I of the fibroblasts printed on the interface (c, d) and the foam HPU (e, f) moistened and control fibroblasts at the bottom of the culture wells (a, b). . The cells printed on the interface and the HPU foam as well as the control cells express collagen I. The intensity of the labeling is stronger in the cytoplasm of some cells, which could be explained by the higher synthesis of collagen I This difference in intensity is observed in the population of fibroblasts printed on both types of dressings (interface and HPU foam) and controls.

Figure 11 shows the immunolabeling results of the fibronectin synthesized by the fibroblasts printed on the humidified interface (c, d) and the foam HPU (e, f) and by the control fibroblasts at the bottom of the culture wells (a). , b). No difference immunolabeling targeting the synthesis of this protein is observed between the printed fibroblasts, and control fibroblasts. The impression on the interface and the HPU foam does not interfere with fibronectin synthesis by fibroblasts.

FIG. 12 shows the immunolabeling results of collagen III synthesized by the fibroblasts printed on the interface (c, d) and the foam HPU (e, f) moistened and by the control fibroblasts at the bottom of the culture wells (a). , b). As with the previous immunolabeling results of collagen I and fibronectin present and synthesized in the cytoplasm of fibroblasts, collagen III is also correctly present in the fibroblasts printed on the interface and the HPU foam and controls. Immunolabeling results, fibroblasts printed on both interface and HPU foam dressings as well as control fibroblasts, are similar. The impression on these two materials does not interfere with the fibroblasts' synthesis of these proteins, which play an essential role in the formation of the extracellular matrix in the dermis. The Ki67 antigen is present only in the nucleus of proliferative cells. Its labeling will make it possible to compare the level of proliferative cells between the fibroblasts printed on the interface and the HPU foam and the control fibroblasts.

Figure 13 shows the immunolabeling results of the Ki67 antigen present in the nucleus of the proliferative fibroblasts printed on the humidified interface (c, d) and the HPU foam (e, f) and by the control proliferative fibroblasts (a). , b). Whatever the condition tested, cells in the quiescence phase (unlabeled nucleus) are observed. In some cases, contact inhibition may explain this non-proliferative state of the cells. From a qualitative point of view, the printed fibroblasts as well as the control fibroblasts express the Ki67 antigen and are therefore, for the most part, in the proliferation phase.

The percentage of labeled cells is calculated in FIG. 14 in order to be able to quantify the expression of the Ki67 antigen and to compare the cells printed with the control cells. The control fibroblasts are mostly proliferative with 83% of the counted cells that express the Ki67 antigen. The results of the printed cells oscillate between 65% and 90% expression of the Ki67 antigen according to the samples. The average level of proliferating cells among the printed cells having migrated from the interface (76% ± 15%) or the HPU foam (81% ± 11%) is comparable to that of the control cells (83% ± 5%). Indeed, the standard deviations of the Ki67 antigen expression percentages of the The fibroblasts printed on the two dressings are relatively large, which brings the results of the printed cells closer to the results of the control cells.

No qualitative and quantitative difference is therefore noted between the cells printed on the interface and the fibroblasts printed on the HPU foam. The proliferative metabolism of fibroblasts printed on these two wet dressing materials works properly. The normalized viability of fibroblasts is very high (greater than 95%) when cells are printed on wet dressings. When the dressings are dry, the viability remains high for the interface but drops significantly for the absorbent dressing and the HPU foam.

Migration test results from wet dressings with a retention time of 30 minutes are the most conclusive. These parameters appear to be the most suitable for the survival and migration of fibroblasts from the dressings on which they were printed.

Markings and immunolabelings give similar results between the printed cells and the control cells. The impression of the fibroblasts on the interface and the wet HPU foam does not modify the synthesis of actin, collagen I and III, fibronectin and Ki67 antigen by the fibroblasts. The metabolism of the primary fibroblasts printed on the interface and the wet HPU foam is thus not modified and remains comparable to the metabolism of the primary non-printed fibroblasts that grow on the surface of a culture well.

All primary keratinocyte prints were made on the humidified interface and HPU foam with a 30 minute storage time after printing in an incubator at 37 ° C with 5% CO ₂ .

The results of the primary keratinocyte priming prints and seeding on the interface and the HPU foam are shown in Figure 15. The cells printed on the interface have viability close to that of the control cells on the interface, with about 70 % ± 7% viability. The viabilities between the control cells and the printed cells being close, printing on this medium is therefore not the cause of the 30% of dead cells. The percentage of viability of primary keratinocyte impressions on the HPU foam is 81% ± 6%. The control cells on this same dressing have a viability percentage of 90% ± 7%. The difference between these two values is significant.

The standardization of the results shows that the viability of the keratinocytes printed on the interface and on the HPU foam are very close to the viability of the controls.

Figure 16 shows the number of days it took the keratinocytes to migrate from the dressings (the interface and the HPU foam) on which they were printed or witnessed. For 50% of the interface dressings on which the keratinocytes were printed and pipetted, no migration is observed. From the remaining 50% of the samples, keratinocyte migration was observed between 2 and 4 days after manual cell printing or seeding. The migration time from the interface is relatively short but this migration is observed only from too few interface samples. Since 19 samples of HPU foam on which the keratinocytes were printed or controls, the migration was observed between 2 and 4 days. The migration of printed keratinocytes has not been observed since only a sample of HPU foam which is negligible. The migration time of the printed and control keratinocytes from the HPU foam is short and concerns almost all the samples.

FIG. 17 shows the immunolabeling results of the Ki67 antigen present in the nucleus of the proliferative keratinocytes printed on the humidified interface (c, d) and the HPU foam (e, f) and by the control proliferative keratinocytes (a). , b). From the observations of Figure 17, the keratinocytes printed on the interface and the HPU foam as well as the control keratinocytes express for the most part the Ki67 antigen. Some cells whose nucleus is blue do not express the K647 antigen and are observed among the printed keratinocytes but also among the control keratinocytes.

After 8 days of migration from samples of the HPU foam, surprisingly, it was observed (for 2 out of 4 wells) a keratinocyte mat at 100% confluence only below the dressing (Figure 18). On the surface of this carpet, some keratinocytes begin to proliferate and stratify. In this case, the keratinocytes proliferated a lot. By contact inhibition, some of these cells do not express the Ki67 antigen and return to the quiescent phase. Other cells remained proliferative, and continued to divide on the surface of the confluent mat. The percentage of proliferation (cells which express the Ki67 antigen) is calculated in FIG. 18 in order to be able to quantify the expression of the Ki67 antigen and to compare the cells printed with the control cells. The control keratinocytes have a proliferation percentage of 68% ± 18%. This great variability can be explained by a keratinocyte seeding density that is too low (2000 cells / cm ² ). The percentage of proliferation of keratinocytes printed on the interface is 92%. The percentage of proliferation of keratinocytes printed on the HPU foam is 80% ± 18%. This result is comparable to the percentage of proliferation of control keratinocytes. The keratinocytes printed on the HPU foam therefore do not undergo any change in their ability to proliferate. The viability of the keratinocytes printed on the interface is close to the viability of the control keratinocytes. After studying the migration of keratinocytes (printed or control) from the interface, it is observed that keratinocytes migrate from the interface only once in two. In cases where the keratinocytes migrated from the interface, the cells grew very well during the 8 to 10 days of migration time and began to cover the surface of the culture well. The results of the percentage proliferation calculations following immunolabelings of the Ki67 antigen indicate that the proliferation of viable keratinocytes is very good. The keratinocytes are for the most part in proliferation phase 8 days after printing on the interface. Seeding by impression or pipetting on the interface appears to affect primary keratinocytes since their viability is lower than that on HPU foam. In contrast, cells migrating from the dressing have a high rate of proliferation. Two hypotheses can explain this phenomenon:

- The first hypothesis is that primary keratinocytes are a fragile cell type. The stress induced by seeding or printing on this material can therefore cause significant mortality, the time that the cells adapt to this material.

Primary keratinocytes certainly have difficulty adhering to this type of support. Differentiated, nonproliferative keratinocytes that have more difficulty adhering may not survive manual printing or seeding on this dressing. The second hypothesis is that the interface selects the keratinocytes whose metabolism is the most efficient with a proliferation capacity most important.

With a viability percentage of 81%, keratinocytes largely survive printing on HPU foam. This result is comparable to the percentage of viability of the control keratinocytes on this same material. Printing as pipetting on this support does not interfere with the survival of primary keratinocytes. The keratinocytes migrate after 2 to 4 days from the HPU foam after the printing or deposition step. the pipette. The cells are not affected by the culture over several days in this dressing. They migrate quickly and colonize the entire surface of the culture well covered by HPU foam. Proliferation of printed keratinocytes proceeds smoothly and appears to increase in contact with HPU foam.

Claims

A cellularized dressing intended to be applied transiently to a wound, said dressing comprising cells on a non-absorbable material.

The cellularized dressing of claim 1, wherein said non-absorbable material is selected from:

an interface dressing,

an absorbent dressing, or

- A hydrophilic polyurethane foam, preferably a hydrophilic polyurethane foam for the absorption of exudates.

The cellularized dressing of any of claims 1-2, wherein the cells are selected from fibroblast-like cells and / or epithelial-like cells.

4. A cellularized dressing according to any one of claims 1-3, wherein the cells are selected from fibroblasts and / or keratinocytes, in particular primary fibroblasts and / or primary keratinocytes.

The cellularized dressing of any of claims 1-4, wherein the cells are bio-printed on the non-resorbable material.

6. A cellularized dressing according to any one of claims 1-5, wherein the cells are present at the fiber of said material or within a pattern or units defined by said fibers.

7. A cellularized dressing according to claim 6, wherein the cells are present at the level of the fibers of a concentric pattern, a radial pattern, a geometric pattern or a non-geometric random pattern, or within at least one of these reasons.

8. The cellularized dressing according to any one of claims 6-7, wherein the cells are present at the level of the fibers of said material, at the intersection of the fibers of said material and / or at the center of each quadrangle of said material.

9. A cellularized dressing according to any one of claims 1-5, said dressing being saturated with liquid up to 90% of its absorption capacity.

10. A cellularized dressing according to any one of claims 1-9, said dressing comprising a cell concentration of between 50 and 30,000 cells / cm ² , preferably between 200 and 20,000 cells / cm ² .

11. A cellularized dressing according to any one of claims 1-10, said dressing further comprising an active agent, preferably selected from an antiseptic, an antibacterial, an antibiotic, a painkiller, an anti-inflammatory, anesthetic or a compound which promotes healing of the wound.

12. A method of manufacturing a cellularized dressing according to any one of claims 1-11, comprising a step of contacting the cells with a non-resorbable material, preferably the contacting step is a step of bio printing cells on said non-absorbable material.

13. A method of manufacturing a cellularized dressing according to claim 12 wherein the bio-printing of cells is carried out from a bio-ink in which the cells are in suspension or in the form of aggregates.

A method of manufacturing a cellularized dressing according to any of claims 12-13, wherein the non-absorbable material is wet or dry.

15. A kit for obtaining a cellularized dressing according to any one of claims 1-11, said kit comprising:

(a) a non-absorbable material, and

(b) cells intended to be brought into contact with the non-resorbable material, said cells being in a medium suitable for cell survival.