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    • 专利摘要:
    The invention relates to a composite biomaterial based on collagen, at least one hydrophobic organic polymer and at least one active principle, its preparation method, a dressing comprising such a composite biomaterial, an abdominal wall reinforcement comprising such a composite biomaterial, as well as the uses of said composite biomaterial, particularly in the therapeutic field.
    公开号:FR3047901A1
    申请号:FR1651432
    申请日:2016-02-22
    公开日:2017-08-25
    发明作者:Christophe Helary；Thibaud Coradin
    申请人:Centre National de la Recherche Scientifique CNRS；Universite Pierre et Marie Curie Paris 6；
    IPC主号:
    专利说明:

    Exemple 2 : préparation d'autres biomatériaux composites synthétiques conformes au premier objet de l'invention
    Des biomatériaux composites M2.Hr, M3.Hr et M4.Hr ont été préparés en imprégnant l'hydrogel déshydraté de collagène pur Ma-hd tel qu'obtenu dans l'exemple 1.3 ci-dessus avec les solutions d'imprégnation suivantes : - une solution comprenant 10'2 M environ de spironolactone et 160 mg/ml environ de PLGA 7-17 KDa, - une solution comprenant 10'2 M environ de spironolactone et 160 mg/ml environ de PLGA 24-38 KDa, et - une solution comprenant 10'2 M environ de spironolactone et 160 mg/ml environ de PCL 14 KDa.
    Les étapes d'imprégnation, de rinçage, de lyophilisation et de réhydratation sont identiques à celles décrites dans l'exemple 1.4 ci-dessus.
    La figure 2 représente le diamètre (en mm) des biomatériaux composites sous la forme d'hydrogels composites conformes à l'invention Mi-r, M2-hr, M3.Hr et M4.hr et à titre comparatif le diamètre d'un hydrogel de collagène pur non conforme à l'invention Ma-h tel qu'obtenu dans l'exemple 1.2 et d'un hydrogel de collagène pur Ma-hr tel qu'obtenu dans l'exemple 1.3.
    La figure 2 montre que la présence dans l'hydrogel d'un principe actif et d'un polymère organique hydrophobe tels que définis dans l'invention n'a que peu ou pas d'influence sur le diamètre de l'hydrogel obtenu. Les hydrogels composites ont donc une bonne capacité à s'hydrater, proche de celle du collagène pur et ne se rétractent pas.
    La figure 3 représente la masse de polymère organique (en mg) présente dans les biomatériaux composites sous la forme de matériaux composites secs conformes à l'invention Mi-S/ M2-s, M3.s et M4.s.
    La figure 3 montre que le biomatériau composite de l'invention peut incorporer une quantité de polymère organique plus importante lorsque le PLGA 7-17 KDa est utilisé (M2-s)· Les capacités d'intégration des polymères organiques testés sont toutefois convenables et sont supérieures à 15 mg environ, et vont de préférence de 20 à 31 mg environ, pour une masse de collagène de l'ordre de 25 mg.
    La figure 4 représente le gonflement en volume des biomatériaux composites sous la forme d'hydrogels composites conformes à l'invention Mi-hr, M2.Hr, M3.Hr et M4-hr et à titre comparatif d'un hydrogel de collagène pur Ma-Hr tel qu'obtenu dans l'exemple 1.3 (en % par rapport au volume initial de l'hydrogel de collagène pur MA.H).
    La figure 4 montre une bonne capacité de gonflement des hydrogels composites conformes à l'invention (55% à 80% environ), voire similaire à celle d'un hydrogel de collagène pur lorsque le PLGA 7-17 KDa est utilisé.
    La figure 5 représente l'élongation à la rupture des biomatériaux composites sous la forme d'hydrogels composites conformes à l'invention Mi-hr, M2-Hr, M3.Hr et M4_hr, et à titre comparatif celle d'un hydrogel de collagène pur Ma.Hr tel qu'obtenu dans l'exemple 1.3 (en % de la longueur initiale de chacun des matériaux testés).
    La figure 6 représente la résistance à la rupture (en MPa) des biomatériaux composites sous la forme d'hydrogels composites conformes à l'invention Mi-Hr, M2-hr, M3.Hr et M4.Hr, et à titre comparatif celle d'un hydrogel de collagène pur Ma-hr tel qu'obtenu dans l'exemple 1.3.
    La figure 7 représente l'élasticité (en Pa) des biomatériaux composites (Module de Young) sous la forme d'hydrogels composites conformes à l'invention Mi-Hr, M2-hr, M3.Hr et M4.Hr, et à titre comparatif celle d'un hydrogel de collagène pur Ma-hr tel qu'obtenu dans l'exemple 1.3.
    La figure 8 représente le module de cisaillement (en Pa) à différentes fréquences (1 Hz et 10 Hz environ) du biomatériau composite sous la forme d'un hydrogel composite conforme à l'invention Mi-Hr, et à titre comparatif celui d'un hydrogel de collagène pur Ma.Hr tel qu'obtenu dans l'exemple 1.3 et celui d'un hydrogel de collagène imprégné par de la spironolactone (i.e. sans polymère organique) Mb-hr obtenu à partir du matériau déshydraté de collagène pur Ma.Hd tel qu'obtenu dans l'exemple 1.3 qui a été imprégné avec une solution comprenant 10'2 M environ de spironolactone dans du THF et rincé, lyophilisé et réhydraté conformément à l'exemple 1.4 ci-dessus. On constate donc une nette amélioration de la tenue mécanique du biomatériau composite de l'invention par rapport à un hydrogène de collagène pur ou un hydrogène de collagène pur comprenant la spironolactone.
    La figure 9 représente le profil de libération de la spironolactone des biomatériaux composites sous la forme de matériaux composites secs conformes à l'invention Mi-S (courbe avec les triangles), Mi-Si (courbe avec les losanges) et Mi-S2 (courbe avec les carrés). Les différentes courbes montrent la quantité cumulative de spironolactone libérée (en nmole) en fonction du temps (en heures).
    Il convient de noter que les biomatériaux initialement utilisés dans cette expérience sont sous la forme de matériaux composites secs. Toutefois, ils sont réhydratés instantanément lors de la mesure du suivi de libération du principe actif. Il en est de même pour les mesures de la toxicité des biomatériaux et de l'activité biologique de la spironolactone telles que décrites ci-après.
    Les biomatériaux composites Mi-Si et Mi-S2 ont été préparés comme dans l'exemple 1, sauf en ce qui concerne la concentration en PLGA dans la solution d'imprégnation qui était de 40 mg/ml environ pour Mi-Si et de 80 mg/ml environ pour Mi-S2 (au lieu de 160 mg/ml environ pour Mi-S).
    On constate qu'une concentration de 160 mg/ml environ est préférée pour favoriser la libération d'une dose constante de spironolactone en fonction du temps et obtenir un meilleur contrôle de la libération de la spironolactone. Il convient de noter que les trois biomatériaux composites conformes à l'invention n'ont libéré que de 30 à 60% environ de spironolactone en 400h (environ 2 semaines).
    La figure 10 représente le profil de libération de la spironolactone des biomatériaux composites de l'invention Mi-S (courbe avec les croix), M2-s (courbe avec les losanges), M3.s (courbe avec les carrés) et M4.s (courbe avec les triangles). Les différentes courbes montrent la dose quotidienne de spironolactone libérée (en nanomole par jour) en fonction du temps (en jours).
    La figure 11 représente la toxicité du biomatériau composite de l'invention Mi-S sur des cellules (fibroblastes) et à titre comparatif : - celle d'un matériau sec de collagène imprégné de spironolactone MB-s obtenu à partir du matériau déshydraté de collagène pur Ma-hd tel qu'obtenu dans l'exemple 1.3 qui a été imprégné avec une solution comprenant 10'2 M environ de spironolactone dans du THF, rincé et lyophilisé conformément à l'exemple 1.4 ci-dessus, - celle d'un matériau sec de collagène imprégné de PLGA Mc-s obtenu à partir du matériau déshydraté de collagène pur Ma-hd tel qu'obtenu dans l'exemple 1.3 qui a été imprégné avec une solution comprenant 160 mg/ml environ de PLGA 30-60 KDa dans du THF, rincé et lyophilisé conformément à l'exemple 1.4, et - celle d'un matériau sec de collagène pur MA.S tel qu'obtenu dans l'exemple 1.3.
    Par ailleurs, C représente un contrôle négatif (puits contenant uniquement les cellules sans hydrogel). Les différents diagrammes montrent la viabilité cellulaire des matériaux Mi-S, MB-s, Mc-s et MA-s tels que définis ci-dessus (en %) à 1 jour et à 6 jours, par rapport au contrôle négatif C.
    Au sixième jour les points sont tous au-dessus de 100 % car les cellules ont proliféré et l'étude est cumulative. On remarque que la spironolactone a un léger effet toxique sur les cellules puisque les deux triangles sont légèrement en-dessous du contrôle (ronds). En effet, au premier jour seulement 74 % des cellules ont survécu. Toutefois, cela n'empêche pas la prolifération cellulaire (les droites sont croissantes). La figure 11 montre également la non-toxicité liée au polymère organique hydrophobe PLGA puisque les carrés sont au même niveau que le contrôle (ronds).
    La figure 12 représente l'activité biologique de la spironolactone libérée à partir d'un hydrogel après 15 jours d'incubation dans le PBS (avec changement du tampon tous les jours) ; lorsque l'on utilise : (1) un mélange d'un biomatériau composite conforme à l'invention Mi-S avec de l'aldostérone à 10'8 M environ, et à titre comparatif : (2) un mélange d'un matériau MB-s avec de l'aldostérone à 10'8 M environ, (3) un mélange d'un matériau Mc-s avec de l'aldostérone à 10'8 M environ, (4) un mélange d'un matériau MA-s avec de l'aldostérone à 10'8 M environ, (5) un contrôle négatif (puits contenant le milieu de culture sans aldostérone), (6) de l'aldostérone à 10'8 M environ, et (7) un mélange d'aldostérone à 10'8 M environ et de la spironolactone à ΙΟ'6 M environ.
    La figure 12 montre la production de la luciférase (en unité relative de lumière ou RLU) selon le type de milieu utilisé. On constate que l'activité de la luciférase est plus importante dans les conditions (6) que dans les conditions (5), ce qui signifie que l'aldostérone s'est bien fixée au récepteur minéralocorticoïde et a engendré une transcription forte. Lorsque l'on ajoute de la spironolactone à 10'6 M environ (conditions (7)), l'effet de l'aldostérone est inhibé, il n'y a pas d'activation du récepteur. Les conditions (1) utilisant le biomatériau composite Mi-S conforme à l'invention montrent une activité comparable à celle obtenue dans les conditions (7), ce qui indique que la spironolactone a bien été relarguée par le biomatériau composite Mi_s, tout en conservant son activité. Lorsqu'il n'y a pas de PLGA (conditions (2)), le collagène ne retient pas la spironolactone et la quantité restante de spironolactone est inactive ou peu concentrée pour agir contre l'aldostérone. Enfin, les deux dernières conditions (3) et (4) correspondent à des contrôles sans spironolactone et sont à la même hauteur que celle associée aux conditions (6) contenant uniquement de l'aldostérone à 10'8 M ; il n'y a donc pas d'effet de l'hydrogel de collagène, ni de l'hydrogel de collagène comprenant du PLGA.
    The invention relates to a composite biomaterial based on collagen, at least one hydrophobic organic polymer and at least one active principle, its preparation method, a dressing comprising such a composite biomaterial, an abdominal wall reinforcement comprising such a composite biomaterial, as well as the uses of said composite biomaterial, particularly in the therapeutic field. The invention applies in particular to the field of biomaterials that deliver active ingredients, especially to treat chronic wounds.
    The skin that completely covers the human body ensures a protective barrier function of organs and tissues against external aggressions. Its role is therefore fundamental for the proper functioning of the body. The skin is composed of three layers: the epidermis, the dermis and the hypodermis or subcutaneous layer. After an injury, tissue repair or healing is immediately put in place. It consists of four phases: hemostasis, the inflammatory phase, the proliferation phase and the remodeling phase. A chronic injury is defined as a skin wound that does not close beyond 42 days. These wounds can take the form of leg ulcers, foot ulcers in diabetic patients or eschars. These chronic wounds result from complications in patients with diabetes, vascular insufficiency or nutritional deficiencies. The prevalence of foot lesions in diabetics is about 20% each year. This problem affects millions of people around the world each year, most often over the age of 65, and is therefore a major challenge for research in regenerative medicine. The major difference with so-called acute wounds is that the healing process is not complete and remains blocked in the inflammatory phase. Chronic injury treatments must both stop inflammation and promote tissue regeneration to achieve wound closure and healing.
    The technique commonly used to treat chronic wounds is debridement of the wound followed by compression. This involves removing the necrotic tissue and fibrous adhesions that have developed between the tissues in order to clean the wound. In some cases, this technique is ineffective, it is then necessary to cover the wound with a dressing based on biomaterials such as hydrocolloids or hydrogels. The dressing has the function of protecting the wound from possible infections, while allowing its closure. Hydrocolloids are defined in the assessment report of the High Authority of Health as dressings made of absorbent polymers, whose physicochemical properties are related to the presence of carboxymethylcellulose. However, this type of dressing may induce a risk of superinfection related to the excessively wet micro-environment that it generates. Hydrogels are physical or chemical gels composed of polymer chains swollen with a large amount of water (about 70% of the total volume of the hydrogel). It is known to use hydrogels based on alginate or collagen. However, alginate degrades quite rapidly in contact with the enzymes of the human body. As for collagen, it has limited intrinsic mechanical properties which can be improved by crosslinking with the aid of chemical agents (aldehydes, carbodiimides, etc.). However, the toxicity of these chemical agents is problematic, as they can lead to undesirable side effects.
    The dressings may be used in combination with an oral treatment or a topical composition based on at least one active ingredient of the antibiotic, anti-inflammatory or healing molecule type. However, oral treatment may be ineffective due to limited access of the active ingredient to the epidermis; and the topical composition generally requires frequent skin applications and direct contact between the wound and the air, increasing the risk of infection.
    Thus, the solutions proposed above are either ineffective and the treatment of the wound then requires the use of a skin substitute which is very expensive, or they do not act on all stages of healing and require to combine several of these solutions.
    Another solution is the prolonged release of an active ingredient using a suitable support to allow the regular absorption and sustained over time of said active ingredient. The release must be controlled, so that the drug is as effective as possible, with the least side effects and a long duration of action. In addition, pure collagen hydrogels are described as a carrier for the controlled release of an active ingredient. However, the pure collagen hydrogels do not sufficiently retain the active ingredient and have insufficient mechanical strength [Wallace et al., Advanced Drug Delivery Reviews, 2003, 55, 1631-1649].
    In addition, composite collagen-organic polymer materials have been proposed for the sustained release of drugs. In particular, Ruszczak et al. [Advanced Drug Delivery Reviews, 2003, 55, 1679-1698] have described a composite material based on collagen, poly (lactic-co-glycolic acid) microparticles (PLGA) and an antibiotic as the active ingredient. The composite material in the form of a composite collagen sponge was prepared in the following manner: PLGA microparticles comprising the antibiotic were prepared by the double emulsion technique. The resulting microparticles were then mixed with an aqueous solution of collagen and antibiotic, and the resulting dispersion was lyophilized. Other known organic collagen-polymer composite materials are in the form of fibers of said organic polymer coated with a thin layer of collagen.
    However, the composite materials of the prior art do not exhibit an optimized release profile of the active ingredient (eg release too fast and / or nonlinear). Moreover, they do not have suitable mechanical properties, in particular elasticity, elongation at break and / or adapted tensile strength (s), to be used in therapeutic dressings (eg extremely rigid materials or too much loose and / or too fragile).
    The object of the present invention is to overcome the disadvantages of the prior art and to provide a biocompatible, biodegradable material which has suitable mechanical properties to be used in the field of therapeutic dressings, which can promote several steps of the process of cicatrisation, in particular in the case of chronic wounds, and which makes it possible to release in a controlled manner at least one active principle participating in said healing process or tissue repair. The subject of the invention is therefore a synthetic composite biomaterial comprising collagen, at least one organic polymer and at least one active ingredient, characterized in that: the organic polymer is biodegradable, biocompatible, hydrophobic and has a glass transition temperature; less than or equal to approximately 45 ° C and a mean molar mass ranging from approximately 5 to 120 KDa, - the collagen is in the form of striated fibrils in which the periodicity of the striations is 67 nm, - the collagen / organic polymer mass ratio is from 10/1 to approximately 1/3, preferably from 5/1 to approximately 1/2, and more preferably from 2/1 to 2/3 approximately, - the active principle is a hydrophobic active ingredient chosen from anti-inflammatory agents antibiotics, compounds promoting tissue repair or scarring and one of their mixtures.
    The synthetic composite biomaterial of the invention is biocompatible, biodegradable and has comparable or improved mechanical properties compared to a pure collagen hydrogel, while allowing the prolonged release of one or more active ingredient.
    In particular, the composite biomaterial of the invention can release a substantially constant active principle over a period of at least 7 days, and preferably over a period of at least 3 weeks, or even 1 month.
    The composite biomaterial of the invention is preferably free of any organic solvent.
    The composite biomaterial of the invention may be in the form of a composite hydrogel or in the form of a dry composite material.
    When the composite biomaterial is in the form of a dry composite material, it comprises at most about 10% by weight of water, and preferably at most about 5% by weight, relative to the total mass of the composite biomaterial. According to a preferred embodiment of the invention, the dry composite material does not comprise solvating water.
    When the composite biomaterial is in the form of a composite hydrogel, it comprises from 70 to 95% by weight of water, and preferably from 80 to 90% by weight of water, relative to the total mass of the composite biomaterial.
    The composite biomaterial of the invention preferably consists of the active ingredient (s), the organic polymer (s), collagen and optionally water or an aqueous solution with a pH ranging from about 7 to 8 (eg phosphate buffered saline). .
    In the present invention, the term "biodegradable organic polymer" means an organic polymer that can be degraded or digested by microorganisms (eg bacteria, fungi, algae), in particular by the action of enzymes. The reactions occurring during biodegradation in humans are hydrolysis reactions, that is to say, covalent bond cuts by reaction with water (see current standard NFEN 13,432).
    In the present invention, the term "biocompatible organic polymer" means an organic polymer having the ability not to interfere and degrade the biological medium in which they are used. In particular, they must not cause a strong inflammatory reaction (eg allergies) and / or must not be toxic to humans.
    In the present invention, the expression "hydrophobic organic polymer" means an organic polymer for which a contact angle is measured between approximately 60 ° and 150 °, preferably between 60 and 110 °, and more preferably between 65 and Approximately 90 °, for example according to the Wihelmy measurement method detailed in the examples below.
    Thus, the organic polymers in accordance with the invention are insoluble in biological fluids.
    The presence of at least one hydrophobic organic polymer in the composite biomaterial of the invention allows the encapsulation of one or more active ingredients and their controlled release.
    The organic polymer preferably has a glass transition temperature of less than or equal to about 40 ° C, and more preferably less than or equal to about 38 ° C.
    Furthermore, it is advantageous for the organic polymer to have a glass transition temperature when associated with the collagen, which is less than or equal to approximately 37 ° C., preferably less than or equal to approximately 32 ° C., and more preferably less than or equal to approximately 28 ° C.
    Indeed, a temperature of about 28 ° C corresponds to the equilibrium temperature of a dressing on the skin (temperature at the ambient air-physiological temperature interface). A glass transition temperature of the organic polymer when it is associated with collagen less than or equal to approximately 40 ° C. makes it possible to obtain a composite biomaterial having comparable mechanical properties, even improved over pure collagen (elasticity, elongation at the breaking and / or breaking strength).
    The organic polymer may be chosen from aliphatic polyesters, polyethylene glycols, polyanhydrides and poly (ortho esters).
    Preferably, the organic polymer is an aliphatic polyester, chosen in particular from a polyglycolide [ie poly (glycolic acid) or PGA], a polylactide [ie poly (lactic acid or PLA), a glycolide and lactide copolymer (PLGA), a polylactone (eg poly (ε-caprolactone)), and a polyhydroxyalkanoate (eg polyhydroxyvalerate, poly (hydroxybutyrate)).
    According to a particularly preferred embodiment of the invention, the organic polymer is a copolymer of glycolide and lactide.
    The glycolide and lactide copolymer may have a lactide / glycolide molar ratio of from about 50:50 to about 85:15, preferably from about 50:50 to about 65:35.
    The organic polymer may have an average molar mass ranging from about 5 KDa to 60 KDa, and more preferably from about 7 KDa to about 20 KDa.
    The organic polymer content of the composite biomaterial when it is in the form of a composite hydrogel is at least about 10 mg / ml, preferably from about 20 to 100 mg / ml, and more preferably from About 30 to 60 mg / ml.
    The organic polymer of the composite biomaterial of the invention is in the form of nanodomains, especially having a mean size of less than or equal to about 700 nm, preferably less than or equal to about 600 nm, and more preferably ranging from 100 to 500 nm. about.
    The average size of these nanodomains is determined by transmission electron microscopy (TEM).
    In other words, the organic polymer is not in the form of fibers or microparticles as is the case in the composite materials of the prior art.
    The collagen is preferably a type I or III collagen, and more preferably a type I collagen. Type I fibrillar collagen is a natural protein present at about 70% by mass in the extracellular matrix of the skin, it contributes to the mechanical structure of connective tissues and is the substrate for cell adhesion.
    The collagen content of the composite biomaterial when it is in the form of a composite hydrogel is at least about 10 mg / ml, preferably about 20 to 100 mg / ml, and more preferably 30 to 60 mg / ml. mg / ml approximately.
    A minimum collagen content of at least about 10 mg / ml in the composite biomaterial prevents its rapid degradation in the physiological medium during dermal application (eg in the form of a dressing).
    In the material of the invention, said fibrils are generally not ordered. The material therefore mainly or even only consists of isotropic domains.
    The active ingredient of antibiotic type may be gentamycin, ryfamicin or amoxicillin.
    The active ingredient of the anti-inflammatory type may be dexamethasone or an analgesic such as ibuprofen.
    The active ingredient promoting healing or tissue repair may be spironolactone.
    Spironolactone is known to be a mineralocorticoid receptor antagonist and to promote repair of skin wounds.
    According to a particularly preferred embodiment of the invention, the active ingredient is spironolactone.
    In the present invention, the expression "hydrophobic active ingredient" means an active ingredient having a partition coefficient P in an octanol / water system such that log P> 0, preferably log P> 2, and more preferably log P> 3.
    The partition coefficient P is well known to those skilled in the art. It is equal to the ratio of the concentrations of a solute (active principle) in two phases: P = C '/ C, C' corresponding to the concentration of the solute in an organic solvent saturated with water, and C corresponding to the concentration of the solute in water saturated with organic solvent.
    The dissolution capacity in water of a hydrophobic active ingredient such as that used in the composite biomaterial of the invention is generally less than about 50 mg / l.
    The content of the biomaterial composite active ingredient depends on the active ingredient that is to be incorporated and therefore the intended application.
    In particular, the composite biomaterial in the form of a composite hydrogel may comprise from 0.1 to 10 mg / ml of spironolactone.
    The composite biomaterial of the invention in the form of a composite hydrogel may have an elasticity (ie a Young's modulus) of at least about 10,000 Pa, preferably at least about 40000 Pa, and more preferably from 50000 to 100000 Pa approximately.
    The composite biomaterial of the invention in the form of a composite hydrogel may have a shear modulus of at least about 3,000 Pa, preferably at least about 5,000 Pa, and more preferably from about 10,000 to about 60,000 Pa. , at a frequency between 1 and 10 Hz.
    The composite biomaterial of the invention may exhibit an elongation at break of at least about 10%, preferably at least about 30%, and more preferably from about 40% to about 75%.
    The composite biomaterial of the invention may have a breaking strength of at least about 2.5 MPa, preferably at least about 3 MPa, and more preferably from about 3.5 to 12 MPa.
    The composite biomaterial of the invention is in the form of a matrix of striated collagen fibrils in which nanodomains consisting of said organic polymer are dispersed homogeneously (homogeneous three-dimensional network). In particular, the nanodomains of said organic polymer have an average size of less than or equal to about 700 nm, preferably less than or equal to about 600 nm and more preferably from about 100 to about 500 nm. The subject of the invention is also a method for preparing a composite biomaterial as defined in the first subject of the invention, characterized in that it comprises at least the following stages: i) the preparation of a hydrogel of collagen in the form of striated fibrils in which the periodicity of the striations is 67 nm, the concentration of collagen in the hydrogel being at least about 10 mg / ml, ii) the dehydration of the hydrogel as prepared in FIG. step i) by incubating said hydrogel in several successive mixed solutions [organic solvent / aqueous solvent] having an increasing proportion of organic solvent, said aqueous and organic solvents being miscible, followed by a final incubation in a pure solution of said organic solvent iii) contacting the dehydrated hydrogel of step ii) with an impregnating solution comprising at least one organic polymer and at least one active ingredient such as ue defined in the first subject of the invention, the volume ratio: volume of the impregnating solution / volume of the dehydrated hydrogel being greater than or equal to about 3, iv) rinsing of the hydrogel impregnated step iii) with an organic solvent and then with an aqueous solvent.
    Preferably, the collagen concentration of the hydrogel from step i) ranges from 20 to 100 mg / ml, and more preferably from 30 to 60 mg / ml. A concentration of about 30 to 60 mg / ml is particularly suitable for obtaining a composite biomaterial having high hydration capacity and swelling capacity, good elasticity and good stability against enzymatic degradation. Step i) can be carried out according to the following substeps: i-1) the preparation of a solution of acid-soluble collagen whose collagen content varies from 1 to 5 mg / ml approximately, and is preferably from the order of 5 mg / ml, i-2) the evaporation in air of the solution of step i-1), and i-3) contacting the solution of step i- 2) with a base. Step i-1) may be carried out by any means known to those skilled in the art, in particular by extraction of collagen in a bovine, murine or porcine species.
    An acid-soluble solution is an acidic aqueous solution in which the collagen can be dissolved. Its pH is generally less than about 4, preferably less than about 3, in the presence of acids, preferably acetic acid, for example at about 0.5 M. Step i-2) is progressive and allows the concentration of collagen within the solution. It can last from 3 days to 2 weeks approximately. At the end of the sub-step i-2), the concentration of the collagen solution is at least about 10 mg / ml, preferably from about 20 to 100 mg / ml, and more preferably from 30 to About 60 mg / ml (ie the collagen has the desired concentration to form the composite biomaterial of the invention). Stage i-3) makes it possible to subject the concentrated collagen solution to an increase in pH which induces the gelation of the collagen and its fibrillogenesis.
    It is advantageously carried out by contacting the solution of step i-2) with a basic gaseous atmosphere, in particular an NH 3 or (NH 4) 2 CO 3 atmosphere. Stage i-3) generally lasts between 12h and 24h approximately. Step i) may furthermore comprise a sub-step i-4) of rinsing the hydrogel of step i-3), in particular with several solutions of phosphate buffered saline (PBS), in order to eliminate the base . Step i) may further comprise a substep i-2 ') between substeps i-2) and i-3) during which the solution of step i-2) is transferred to a mold and centrifuged. This step i-2 ') makes it possible to smooth out the surface irregularities of the concentrated (and viscous) collagen solution of step i-2). Step i) can also be performed according to the procedure as described in Wang et al., Nature Materials, 2012, 11, 724-733. Step ii) leads to a dehydrated material (ie without water) of pure collagen.
    In a particular embodiment, the organic solvent is chosen from tetrahydrofuran (THF) and dimethylsulfoxide (DMSO).
    In a particular embodiment, the aqueous solvent is selected from water and a phosphate buffered saline (PBS). Step ii) can be carried out by incubating the hydrogel of step i) in a mixed organic solvent / aqueous solvent solution whose content of organic solvent is approximately 20 to 30% by volume, and then in a mixed solvent solution. organic / aqueous solvent whose content of organic solvent is 40 to 50% by volume approximately, then in a mixed organic solvent / aqueous solvent solution whose content of organic solvent is 60 to 70% by volume approximately, then in a solution mixed organic solvent / aqueous solvent whose organic solvent content is 80 to 95% by volume, and then in a pure solution of said organic solvent. Incubation in each of the solutions may last from about 30 minutes to about 2 hours, and preferably from about 45 minutes to about 1:15, and more preferably in the order of about 1 hour. Stage ii) makes it possible to create and / or maintain a homogeneous porosity within the collagen. This step ii) is essential in the process of the invention since it makes it possible on the one hand to have reproducible results, and on the other hand to obtain good impregnation of the hydrogel during the next step iii ). This gives a homogeneous composite biomaterial (eg absence of microscopic domains of organic polymer in the composite biomaterial and retention of the striated fibrillar structure of collagen) which has good properties of sustained release of the active ingredient while ensuring good mechanical properties. Step iii) makes it possible to impregnate the hydrogel of step ii) with a solution of organic polymer and of active principle.
    Due to the porosity of the hydrogel and a medium conducive to the diffusion of the organic polymer, a significant amount of organic polymer can be incorporated in the hydrogel. Step iii) is preferably carried out by preparing an impregnating solution comprising the active ingredient and the organic polymer, and then incubating the dehydrated pure collagen hydrogel of step ii) in the impregnating solution.
    In particular, the impregnating solution is prepared by dissolving the active ingredient at the desired concentration in an organic solvent, then adding to the preceding solution the organic polymer at the desired concentration, and finally dissolving it in said previous solution. Step iii) is preferably carried out at room temperature.
    The organic solvent may be chosen from all the solvents which make it possible to solubilise both the active ingredient and the organic polymer. By way of example, the organic solvent may be THF or DMSO.
    The organic polymer concentration of the impregnating solution may range from about 20 to about 500 mg / ml, and preferably from about 80 to about 160 mg / ml.
    The concentration of the active ingredient of the impregnating solution may vary from about 0.1 to 100 mg / ml, and preferably from 1 to 10 mg / ml.
    The volume ratio: volume of the impregnating solution / volume of the dehydrated hydrogel preferably varies from 3/1 to 20/1, and is preferably of the order of 5/1. Step iii) generally lasts from 12 to about 24 hours.
    The organic solvent of step iv) may be THF or DMSO.
    The aqueous solvent of step iv) can be a phosphate buffered saline (PBS), a fluid simulating body fluids (well known under the Anglicism "Body Fluid Serum" or SBF), water or a culture medium cellular. Step iv) is preferably carried out by rinsing the impregnated hydrogel of step iii) in several organic solvent baths, especially for about 15 seconds to about 1 minute, and preferably for about 30 seconds; and rinsing the impregnated hydrogel of step iii) in several aqueous solvent baths, especially for about 15 minutes to 1 hour, and preferably for about 30 minutes.
    Rinsing with an organic solvent makes it possible to remove the excess of polymer which has not been absorbed by the collagen hydrogel.
    Rinsing with an aqueous solvent makes it possible to freeze the polymer within the collagen hydrogel. Without this last rinsing step with an aqueous solvent, the composite biomaterial does not have sufficient mechanical strength and is fragile. At the end of step iv), the composite biomaterial is in the form of a composite hydrogel.
    The method may further comprise a step v) of lyophilizing the composite biomaterial of step iv). At the end of step v), the composite biomaterial is in the form of a dry composite material. The dry composite material is preferably free of any organic or aqueous solvent.
    The dry composite material can then be preserved in the long term.
    The method may further comprise a step vi) of hydration of the dry composite material. This step vi) makes it possible to reform the composite material in the form of a composite hydrogel. Step vi) may be carried out in the presence of an aqueous solution selected from water, a phosphate buffered saline, a fluid simulating body fluids and a cell culture medium.
    The steps v and vi) completely eliminate any organic solvent that could have possibly still be present in the composite hydrogel obtained at the end of the rinsing step iv). The third object of the invention is a composite biomaterial according to the first subject of the invention for its medical use. The fourth object of the invention is a composite biomaterial according to the first subject of the invention for its use in the treatment of chronic wounds. These chronic wounds are usually found in foot ulcers, venous ulcers or pressure ulcers.
    In a particular embodiment, the composite biomaterial according to the invention is left in contact with the skin for at least one week. The subject of the invention is a composite biomaterial according to the first subject of the invention, for its use in the preventive treatment of infections after cardiac or colorectal surgery. The sixth subject of the invention is a therapeutic dressing comprising an inner layer and an outer layer consisting of a secondary dressing chosen from an adhesive, a compress, a bandage and one of their mixtures, characterized in that the inner layer comprises a biomaterial. composite according to the first object of the invention.
    The secondary dressing may in particular be a porous dressing, sterile gauze, a film or a polyurethane foam, or a nonwoven polyamide / polyester film.
    The inner layer is in contact with the skin and the outer layer maintains the inner layer.
    The dressing may release the active ingredient, and in particular spironolactone, at a constant dose for at least one week. The subject of the invention is a composite biomaterial according to the first subject of the invention for use in the treatment of hernias of the abdominal wall or evenifices. The invention has for eighth object an abdominal wall reinforcement comprising a composite biomaterial according to the first subject of the invention.
    Examples
    The raw materials used in the examples are listed below: PLGA 7-17 KDa, 719897-5G, Sigma, 50:50 (Lactide: Glycolide), PLGA 24-38 KDa, 719870-5G, Sigma, 50 : 50 (Lactide: Glycolide), - PLGA 30-60 KDa, P2191-5G, Sigma, 50:50 (Lactide: Glycolide), - PCL 14 KDa, 440752-5G, Sigma, - Rat tail tendons, Gibco A1048301 Spironolactone, S3378-1G, Sigma, and Aldosterone, A9477-25MG, Sigma.
    Unless otherwise indicated, all materials have been used as received from manufacturers.
    Prepared or commercial materials were characterized by transmission electron microscopy (TEM, hydroxyproline assay, contact angle measurement, elongation at break measurement, breaking strength measurement, measurement of elasticity, measurement of toxicity, measurement of monitoring of release of the active principle by UV spectroscopy and measurement of the biological activity of spironolactone Transmission Transmission Electron Microscopy (TEM) analysis was carried out with the aid of an apparatus sold under the trade name Technai Spirit G2, by the company FEI.
    The measurement of the contact angle was carried out using a tensiometer sold under the trade name DSA 30 by Kussel. This measurement was carried out according to the following Wihelmy method: a solution of organic polymer of concentration 160 mg / ml approximately dissolved in tetrahydrofuran (THF) is deposited on a clean and dry glass slide. Dry to form a film of the organic polymer on the surface of the glass slide. Then, a drop of water is deposited on the glass slide covered with organic polymer. This operation is carried out at about 25 ° C. The tensiometer measures the surface tension between the water and the glass slide covered with organic polymer, and calculates the angle of contact with the water that results. The greater the contact angle, the more hydrophobic the organic polymer. The elasticity was determined at 25 ° C. using an apparatus sold under the trade name Electroforce 3220 by the company Bose.
    The shear modulus was determined at 25 ° C using an apparatus sold under the trade name MCR 301 by the company Anton Paar. Elongation at break was determined at 25 ° C. using an apparatus sold under the trade name Electroforce 3220 by the company Bose.
    The breaking strength was determined at 25 ° C using an apparatus sold under the trade name Electroforce 3220 by the company Bose.
    The determination of the hydroxyproline makes it possible to determine the concentration of collagen in the different solutions or materials. Hydroxyproline is an amino acid very present in the polypeptide chain of collagen. The assay was carried out as follows: the collagen solution to be tested was hydrolysed under acidic conditions (6M HCl) at approximately 108 ° C. Hydroxyproline was thus released, and the resulting mixture was dried. Hydroxyproline was oxidized with Chloramine T and then complexed with dimethylamino-4-benzaldehyde (DMBA) to give a colored product. The determination of the concentration was made by spectrophotometric measurement at 557 nm compared to a standard range.
    The measurement of the toxicity of the materials was carried out in the following manner: human primary dermal fibroblasts were inoculated on a culture plate. The composite hydrogels were then incubated with the fibroblasts for 1 or 6 days. Cell viability was measured by a metabolic test (AlamarBlue Assay®) and compared with that of control fibroblasts (without addition of composite hydrogel).
    The measurement of the release of the active principle was carried out by UV spectroscopy using an apparatus sold under the trade name Uvikon XL by NorthStar Scientific.
    The measurement of the biological activity of spironolactone was carried out as follows: the H9C2 cells are genetically modified myoblasts by incorporation of the reporter gene coding for luciferase downstream of the promoter having the sequences binding the complex: aldosterone with its receiver. When aldosterone was added, luciferase was expressed and detected by bioluminescence. When the spironolactone released by the hydrogels tested is active, its incubation with H9C2 inhibits aldosterone and makes the bioluminescence signal null. This inhibition accounts for the biological activity of spironolactone on H9C2 incubated with aldosterone. The activity of spironolactone was measured directly on the spironolactone release liquid from the different types of hydrogels tested.
    Example 1 Preparation of a Synthetic Composite Biomaterial According to the First Object of the Invention 1.1 Extraction of Collagen
    The collagen was extracted from rat tail tendons, known to be very rich in type I collagen. To do this, rat tendons were rinsed with phosphate buffered saline (PBS) by centrifugation for about 5 minutes, at about 4 ° C and 3000 G (G is the unit of measurement for centrifugation speed and corresponds to the acceleration of gravity) until the solution becomes clear, free of cells and blood. They were then rinsed with approximately 4M NaCl solution to destroy all remaining cells. After further rinsing with PBS to remove all traces of NaCl, the washed tendons were mixed with about 0.5M acetic acid solution for about 24 hours and then the resulting mixture was centrifuged at about 3000 G for about 20 minutes. . The resulting solution then included triple helices of collagen I in the presence of other proteins. Collagen I was selectively precipitated by dropwise addition of approximately 4M NaCl solution to the resulting solution to obtain a final NaCl concentration of 0.7M. The resulting mixture was then centrifuged at 3000 g and the resulting precipitate was solubilized in 0.5 M acetic acid to form a solution comprising mainly collagen I. This solution was dialyzed against the same solvent to eliminate totally NaCl (4 baths of about 24 hours) and centrifuged at about 21,000 G for about 3 hours to remove the last colloidal aggregates.
    The collagen solution thus prepared was stored at about 4 ° C. in order to preserve its triple helix structure. The determination of hydroxyproline in this solution made it possible to determine its collagen concentration which was about 5 mg / ml. 1.2 Preparation of a concentrated collagen hvdroael I
    The collagen solution at about 5 mg / ml as prepared above was dissolved in about 0.5 M acetic acid solution. The solution was evaporated in the air under a sterile hood until a final collagen concentration of about 40 mg / ml was reached. Evaporation of the solvent was monitored by weighing. The final concentration of the solution was then confirmed by a hydroxyproline assay.
    The resulting collagen solution at 40 mg / ml was introduced into a mold, and then the mold / collagen solution assembly was centrifuged to smooth out the irregularities. The resulting mold was then placed under ammonia vapors for about 12 hours to allow gelation and fibrillogenesis of collagen I. The resulting hydrogel was rinsed several times in sterile PBS (ie, wet-sterilized) baths. in an autoclave) to remove the ammonia and bring the pH back to 7. The pH was checked before each wash. The pure collagen hydrogel obtained is called MA.H. 1.3 Dehydration of a concentrated collagen hvdroael I The collagen I hydrogel as obtained above was dehydrated progressively by incubation in several successive THF / water mixing baths: a THF / water bath having a THF volume concentration of 30%, a THF / water bath having a THF volume concentration of 50%, a THF / water bath having a 70% THF volume concentration, a THF / water bath having a 95% THF volume concentration and finally a pure THF bath. Incubation in each of the baths lasted about 1 hour.
    Following the dehydration step, a dehydrated hydrogel of pure collagen Ma-hd-
    The dehydrated pure collagen Ma.Hd hydrogel can be rinsed with PBS and lyophilized to give a dry material of pure MA.S collagen and then MA.S can be rehydrated with PBS to reform a pure collagen hydrogel called Ma_Hr. 1.4 Preparation of Synthetic Composite Biomaterial Mi ^
    A solution comprising 160 mg / ml 30-60 KDa PLGA and 10 -2 M (ie 4.16 mg / ml) spironolactone was prepared by dissolving the spironolactone in THF and then dissolving the PLGA in the above mixture. The dehydrated pure collagen Ma-hd hydrogel as prepared above was impregnated for approximately 12 hours with the solution comprising PLGA and spironolactone, the volume of the solution being at least 5 times larger than that of the hydrogel. of pure dehydrated collagen.
    After the impregnation, the composite hydrogel obtained was rinsed 3 times approximately 30 seconds with pure THF in order to eliminate the excess PLGA, then 3 times approximately 30 minutes with sterile PBS in order to freeze the polymer within the collagen network. fibrillated and form the composite biomaterial of the invention Mi-h in the form of a composite hydrogel.
    A dry form of said composite biomaterial has also been obtained. To do this, the composite biomaterial Mi-h was immersed in liquid nitrogen for about 10 minutes to prevent the formation of crystalline ice, then lyophilized for about 24 hours (temperature below -40 ° C, vacuum at 100 pBarr) to form the composite biomaterial of the invention Mi-s in the form of a dry composite material.
    The dry composite material was then rehydrated by adding a phosphate buffered saline to form a Mi-Hr material in the form of a composite hydrogel.
    The mass ratio collagen / organic polymer in said biocomposite material was 1/1.
    FIG. 1 shows an image by transmission electron microscopy of a pure collagen hydrogel MA.H as obtained in Example 1.2 (FIG. 1a) and which does not form part of the invention, and an image of the biomaterial. composite Mi_Hr in the form of a composite hydrogel according to the invention obtained in Example 1.4 (Figure 1b).
    Figure 1 shows a homogeneous composite biomaterial in which the fibrillar and striated structure of the collagen has been preserved. Moreover, microscopic domains of PLGA polymer are not distinguished, which means that the polymer is uniformly distributed within the collagen.
    The table below lists the values of the contact angles of the various organic polymers used in Example 1 and the examples below:
    Example 2 Preparation of Other Synthetic Composite Biomaterials According to the First Object of the Invention
    Composite biomaterials M2.Hr, M3.Hr and M4.Hr were prepared by impregnating the dehydrated hydrogel with pure collagen Ma-hd as obtained in Example 1.3 above with the following impregnating solutions: a solution comprising about 10.2 M spironolactone and about 160 mg / ml PLGA 7-17 KDa; a solution comprising about 10.2 M spironolactone and about 160 mg / ml PLGA 24-38 KDa; and solution comprising about 10'2 M spironolactone and about 160 mg / ml PCL 14 KDa.
    The steps of impregnation, rinsing, lyophilization and rehydration are identical to those described in Example 1.4 above.
    FIG. 2 represents the diameter (in mm) of the composite biomaterials in the form of composite hydrogels according to the invention Mi-r, M2-hr, M3.Hr and M4.hr and, by way of comparison, the diameter of a hydrogel pure collagen not according to the invention Ma-h as obtained in Example 1.2 and a pure collagen hydrogel Ma-hr as obtained in Example 1.3.
    Figure 2 shows that the presence in the hydrogel of an active ingredient and a hydrophobic organic polymer as defined in the invention has little or no influence on the diameter of the hydrogel obtained. Composite hydrogels therefore have a good ability to hydrate, close to that of pure collagen and do not shrink.
    FIG. 3 represents the mass of organic polymer (in mg) present in the composite biomaterials in the form of dry composite materials according to the invention Mi-S / M2-s, M3.s and M4.s.
    FIG. 3 shows that the composite biomaterial of the invention can incorporate a larger amount of organic polymer when PLGA 7-17 KDa is used (M2-s) · The integration capacities of the organic polymers tested are however suitable and are greater than about 15 mg, and preferably from about 20 to about 31 mg, for a collagen mass of about 25 mg.
    FIG. 4 shows the volume swelling of the composite biomaterials in the form of composite hydrogels according to the invention Mi-hr, M2.Hr, M3.Hr and M4-hr and, for comparison, a pure collagen hydrogel. -Hr as obtained in Example 1.3 (in% relative to the initial volume of the pure collagen hydrogel MA.H).
    FIG. 4 shows a good swelling capacity of the composite hydrogels according to the invention (approximately 55% to 80%), or even similar to that of a pure collagen hydrogel when PLGA 7-17 KDa is used.
    FIG. 5 shows the elongation at break of the composite biomaterials in the form of composite hydrogels according to the invention Mi-hr, M2-Hr, M3.Hr and M4_hr, and for comparison that of a collagen hydrogel pure Ma.Hr as obtained in Example 1.3 (in% of the initial length of each of the materials tested).
    FIG. 6 represents the breaking strength (in MPa) of the composite biomaterials in the form of composite hydrogels according to the invention Mi-Hr, M2-hr, M3.Hr and M4.Hr, and by way of comparison that of a pure collagen hydrogel Ma-hr as obtained in Example 1.3.
    FIG. 7 represents the elasticity (in Pa) of the composite biomaterials (Young's modulus) in the form of composite hydrogels according to the invention Mi-Hr, M2-hr, M3.Hr and M4.Hr, and comparing that of a pure collagen hydrogel Ma-hr as obtained in Example 1.3.
    FIG. 8 represents the shear modulus (in Pa) at different frequencies (about 1 Hz and 10 Hz) of the composite biomaterial in the form of a composite hydrogel according to the invention Mi-Hr, and by way of comparison that of a pure collagen hydrogel Ma.Hr as obtained in Example 1.3 and that of a collagen hydrogel impregnated with spironolactone (ie without organic polymer) Mb-hr obtained from the dehydrated material of pure collagen Ma. Hd as obtained in Example 1.3 which was impregnated with a solution comprising about 10.2 M spironolactone in THF and rinsed, lyophilized and rehydrated according to Example 1.4 above. There is therefore a marked improvement in the mechanical strength of the composite biomaterial of the invention compared to a pure collagen hydrogen or a pure collagen hydrogen including spironolactone.
    FIG. 9 represents the spironolactone release profile of the composite biomaterials in the form of dry composite materials in accordance with the invention Mi-S (curve with the triangles), Mi-Si (curve with the diamonds) and Mi-S2 ( curve with the squares). The different curves show the cumulative amount of spironolactone released (in nmol) as a function of time (in hours).
    It should be noted that the biomaterials initially used in this experiment are in the form of dry composite materials. However, they are rehydrated instantly when measuring the release of the active ingredient. It is the same for measurements of the toxicity of biomaterials and the biological activity of spironolactone as described below.
    The composite biomaterials Mi-Si and Mi-S2 were prepared as in Example 1, except for the concentration of PLGA in the impregnating solution which was about 40 mg / ml for Mi-Si and 80 mg / ml for Mi-S2 (instead of approximately 160 mg / ml for Mi-S).
    It is found that a concentration of about 160 mg / ml is preferred to promote the release of a constant dose of spironolactone as a function of time and to obtain better control of the release of spironolactone. It should be noted that the three composite biomaterials according to the invention have released only about 30 to 60% of spironolactone in 400h (about 2 weeks).
    Figure 10 shows the spironolactone release profile of composite biomaterials of the invention Mi-S (curve with crosses), M2-s (curve with diamonds), M3.s (curve with squares) and M4. s (curve with the triangles). The different curves show the daily dose of spironolactone released (in nanomole per day) as a function of time (in days).
    FIG. 11 represents the toxicity of the composite biomaterial of the invention Mi-S on cells (fibroblasts) and for comparison: that of a collagen dry material impregnated with spironolactone MB-s obtained from the dehydrated collagen material pure Ma-hd as obtained in Example 1.3 which was impregnated with a solution comprising approximately 10'2 M spironolactone in THF, rinsed and lyophilized in accordance with Example 1.4 above, - that of a dry collagen material impregnated with PLGA Mc-s obtained from the dehydrated material of pure collagen Ma-hd as obtained in Example 1.3 which was impregnated with a solution comprising approximately 160 mg / ml of PLGA 30-60 KDa in THF, rinsed and lyophilized according to Example 1.4, and - that of a dry material of pure MA.S collagen as obtained in Example 1.3.
    Moreover, C represents a negative control (well containing only cells without hydrogel). The various diagrams show the cellular viability of Mi-S, MB-s, Mc-s and MA-s materials as defined above (in%) at 1 day and at 6 days, compared to the negative control C.
    By the sixth day the points are all above 100% because the cells have proliferated and the study is cumulative. Note that spironolactone has a slight toxic effect on cells since both triangles are slightly below control (round). Indeed, at the first day only 74% of the cells survived. However, this does not prevent cell proliferation (the rights are increasing). Figure 11 also shows the non-toxicity related to the PLGA hydrophobic organic polymer since the squares are at the same level as the control (round).
    Figure 12 shows the biological activity of spironolactone released from a hydrogel after 15 days of incubation in PBS (with buffer change daily); when using: (1) a mixture of a composite biomaterial according to the invention Mi-S with aldosterone to 10'8 M approximately, and for comparison: (2) a mixture of a material MB-s with aldosterone at about 10'8 M, (3) a mixture of Mc-s material with aldosterone at about 10'8 M, (4) a mixture of MA-material s with aldosterone at approximately 10'8 M, (5) a negative control (wells containing the culture medium without aldosterone), (6) aldosterone at approximately 10'8 M, and (7) a mixture of aldosterone at about 10'8 M and spironolactone at about ΙΟ'6 M.
    Figure 12 shows the production of luciferase (in relative light unit or RLU) according to the type of medium used. Luciferase activity is found to be more important under conditions (6) than under conditions (5), which means that aldosterone is well attached to the mineralocorticoid receptor and has resulted in strong transcription. When spironolactone is added at about 10 -6 M (conditions (7)), the effect of aldosterone is inhibited, there is no activation of the receptor. The conditions (1) using the Mi-S composite biomaterial according to the invention show an activity comparable to that obtained under the conditions (7), which indicates that the spironolactone has been released by the composite biomaterial Mi.sub.s while retaining his activity. When there is no PLGA (conditions (2)), collagen does not retain spironolactone and the remaining amount of spironolactone is inactive or poorly concentrated to act against aldosterone. Finally, the last two conditions (3) and (4) correspond to controls without spironolactone and are at the same height as that associated with the conditions (6) containing only 10'8 M aldosterone; therefore, there is no effect of the collagen hydrogel or the collagen hydrogel comprising PLGA.
    权利要求:
    Claims (20)
    [1" id="c-fr-0001]
    1. Synthetic composite biomaterial comprising collagen, at least one organic polymer and at least one active principle, characterized in that: the organic polymer is biodegradable, biocompatible, hydrophobic and has a glass transition temperature of less than or equal to 45 ° C. C and an average molar mass ranging from 5 to 120 KDa, the collagen is in the form of striated fibrils in which the periodicity of the striations is 67 nm, the collagen / organic polymer mass ratio ranges from 10: 1 to 1: 1. 3, - the active principle is a hydrophobic active ingredient chosen from anti-inflammatories, antibiotics, compounds promoting tissue repair or healing and one of their mixtures.
    [2" id="c-fr-0002]
    2. Biomaterial according to claim 1, characterized in that it is in the form of a composite hydrogel and comprises from 70 to 95% by weight of water, relative to the total mass of the composite biomaterial.
    [3" id="c-fr-0003]
    3. Biomaterial according to claim 2, characterized in that it has an elasticity ranging from 50000 Pa to 100000 Pa.
    [4" id="c-fr-0004]
    4. Biomaterial according to claim 2 or 3, characterized in that it has an elongation at break ranging from 40 to 75%.
    [5" id="c-fr-0005]
    5. Biomaterial according to claim 1, characterized in that it is in the form of a dry composite material and comprises at most 10% by weight of water, relative to the total mass of the composite biomaterial.
    [6" id="c-fr-0006]
    6. Biomaterial according to any one of the preceding claims, characterized in that the organic polymer is chosen from aliphatic polyesters, polyethylene glycols, polyanhydrides and poly (ortho-esters).
    [7" id="c-fr-0007]
    7. Biomaterial according to any one of the preceding claims, characterized in that the organic polymer is an aliphatic polyester selected from a polyglycolide, a polylactide, a glycolide and lactide copolymer, a polylactone, and a polyhydroxyalkanoate.
    [8" id="c-fr-0008]
    8. Biomaterial according to any one of the preceding claims, characterized in that the organic polymer is in the form of nanodomains having an average size of less than or equal to 700 nm.
    [9" id="c-fr-0009]
    9. Process for preparing a composite biomaterial as defined in any one of claims 1 to 8, characterized in that it comprises at least the following steps: i) the preparation of a collagen hydrogel in the form striated fibrils in which the periodicity of the striations is 67 nm, the concentration of collagen in the hydrogel being at least 10 mg / ml, ii) the dehydration of the hydrogel as prepared in step i) by incubating said hydrogel in several successive mixed solutions [organic solvent / aqueous solvent] having an increasing proportion of organic solvent, said aqueous and organic solvents being miscible, followed by a final incubation in a pure solution of said organic solvent, iii) the implementation contacting the dehydrated hydrogel of step ii) with an impregnation solution comprising at least one organic polymer and at least one active ingredient as defined in any one of the 1 to 8, the volume ratio: volume of the impregnating solution / volume of the dehydrated hydrogel being greater than or equal to 3; iv) rinsing the impregnated hydrogel of step iii) with an organic solvent; then with an aqueous solvent.
    [10" id="c-fr-0010]
    10. Process according to claim 9, characterized in that step i) is carried out according to the following sub-steps: i-1) the preparation of an acid-soluble collagen solution whose collagen content varies from 1 to 5 mg / ml, i-2) air evaporation of the solution of step i-1), and i-3) contacting the solution of step i-2) with a based.
    [11" id="c-fr-0011]
    11. The method of claim 9 or 10, characterized in that step ii) is conducted by incubating the hydrogel of step i) in a mixed organic solvent / aqueous solvent solution whose organic solvent content is 20. at 30% by volume, then in a mixed organic solvent / aqueous solvent solution whose content of organic solvent is 40 to 50% by volume, then in a mixed organic solvent / aqueous solvent solution whose organic solvent content is 60 at 70% by volume, then in a mixed organic solvent / aqueous solvent solution whose organic solvent content is 80 to 95% by volume, then in a pure solution of said organic solvent.
    [12" id="c-fr-0012]
    12. Method according to any one of claims 9 to 11, characterized in that the incubation of step ii) in each of the solutions lasts from 30 minutes to 2 hours.
    [13" id="c-fr-0013]
    13. Method according to any one of claims 9 to 12, characterized in that the organic polymer concentration of the impregnating solution is from 20 to 500 mg / ml.
    [14" id="c-fr-0014]
    14. Method according to any one of claims 9 to 13, characterized in that it further comprises a step v) lyophilization of the composite biomaterial of step iv).
    [15" id="c-fr-0015]
    15. Composite biomaterial as defined in any one of claims 1 to 8 for its medical use.
    [16" id="c-fr-0016]
    16. Composite biomaterial as defined in any one of claims 1 to 8 for its use in the treatment of chronic wounds.
    [17" id="c-fr-0017]
    17. A composite biomaterial as defined in any one of claims 1 to 8, for its use in the preventive treatment of infections after cardiac or colorectal surgery.
    [18" id="c-fr-0018]
    18. A composite biomaterial as defined in any one of claims 1 to 8 for use in the treatment of hernias of the abdominal wall or eventrations.
    [19" id="c-fr-0019]
    19. Therapeutic dressing comprising an inner layer and an outer layer consisting of a secondary dressing chosen from an adhesive, a compress, a bandage and one of their mixtures, characterized in that the inner layer comprises a composite biomaterial as defined in US Pat. any of claims 1 to 8.
    [20" id="c-fr-0020]
    20. Abdominal wall reinforcement, characterized in that it comprises a composite biomaterial as defined in any one of claims 1 to 8.
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    同族专利:
    公开号 | 公开日
    WO2017144806A1|2017-08-31|
    US20190151495A1|2019-05-23|
    FR3047901B1|2018-02-23|
    JP2019505338A|2019-02-28|
    CA3015030A1|2017-08-31|
    EP3419684A1|2019-01-02|
    EP3419684B1|2019-12-11|
    引用文献:
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    US8632839B2|2010-10-19|2014-01-21|Covidien Lp|Methods of forming self-supporting films for delivery of therapeutic agents|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|
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    US11135336B2|2019-09-30|2021-10-05|L'oreal|Thin-film vehicles that stabilize highly reactive active ingredients|
    法律状态:
    2017-02-17| PLFP| Fee payment|Year of fee payment: 2 |
    2017-08-25| PLSC| Publication of the preliminary search report|Effective date: 20170825 |
    2018-02-23| PLFP| Fee payment|Year of fee payment: 3 |
    2020-02-19| PLFP| Fee payment|Year of fee payment: 5 |
    2021-11-12| ST| Notification of lapse|Effective date: 20211005 |
    优先权:
    申请号 | 申请日 | 专利标题
    FR1651432A|FR3047901B1|2016-02-22|2016-02-22|BIOMATERIAL COMPOSITIONS WITH CONTROLLED RELEASE OF ACTIVE INGREDIENTS|
    FR1651432|2016-02-22|FR1651432A| FR3047901B1|2016-02-22|2016-02-22|BIOMATERIAL COMPOSITIONS WITH CONTROLLED RELEASE OF ACTIVE INGREDIENTS|
    EP17710334.8A| EP3419684B1|2016-02-22|2017-02-20|Composite biomaterials with controlled release of the active ingredient, preparation method, and uses|
    CA3015030A| CA3015030A1|2016-02-22|2017-02-20|Composite biomaterials with controlled release of the active ingredient, preparation method, and uses|
    PCT/FR2017/050377| WO2017144806A1|2016-02-22|2017-02-20|Composite biomaterials with controlled release of the active ingredient, preparation method, and uses|
    JP2018544211A| JP2019505338A|2016-02-22|2017-02-20|Composite biomaterials with controlled release of active ingredients, preparation methods and uses|
    US16/350,064| US20190151495A1|2016-02-22|2017-02-20|Composite biomaterials with controlled release of active ingredient, preparation process and uses|
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