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    • 专利摘要:
    The present invention relates to a cellularized dressing and to the method of manufacturing such a dressing, this method preferably comprising a step of bio-printing of cells.
    公开号:FR3082123A1
    申请号:FR1854944
    申请日:2018-06-07
    公开日:2019-12-13
    发明作者:Delphine Fayol；Fabien Guillemot；Catherine Van Der Mee；Christelle Laurensou
    申请人:Poietis；Urgo Recherche Innovation et Developpement；
    IPC主号:
    专利说明:

    CELLULARIZED DRESSING AND MANUFACTURING METHOD THEREOF
    The present invention relates to a cellularized dressing and to the method of manufacturing such a dressing, this method preferably comprising a step of bio-printing of cells.
    Cellularized dressings or skin substitutes have been known and marketed for a long time. The advantage of cellularized dressings lies in the fact that the exogenous supply of living cells participates in wound healing. The cells provided by the dressing participate directly or indirectly (via the secretion of factors) in the healing process.
    Such dressings are generally in the form of at least one absorbable material (i.e. at least one material naturally present in the cellular environment). By way of example, mention may be made of the GRAFIX® products sold by the company OSIRIS. These products are composed of a placental membrane containing an extracellular matrix (ECM) rich in collagen, growth factors, fibroblasts, mesenchymal stem cells and epithelial cells. Mention may also be made of the product APLIGRAF®, marketed by Organogenesis, composed of keratinocytes, fibroblasts and bovine collagen. The DERMAGRAFT® product, marketed by the company Advanced Biohealing, contains dermal derivatives and human fibroblasts.
    Such cellularized dressings, although having proven efficacy on wound healing, however, present risks of transmission of viruses such as, for example, prions (in particular for cellularized dressings containing compounds of animal origin).
    Furthermore, in a cellularized dressing, cell density, the area of localization of cells or the homogeneous distribution of cells are parameters that are poorly controlled.
    There is therefore a need for new cellularized dressings which do not have the drawbacks of the prior art.
    The printing of cells, also called bio-printing, directly on bio-absorbable materials in order to reconstitute a dermis or an epidermis is known and described in patent application WO2016 / 115034 of Wake Forest University or in patent application W020160 / 073782 Organgan. These patent applications relate to the printing of cells and extracellular 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.).
    Biomaterials and tissue engineering are also known to replace a part or function of an organ or tissue.
    Biomaterials are materials, synthetic or living, that can be used for medical purposes to replace a part or function of an organ or tissue. Said biomaterials must comply with several obligations:
    be well tolerated by the recipient, ie do not cause infection, inflammation, allergy, or even rejection reaction if it is living material; do not contain toxic substances, such as endocrine disruptors or carcinogens;
    respond to mechanical constraints to adapt to the pressures exerted by the environment (blood pressure for vascular prostheses, millions of openings and closings for a heart valve, body weight for hip or knee prostheses, ...);
    be able to be shaped, be implantable or injectable, degradable (resorbable) or not depending on the case, possibly porous if they must be colonized once implanted, ...
    Tissue engineering consists of fabricating tissue by multiplying cells around a matrix or scaffold ("scaffold" type). Concrete implementation comes up against various problems, however. For example, in an artificial environment cells tend to lose their ability to differentiate. In addition, cells sometimes express atypical proteins which, after implantation, can cause inflammation or rejection reactions.
    The use for therapeutic purposes 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). However, when this matrix is not absorbable it is intended to be held in place within the body at least for a long period, it is not intended to be removed. The same is true for the use of non-absorbable biomaterials.
    On the contrary, the present invention relates to the use for therapeutic purposes of non-absorbable materials (preferably synthetic materials), but said materials are not intended to replace a part or a function of an organ or tissue. They are not intended to be kept in place within the body, they are intended to be removed after regeneration of the organ or tissue to which they have been applied. According to the invention, the materials thus have a role of transient dressing.
    The bio-impression of cells as described in applications WO2016 / 115034 and W020160 / 073782 is carried out on bio-absorbable materials. Bioprinting methods are also described in applications WO2011 / 107599, WO2016 / 097619 and WO2016 / 097620. These applications describe in particular that bio-printing can be used to produce tissues (for example implantable tissues for regenerative medicine).
    Bioprinting on non-absorbable materials intended to be used temporarily is therefore not described in the prior art.
    In the context of the present invention, cells are thus printed on non-absorbable materials. Said materials are used as dressings. Such materials are not naturally present in the cell environment and are not commonly used in cell culture. Surprisingly, the inventors have found that the cells bio-imprinted on such materials were not only viable, capable of proliferating, but were also capable of migration. The advantage of such an impression or bio-impression is that it is possible to personalize or adapt the dressing to each patient and each wound, thus allowing tailor-made treatment in order to optimize wound healing. Thus, depending on the healing phase in which the wound is located, it is possible to integrate dermis cells and epidermis cells 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. Furthermore, 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. Bioprinting 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 with grid fibers, cells can be printed specifically on the grid, or outside the grid. The precision of this technique is around ten pm. The cell density, the area of localization of the cells and / or the homogeneous distribution of the cells are thus better controlled in the dressings according to the invention compared to 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-absorbable material.
    For the purposes of the present invention, by "cellularized dressing" is meant that the dressing comprises cells.
    According to the invention, the expression "intended to be applied transiently to a wound" means that the dressings are intended to be removed from the wound. The dressings according to the invention indeed have 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 several weeks). Advantageously, the dressing covers all or part of the wound, preferably the whole wound.
    For the purposes of the present invention, by "non-absorbable material" is meant that the material does not gradually disappear within the wound, unlike absorbable materials which themselves decompose naturally. The removal / degradation of a non-absorbable material therefore requires physical / mechanical action, unlike the degradation of an absorbable material.
    Said non-absorbable material advantageously has the following properties: (1) it allows the absorption of exudates, (2) it can undergo a dimensional change (by gelation or deformation linked to absorption), (3) it does not does not adhere to tissue, (4) it is preferably partially hydrophilic in the hydrated state, (5) it exhibits slipperiness in the hydrated state, and (6) it is not cytotoxic. According to the invention, "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.
    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-adhesive cohesive gel formed of a hydrophobic elastomeric matrix consisting of a triblock elastomer of the 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 highly plasticized by means of a mineral oil, and containing in dispersion a small quantity of hydrophilic particles of a hydrocolloid, and (ii) a flexible open-mesh fabric, said fabric comprising threads which are coated with the cohesive gel not adherent so as to leave the meshes essentially unsealed, characterized in that the fabric is a thermofixed knit with woven threads, said threads being cone threads continuous with non-elastic filaments, which has in the transverse direction an extensibility measured according to standard EN 13726-4 of between 0.01 and 0.5 N / cm. According to a preferred embodiment, said non-adherent 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 the styrene (ethylene - butylene) or styrene - (ethylene-propylene) type, 1,000 to 2,000 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 patent application EP2696828, that is to say an absorbent adhesive dressing comprising an absorbent nonwoven (6) and a protective support impermeable to fluids and permeable to water vapor (4), characterized in that: (i) the support consists of the assembly of a continuous film (4a) and an openwork reinforcement coated, on at least one of its faces, with adhesive silicone gel (4b), without sealing the openings of the frame, said frame covering the entire surface of the film, (ii) in that said dressing also comprises a non-absorbent veil (5) and a non complementary woven (7) which are fixed to each other on their periphery by wrapping said absorbent nonwoven, preferably without point of attachment with the latter, and (iii) in that said non-absorbent web (5) sticks with adhesive silicone gel (4b) coated on said frame.
    According to one embodiment of the invention, the cells present within the dressing are cells adherent to a substrate (for example polystyrene in a dish or a culture flask). They are chosen in particular from the cells of the dermis or the 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 the formation of fibers. In cell cultures, many other cell types cannot be distinguished morphologically from fibroblasts. In organ and tissue cultures in which cell relationships are maintained, fibroblasts can be identified using accepted histological criteria. The term "epithelial cells" refers to cells opposite one another which form a continuous mosaic-like tissue with very few intercellular substances as can be seen in in vitro cultures, tissues or d organs. The term "fibroblast-like cells" refers to cells which are attached to a substrate and which appear elongated and bipolar. In cell cultures, various cell types exhibit similar morphologies. Cells that take irregular shapes or spindle shapes are often referred to as fibroblasts. The term "epithelial cells" refers to cells which are attached to a substrate and which appear flat and polygonal in shape. In cell cultures, epithelial cells can take many forms, but tend to form a tissue of tight polygonal cells.
    According to one embodiment of the invention, in said dressing, the cells are (or have been previously) bio-printed on said non-absorbable material. Some dressings have the property of not adhering to wounds, and cells do not adhere to the materials generally used in dressings. It is therefore complicated to bring cells to life on the surface of this type of dressing since the cells will not be able to adhere to it. One of the advantages of bioprinting is that it allows the cells to be printed on the surface of this type of dressing, and to keep them there until the dressing is transferred to the wound. For example, applications WO2016 / 115034, W020160 / 073782 WO2011 / 107599, WO2016 / 097619 and WO2016 / 097620 describe bio-printing methods which can be used to bio-print a dressing according to the invention.
    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, "enters 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 cells to the non-absorbable material or when bioprinting cells, only a few picoliters of cellular ink are deposited or printed. The cells must be in an environment saturated with humidity or even liquid to be able to survive and grow. It is therefore necessary to be able to maintain a certain cellular viability, while allowing the dressing to perform these functions. It is therefore important to find a balance between absorption or gelling of exudates by the dressing, and cell survival. Preferably, the dressing should therefore be sufficiently hydrated (but not at saturation) 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 balance 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 concentration of cells of between 50 and 30,000 cells / cm 2 , preferably between 200 and 20,000 cells per cm 2 .
    According to an embodiment of the invention, said dressing further comprises an active, preferably an active having a favorable role in the treatment of wounds. Advantageously, said active ingredient is chosen from an antiseptic, an antibacterial, an antibiotic, a painkiller, an anti-inflammatory, an anesthetic or a compound which promotes wound healing. For example, the antibacterials / antibiotics can 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), metrodinazole, neomycin, Polymyxin B, penicillins (Amoxycillin), clavulanic acid, tetracyclines, Minocycline, chlorotetracycline, aminoglycosides, Amikacin, Gentamicin or probiotics. Antiseptics can be chlorhexidine, triclosan, biguanide, hexamidine, thymol, Lugol, Povidone iodine, Benzalkonium and Benzethonium Chloride. Painkillers can be Paracetamol, Codeine, Dextropropoxyphene, Tramadol, Morphine and its derivatives, Corticoids and derivatives. Anti-inflammatory drugs can be Glucocorticoids, non-steroidal 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 sulfated oligosaccharides having 1 to 4 dosed 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, tripsine or collagenase), peptides or protease inhibitors. Anesthetics such as benzocaine, lidocaine, dibucaine, pramoxine hydrochloride, bupivacaine, mepivacaine, prilocaine, or etidocaine can 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 active ingredient as mentioned above.
    The dressing according to the invention can also comprise any other material conventionally used by a person skilled in the art in the field of dressings, for example at least one protective sachet or a culture dish or any system making it possible to facilitate its handling 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 process 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 bringing the cells into contact with a non-absorbable material. This contacting step can 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 of cells on said non-absorbable material. In particular, the manufacturing process involves 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.
    According to a preferred embodiment, the bio-printing of cells is carried out using 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 can be prepared according to the following protocol: after a cell culture step (for example under conventional 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, then suspended in a culture medium at a cell density of 70 × 10 6 cells / ml. The bio-ink can 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 patent application WO2016 / 089825.
    Advantageously, during the bioprinting step, said non-absorbable material is wet or dry, preferably wet. Even more advantageously, said material is wet or dry when the bioprinting step is carried out on the interface dressing. Alternatively, said material is wet when the bioprinting step is carried out 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 bioprinting step, said dressings can be moistened using a culture medium (for example with 1-2 ml of culture medium for a dressing of approximately 1.5 cm × 1, 5cm), 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 bioprinting step.
    According to a preferred embodiment, the method according to the invention comprises the following steps:
    optionally, a step of cell culture of the cells intended to be bio-printed, optionally, a step of preparing a bio-ink comprising the cells intended to be bio-printed, optionally, a step of humidifying non-absorbable material, using a culture medium, a step of bio-printing of cells on said non-absorbable material.
    According to a preferred embodiment, said non-absorbable material is bioprinted at the fibers of said material, at the intersection of the fibers of said material and / or at the center of each grid 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 intended to be treated, of the cellularized dressing as defined above, optionally, the removal of said dressing.
    According to one embodiment, said method can also include the following steps:
    topical administration, on the wound intended to be treated, of a first cellularized dressing as defined above, removal of the first dressing, topical administration, on the wound intended to be treated, of a second dressing cellularized as defined above, optionally, removal of the second dressing.
    The invention will be better illustrated by the following examples and figures. The examples below aim to clarify the subject of the invention and to illustrate advantageous embodiments. These examples are not intended to limit 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 printing pattern on the Urgotul® interface dressing: printing spots are added to each of the fibers.
    Figure 3 shows the pattern print spots on the absorbent dressing: the print spots are positioned at the intersection of the fibers, on the fibers themselves, and in the center of each grid.
    FIG. 4 represents the results of the impressions and control inoculations of primary fibroblasts on the interface, the absorbent dressing and the HPU foam, when the dressings are wet. *** means that p <0.001.
    FIG. 5 represents the results of the impressions and control seedings of primary fibroblasts on the interface, the absorbent dressing and the HPU foam, when the dressings are dry. *** means that p <0.001.
    Figure 6 represents the standardized results of Figure 4, where a corresponds to the results obtained with the Urgotul® interface dressing, b the absorbent Urgotul Absorb® dressing and c the HPU foam. *** means that p <0.001.
    Figure 7 represents the standardized results of Figure 5, where "corresponds to the results obtained with the Urgotul® interface dressing, b the absorbent Urgotul Absorb® dressing and c the HPU foam. *** means that p <0.001.
    Figure 8 represents the results of the impressions and control inoculations of primary keratinocytes on the wet Urgotul® interface and the wet HPU foam. * means that p <0.05.
    FIG. 9 represents the normalized results for the viability of keratinocytes in which a represents the results obtained with the Urgotul® interface and b with HPU foam. * means that p <0.05.
    FIG. 10 represents the results of immunolabelling of collagen I of the fibroblasts printed on the interface (c, d) and the HPU foam (e, f) humidified and of control fibroblasts at the bottom of the culture wells (a, b).
    FIG. 11 represents the results of immunostaining of fibronectin synthesized by the fibroblasts printed on the interface (c, d) and the HPU foam (e, f) humidified and by the control fibroblasts at the bottom of the culture wells (a, b).
    FIG. 12 represents the results of immunolabelling of collagen III synthesized by the fibroblasts printed on the interface (c, d) and the HPU foam (e, f) humidified and by the control fibroblasts at the bottom of the culture wells (a, b).
    FIG. 13 represents the results of immunostaining of the Ki67 antigen present in the nucleus of proliferative fibroblasts imprinted on the interface (c, d) and the HPU foam (e, f) moistened and by the control proliferative fibroblasts (a, b).
    Figure 14 shows the percentage of cells labeled with Ki67.
    FIG. 15 represents the results of the impressions and control seedings of primary keratinocytes on the interface and the HPU foam. * means that p <0.05.
    Figure 16 represents represents the number of days that it took for the keratinocytes to migrate from the dressings (the interface and the HPU foam) on which they were printed or controls.
    FIG. 17 represents the results of immunostaining of the Ki67 antigen present in the nucleus of the proliferative keratinocytes printed on the interface (c, d) and the HPU foam (e, f) moistened and by the control proliferative keratinocytes (a, b).
    Figure 18 shows the carpet of keratinocytes at 100% confluence 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 (breast plasties and foreskin).
    1. Cell culture
    The culture medium for DMEM fibroblasts, is composed of 10% fetal calf serum, 1% antibiotics: penicillin, streptomycin, amphotericin.
    The culture medium for keratinocytes is the CNT-PR medium sold by the company CellnTec.
    The culture media of these two cell types are changed every 2 to 3 days.
    2. Preparation of 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. A count with trypan blue is carried out to count the population and determine the 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 the density of 70 × 10 6 cells / ml.
    3. Fluorescent labeling of cells
    The cells are optionally labeled with a fluorescent cell tracer, the orange CellTracker ™ CMRA Dye (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., then again centrifuged.
    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 (approximately 1.5cm x 1.5cm) and positioned in the 12 well culture plate wells. In the case where the dressings are moistened, ImL of culture medium is deposited on the Urgotul® interface, and 2 ml is deposited on the absorbent dressing Urgotul Absorb® and the HPU foam which are thicker. After 20 minutes, the culture medium is removed from each of the culture wells containing the dressings in order 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 place the dressing before printing on a sterile pad so that the excess culture medium between the gridded 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 bio-printing of cells carried out in this example uses the laser-assisted bio-printing mode of the printer as described in patent applications WO2011 / 107599, WO2016 / 097619 and WO2016 / 097620. This bio-printing process requires the prior creation of a print file containing all of the instructions to be executed by the machine. The pattern (geometry and dot spacing) is part of the information 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. During printing, the laser beam passes through this cartridge and reaches the bio-ink area. A cavity is formed and propagates to finally generate a jet which causes the formation of a drop of liquid and its deposit on the receiver. By moving on the donor slide, the laser beam generates drops which are deposited on the recipient according to a predefined cellular pattern. This laser-assisted bio-printing process is based on the physical phenomenon of laser matter interaction and involves many parameters. Some are fixed during the design of the machine (such as the laser wavelength), others can be adjusted by the operator depending on the printing conditions (such as the laser energy) .
    In this example, the adjustable parameters in Table 1 below have been kept at a fixed value. The motif was therefore the only variable parameter during bio-printing. This pattern was created from the image of the receiving substrate and personalized 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.
    Energy Pulse duration Cell concentration in bio-ink Distance to focal plane Thickness of the bio-ink layer Donor / recipient distance Volume of ink deposited on the donor slide 30pJ 200ns (T8) 70 millioncells / ml -100 pm 45 pm 500pm Between 8 and8,5pL
    Table 1: The fixed printing parameters during the bioprinting step carried out in the examples
    Imaging system and software tool
    An imaging system and a software tool have been developed in order to automatically create personalized printing patterns based on the grid visible on the dressing materials. This tool makes it possible to match the grids observable on the dressings (Urgotul® interface and absorbent dressing Urgotul Absorb®) with the printing areas. It is also possible to vary the cell density per fiber by modulating the spacing between the printing spots.
    Two patterns have been chosen for the Urgotul® interface. On the first, the printing spots are positioned at the intersection of the fibers (Figure 1). A second pattern is created by adding spots to each of the fibers (Figure 2). For the absorbent dressing, the pattern spots are located at the intersection of the fibers, on the fibers themselves, and in the center of each grid (Figure 3).
    The dressing is deposited at the bottom of a well of a culture plate (12-well plate). Then, the imaging system performs an image acquisition and reconstruction so as to restore the entire surface of the material (12 photos in total). During 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 (= fibers), in white. The coordinates of the centers of the dark areas are then calculated. These points are on a fiber. By iterating analogously with the calculated stitches, it is possible to create patterns with a variable stitch density per fiber. The pattern produced by the algorithm can then be loaded into the printer software in order to use it as a template for printing 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, in order to reproduce the desired pattern. This objective was fulfilled during the development of the software. The imaging of the interface dressing makes it possible to generate images with greater contrast than with the absorbent dressing. Insufficient contrast is a source of errors in the calculation of the positioning of the points, which is the case with the absorbent dressing. An intermediate solution was found: a pattern correction function was added to the software. It allows to manually delete or add points and therefore to correct on a case by case basis errors in the calculation of the pattern.
    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 impression, it is primary fibroblasts and primary keratinocytes marked with fluorescent tracer in orange which have been printed in place of the hydrogel initially intended. 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. Whatever the type of cell printed, the software leaves the choice as to the pattern to be used. For example, the cell spots can be positioned automatically at the intersection of each fiber of the dressing, or the user can position the cell spots himself 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 seeding of the controls, 30,000 cells are deposited on each of the dressings in 7.5 μL of culture medium. Immediately after printing or depositing the cells with the pipette (for the controls), the dressings are immersed in 2 ml of culture medium and are "flushed" (in order to recover the maximum number of cells from the materials, successive "flushes" are using a pipette). The cells are then labeled and counted on a Malassez cell. The marking is carried out on the cells directly after printing. The cells are left in culture (post-printing) minimum 24 hours before doing the cell viability test. The keratinocytes or fibroblasts in solution are then seeded 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 living cells and dead cells in 6 zones per culture well.
    First, the “live dead” technique is performed on primary fibroblasts printed or control on the Urgotul® interface, the absorbent dressing Urgotul Absorb® and dry and wet HPU foam. The “live dead” technique makes it possible to distinguish living cells from dead cells within the same culture. Ubiquitous intracellular esterase activity and the presence of an intact plasma membrane are 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. Ethidium homodimer-1 (EthD-1) enters these cells and binds to nucleic acids, which results in the presence of red fluorescence.
    In a second step, cell viability was studied on primary keratinocytes printed or control on the Urgotul® interface and on wet HPU foam.
    The statistical test used to analyze the cell viability count results is a Student test, the value of a is 0.05.
    A. Result of fibroblast viability
    The results of the impressions and control inoculations of primary fibroblasts on the interface, the absorbent dressing and the HPU foam are shown in FIG. 4 (with wet dressings) and FIG. 5 (with dry dressings). The standardized results are presented in Figures 6 and 7 where a corresponds to the results obtained with the Urgotul® interface dressing, b the Urgotul Absorb® absorbent dressing and c the HPU foam.
    When the fibroblasts are printed on the interface, the absorbent dressing and the HPU foam 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 results of viabilities of the impressions on dry dressings are very variable apart from 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 dry Urgotul Absorb® absorbent dressing and the dry HPU foam give viability results comparable to the results on these same but moist dressings. The viability results of fibroblasts printed on the absorbent dressing Urgotul Absorb® are highly variable. The result is 57% ± 46%. Finally, the cells printed on the dry HPU foam have a cell viability of 36%. Slightly more than half of the cells die after being printed on this dry dressing compared to this same wet dressing. Generally, cells do not support dry environments and print media, which may explain this difference in cell viability.
    B. Result of the viability of keratinocytes
    The results of the impressions and control seedings of primary keratinocytes on the wet Urgotul® interface and the wet HPU foam are represented in FIG. 8. FIG. 9 presents the normalized results of the viability of the keratinocytes in which a represents the results obtained with Urgotul® and b interface with HPU foam.
    The cells printed on the wet Urgotul® interface have a viability close to that of the control cells on the interface, with approximately 70% ± 7% of viability. The viabilities between the control cells and the printed cells being close, printing on this support is therefore not the cause of the 30% of dead cells.
    The percentage of viability of the impressions of primary keratinocytes on the wet HPU foam is 81% ± 6%. The control cells on this same dressing have a percentage of viability of 90% ± 7%. The difference between these two values is significant.
    The normalization 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 seeded on the dressings, with 30,000 cells in 7.5 μL of culture medium. After the printing and pipetting step (controls) of the cells, the dressings are kept either 30 minutes or 3 hours in an incubator at 37 ° C and 5% CO2. This period is called the shelf life. Each dressing is then inverted (printed side against the culture well) and immersed in 2 ml of culture medium. A stainless steel ring is placed on each dressing so that it does not float. The culture medium is changed every 2 to 3 days. The dressings are maintained in culture for 4 days for primary fibroblasts and 8 days for primary keratinocytes (migration time required to reach 50% confluence), in order to be able to subsequently mark and immunolabel the cells which have migrated from the dressings on 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, whereas the control fibroblasts migrate after only one day.
    It takes 4 and 9 days for the printed fibroblasts to migrate from the wet Urgotul Absorb® absorbent dressings after a waiting time of 30 minutes or 3 hours after printing. The control cells on this wet dressing take a comparable time to migrate: 4 days and 5 days with a waiting time of 30 minutes and 3 hours respectively after printing.
    It takes only one day for the printed and control fibroblasts to migrate from the wet 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 have been printed.
    Control fibroblasts generally take longer to migrate from the Urgotul® interface, the absorbent dressing Urgotul Absorb® and wet HPU foam if the storage time is 3 hours. This result seems similar on the absorbent dressing Urgotul Absorb® 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 HPU foam. The storage time of 30 minutes therefore seems more suited to the cells printed on the wet dressings.
    No migration is observed from the Urgotul® interface, the Urgotul Absorb® absorbent dressing and the dry HPU foam when the storage time is 3 hours while the printed and control fibroblasts migrate from all dry dressings when the conservation is 30 minutes. This storage time can be increased (beyond 30 minutes) by using a larger seeding volume (7.5 pL <V <lmL), while remaining below the volume of hydration at saturation for the cellularized dressing. can always play its role of absorbing exudates from the wound.
    The migration time of bioprinted fibroblasts was studied from the Urgotul® interface, and wet HPU foam, after a storage time of 30 minutes. Complementary results were thus acquired on 30 samples per condition. For each sample, it is observed over a period of 4 days whether the bioprinted cells migrate out of the dressing.
    Under these conditions, for all the samples of HPU foam on which the fibroblasts were printed, the migration was observed from 2 days.
    Fibroblast migration results from the Urgotul® interface are more variable. Migration is observed after 2 days after printing for a large part of the interface samples (19 samples out of 30). After 4 days the fibroblasts started to migrate from 4 samples, and no migration was observed from 7 samples.
    B. Results on the migration of keratinocytes
    For 50% of the Urgotul® interface dressings on which the keratinocytes were printed and deposited with a pipette, no migration was observed. From the remaining 50% of samples, migration of keratinocytes was observed between 2 and 4 days after printing or manual seeding of the cells. The migration time from the Urgotul® interface is relatively short, but this migration is only observed from half of the Urgotul® interface samples.
    From 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 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 of the samples.
    The keratinocytes printed on the Urgotul® interface and the HPU foam as well as the control keratinocytes mostly express the Ki67 antigen.
    After 8 days of migration from samples of the HPU foam, surprisingly, it was observed (for 2 wells out of 4) a carpet of keratinocytes 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 have entered the quiescence phase. Other cells remained proliferative, and continued to divide on the surface of the confluent carpet.
    The percentage of proliferation (cells which express the Ki67 antigen) is calculated in order to be able to quantify the expression of the Ki67 antigen and compare the printed cells with the control cells. Control keratinocytes have a proliferation percentage of 68% ± 18%. This great variability can be explained by a too low seeding density of keratinocytes (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 capacity to proliferate.
    EXAMPLE 4 Labeling and Immunolabeling on Cells
    After printing and pipetting the fibroblasts or keratinocytes on the Urgotul® interface and the HPU foam moistened with a storage time of 30 minutes, the dressings are turned over (printing side against the bottom of the culture well) for 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 carried out on the cells.
    The markings made are:
    -Actin, collagen I, collagen III, fibronectin and Ki67 on fibroblasts,
    -The ki67 on keratinocytes.
    The actin filaments are observed using phalloidin labeling. The 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 red.
    The cells are fixed with 4% formaldehyde. The cell membranes are permeabilized using a Triton solution, then treatment with BSA (bovine serum albumin) makes it possible to reduce the non-specific fixation. The cells are then labeled with phalloidin and then observed under a fluorescence microscope.
    A. Immunolabels of collagen I, collagen III and fibronectin
    Collagen I and III are fibrillar polypeptides synthesized and secreted by the primary fibroblasts of the dermis. Their role is to participate in the elasticity and resistance of the extra cellular matrix of the dermis.
    Fibronectin is a glycoprotein also synthesized and secreted by the primary fibroblasts of the dermis. It participates in cell adhesion and migration in the extra cellular matrix.
    The three labeled proteins are located in the cell cytoplasm. If no labeling is observed, 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 BSA solution, then the cells are first labeled with the primary antibody, then secondly with the secondary antibody (which binds to the primary antibody to fluoresce) and Dapi (which marks cell nuclei in blue). The cells are then observed under a fluorescence microscope.
    B. Immunostaining of the Ki67 antigen
    Ki67 is the antigen of a nuclear protein present in proliferative cells in the Gl, S, G2 and M phases. Cells in the GO quiescence phase do not express this nuclear protein. This labeling is located in the nucleus of the cells. If certain 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 the proliferative phase is calculated.
    C. Results
    FIG. 10 presents the results of immunolabelling of collagen I of fibroblasts printed on the interface (c, d) and the humidified HPU foam (e, f) and of 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 greater synthesis of collagen I This difference in intensity is observed in the population of fibroblasts imprinted on the two types of dressings (interface and HPU foam) and controls.
    FIG. 11 presents the results of immunostaining of fibronectin synthesized by the fibroblasts printed on the interface (c, d) and the HPU foam (e, f) moistened and by the control fibroblasts at the bottom of the culture wells (a , b). No difference in immunostaining targeting the synthesis of this protein is observed between the printed fibroblasts and the control fibroblasts. The printing on the interface and the HPU foam therefore does not disturb the synthesis of fibronectin by fibroblasts.
    FIG. 12 presents the results of immunolabelling of collagen III synthesized by the fibroblasts printed on the interface (c, d) and the HPU foam (e, f) moistened and by the control fibroblasts at the bottom of the culture wells (a , b). As with the previous results of immunolabelling of collagen I and fibronectin present and synthesized in the fibroblast cytoplasm, collagen III is also correctly present in fibroblasts printed on the interface and the HPU and control foam. The results of immunolabelling, of fibroblasts printed on the two interface dressings and HPU foam as well as of control fibroblasts, are similar. Printing on these two materials therefore does not disturb the synthesis, by fibroblasts, of these proteins which have an essential role in the formation of the extra cellular matrix in the dermis. The Ki67 antigen is only present in the nucleus of proliferative cells. Its labeling will make it possible to compare the rate of proliferative cells between the fibroblasts printed on the interface and the HPU foam and the control fibroblasts.
    FIG. 13 presents the results of immunostaining of the Ki67 antigen present in the nucleus of proliferative fibroblasts imprinted on the interface (c, d) and the HPU foam (e, f) moistened and by the control proliferative fibroblasts (a , b). Whatever the condition tested, cells in the quiescent phase (unmarked 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 Figure 14 to be able to quantify the expression of the Ki67 antigen and compare the printed cells with the control cells. The control fibroblasts are for the most part proliferative with 83% of the cells counted which express the Ki67 antigen. The results of the printed cells oscillate between 65% and 90% of expression of the Ki67 antigen depending on the samples. The average rate of proliferative cells among printed cells having migrated from the interface (76% ± 15%) or HPU foam (81% ± 11%) is comparable to that of control cells (83% ± 5%). Indeed, the standard deviations of the percentages of expression of the Ki67 antigen of 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 to be noted between the cells printed on the interface and the fibroblasts printed on the HPU foam. The proliferative metabolism of fibroblasts imprinted on these two wet dressing materials is functioning properly. The normalized fibroblast viability is very high (greater than 95%) when the cells are printed on the wet dressings. When the dressings are dry, the viability remains high for the interface but drops significantly for the absorbent dressing and HPU foam.
    The results of migration tests from wet dressings with a storage time of 30 minutes are the most conclusive. These parameters seem to be the most suitable for the survival and migration of fibroblasts from the dressings on which they were printed.
    Labeling and immunostaining give similar results between the printed cells and the control cells. The printing of fibroblasts on the wet interface and HPU foam does not modify the synthesis of actin, collagen I and III, fibronectin and Ki67 antigen by fibroblasts. The metabolism of primary fibroblasts printed on the interface and the wet HPU foam is therefore not modified and remains comparable to the metabolism of primary non-printed fibroblasts which grow on the surface of a culture well.
    All primary keratinocyte prints are made on the interface and the HPU foam moistened with a storage time of 30 minutes after printing in an incubator at 37 ° C with 5% CO2.
    The results of the printing and control seedings of primary keratinocytes on the interface and the HPU foam are shown in Figure 15. The cells printed on the interface have a 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 support is therefore not the cause of the 30% of dead cells. The percentage of viability of the impressions of primary keratinocytes on the HPU foam is 81% ± 6%. The control cells on this same dressing have a percentage of viability of 90% ± 7%. The difference between these two values is significant. The normalization 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 for keratinocytes to migrate from the dressings (interface and HPU foam) on which they were printed or control. For 50% of the interface dressings on which the keratinocytes were printed and deposited with the pipette, no migration was observed. From the remaining 50% of samples, migration of keratinocytes was observed between 2 and 4 days after printing or manual seeding of the cells. The migration time from the interface is relatively short but this migration is observed only from too few interface samples. From 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 was not observed from only one 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 of the samples.
    Figure 17 presents the results of immunostaining of the Ki67 antigen present in the nucleus of proliferative keratinocytes printed on the interface (c, d) and the HPU foam (e, f) moistened and by the control proliferative keratinocytes (a , b). According to the observations in Figure 17, the keratinocytes printed on the interface and the HPU foam as well as the control keratinocytes mostly express the Ki67 antigen. Some cells with a blue nucleus do not express the K647 antigen and are observed among printed keratinocytes but also among control keratinocytes.
    After 8 days of migration from samples of the HPU foam, surprisingly, it was observed (for 2 wells on 4) a carpet of keratinocytes 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 have entered the quiescence phase. Other cells remained proliferative, and continued to divide on the surface of the confluent carpet.
    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 compare the printed cells with the control cells. Control keratinocytes have a proliferation percentage of 68% ± 18%. This great variability can be explained by a too low seeding density of keratinocytes (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 capacity 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 the keratinocytes only migrate from the interface once in two. In cases where the keratinocytes migrated from the interface, the cells grew very well during the 8-10 days of migration time and began to cover the surface of the culture well. The results of the percentage proliferation calculations following the immunolabelling of the Ki67 antigen indicate that the proliferation of viable keratinocytes is very good. The keratinocytes are for the most part in the proliferation phase 8 days after printing on the interface. Seeding by printing or by pipetting on the interface seems to affect the 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 a seeding or an impression on this material can thus cause a significant mortality, the time that the cells adapt to this material.
    - Primary keratinocytes certainly have difficulty adhering to this type of support. Differentiated, non-proliferative keratinocytes that have more difficulty adhering may not survive printing or manual seeding on this dressing. The second hypothesis is therefore that the interface selects the keratinocytes whose metabolism is the most efficient with the greatest proliferation capacity.
    With a viability percentage of 81%, keratinocytes largely survive printing on HPU foam. This result is comparable to the percentage of viability of control keratinocytes on this same material. Printing, just like pipetting on this support, does not disturb the survival of primary keratinocytes. The keratinocytes migrate after 2 to 4 days from the HPU foam after the printing or pipetting step. The cells are therefore 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. The proliferation of printed keratinocytes takes place correctly and seems to increase in contact with the HPU foam.
    权利要求:
    Claims (11)
    [1" id="c-fr-0001]
    1. Cellularized dressing in a form suitable for transient application on a wound, said dressing comprising cells on a non-absorbable material.
    [2" id="c-fr-0002]
    2. A cellularized dressing according to claim 1, in which said non-absorbable material is chosen from:
    an interface dressing, an absorbent dressing, or
    - a hydrophilic polyurethane foam.
    [3" id="c-fr-0003]
    3. A cellularized dressing according to claim 1, in which the cells are chosen from fibroblast type cells and / or epithelial type cells.
    [4" id="c-fr-0004]
    4. Cellularized dressing according to claim 1, in which the cells are chosen from fibroblasts and / or keratinocytes, in particular primary fibroblasts and / or primary keratinocytes.
    [5" id="c-fr-0005]
    5. A cellularized dressing according to any one of claims 1-4, wherein the cells are bio-imprinted on the non-absorbable material.
    [6" id="c-fr-0006]
    6. A cellularized dressing according to any one of claims 1-5, said dressing being saturated with liquid up to 90% of its absorption capacity.
    [7" id="c-fr-0007]
    7. A cellularized dressing according to any one of claims 1-6, said dressing comprising a concentration of cells of between 50 and 30,000 cells / cm 2 , preferably between 200 and 20,000 cells / cm 2 .
    [8" id="c-fr-0008]
    8. A cellularized dressing according to any one of claims 1-7, said dressing further comprising an active, preferably chosen from an antiseptic, an antibacterial, an antibiotic, a painkiller, an anti-inflammatory, an anesthetic or a compound which promotes wound healing.
    [9" id="c-fr-0009]
    9. A method of manufacturing a cellularized dressing according to any one of claims 1-8, comprising a step of bringing the cells into contact with a non-absorbable material, preferably the contacting step being a bio step. printing of cells on said non-absorbable material.
    [10" id="c-fr-0010]
    10. A method of manufacturing a cellularized dressing according to claim 9 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.
    [11" id="c-fr-0011]
    11. A method of manufacturing a cellularized dressing according to any one of claims 9-10, wherein the non-absorbable material is wet or dry.
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    同族专利:
    公开号 | 公开日
    FR3082123B1|2020-10-16|
    WO2019234365A1|2019-12-12|
    US20210220510A1|2021-07-22|
    EP3801652A1|2021-04-14|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2010112678A1|2009-04-01|2010-10-07|Helsingin Yliopisto|Regenerative matrix comprising cells activated by nemosis and/or factors released from such cells|
    WO2016115034A1|2015-01-12|2016-07-21|Wake Forest University Health Sciences|Multi-layer skin substitute products and methods of making and using the same|
    ES2671594T3|2010-03-04|2018-06-07|INSERM |Bioprinting station, assembly comprising said bioprinting station and bioprinting method|
    FR2974005B1|2011-04-15|2014-05-02|Urgo Lab|THIN ABSORBENT THIN ADHESIVE DRESSING, ITS USES FOR THE TREATMENT OF CHRONIC WOUNDS|
    WO2013093213A1|2011-12-19|2013-06-27|Laboratoires Urgo|Adherent interface dressing|
    JP2017537654A|2014-11-05|2017-12-21|オルガノボ インコーポレイテッド|Artificial three-dimensional skin tissue, array thereof, and manufacturing method thereof|
    US10926001B2|2014-12-02|2021-02-23|Polarityte, Inc.|Methods related to minimally polarized functional units|
    FR3030360B1|2014-12-17|2018-07-13|Universite de Bordeaux|LASER PRINTING METHOD AND DEVICE FOR IMPLEMENTING SAME|
    FR3030361B1|2014-12-17|2017-01-20|Univ Bordeaux|METHOD FOR PRINTING BIOLOGICAL ELEMENTS BY LASER AND DEVICE FOR IMPLEMENTING SAID METHOD|GB201020236D0|2010-11-30|2011-01-12|Convatec Technologies Inc|A composition for detecting biofilms on viable tissues|
    EP2648793B1|2010-12-08|2020-03-11|ConvaTec Technologies Inc.|Integrated system for assessing wound exudates|
    ES2748519T3|2010-12-08|2020-03-17|Convatec Technologies Inc|Wound exudate system accessory|
    GB2497406A|2011-11-29|2013-06-12|Webtec Converting Llc|Dressing with a perforated binder layer|
    WO2018009873A1|2016-07-08|2018-01-11|Convatec Technologies Inc.|Fluid collection apparatus|
    法律状态:
    2019-06-25| PLFP| Fee payment|Year of fee payment: 2 |
    2019-12-13| PLSC| Search report ready|Effective date: 20191213 |
    2020-06-22| PLFP| Fee payment|Year of fee payment: 3 |
    2021-05-26| PLFP| Fee payment|Year of fee payment: 4 |
    优先权:
    申请号 | 申请日 | 专利标题
    FR1854944|2018-06-07|
    FR1854944A|FR3082123B1|2018-06-07|2018-06-07|CELLULARIZED DRESSING AND ITS MANUFACTURING PROCESS|FR1854944A| FR3082123B1|2018-06-07|2018-06-07|CELLULARIZED DRESSING AND ITS MANUFACTURING PROCESS|
    US16/972,105| US20210220510A1|2018-06-07|2019-06-06|Cellularised Dressing and Method for Producing Same|
    PCT/FR2019/051367| WO2019234365A1|2018-06-07|2019-06-06|Cellularised dressing and method for producing same|
    EP19740640.8A| EP3801652A1|2018-06-07|2019-06-06|Cellularised dressing and method for producing same|
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