• 专利附录
  • 专利说明
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    • 专利摘要:
    support material for wound care and / or other tissue healing applications. Support material for wound care and / or other tissue healing applications and methods for producing the same is described. The support material is comprised of an extracellular matrix devoid of fish skin cells. The carrier material may also include lipids from the lipid layer of fish skin. Also described are methods for producing and employing the support material.
    公开号:BR112012007597B1
    申请号:R112012007597-7
    申请日:2010-10-06
    公开日:2019-10-15
    发明作者:Gudmundur Fertram Sigurjonsson；Dora Hlin Gisladottir；Gudmundur Phd Gudmundsson
    申请人:Kerecis Ehf；
    IPC主号:
    专利说明:

    [0001] The present invention relates to a support material for wound care and / or other tissue healing applications and methods for producing it. The support material comprises a decellularized extracellular matrix of fish skin.
    BACKGROUND [0002] A variety of human, animal and synthetic materials are currently described or used in medical procedures to augment, repair or correct tissue defects.
    [0003] For example, United States Patent Application No. 2003/0059460 discloses a hybrid polymeric material comprising synthetic and natural polymers that can be used in the regeneration of living body tissue. The hybrid is composed of a naturally occurring cross-linked polymer and a synthetic polymer absorbable by biodegradation. A number of complicated process steps, however, must be taken to produce the water material. In addition, the resulting hybrid material contains synthetics as well as naturally occurring materials.
    [0004] U.S. Patent No. 6,541,023 to the United States describes the use of porous collagen gels derived from fish skin for use as supports in tissue engineering. The preparation of collagen gels involves grinding fish skin. Additionally, Chinese Patent No. 1068703 describes a process for preparing fish skin for burn wound dressings, involving separating the fish skin from the fish body and placing the skin in a preservative solution of iodine, ethanol, borneol , zinc sulfadiazine and hydrochloric acid in sufficient proportions to establish a pH value of 2.5-3. However, these products can be difficult to manipulate because the product of U.S. Patent 6,541,023 is in gel form and the product of Chinese Patent No. 1068703 is stored in a solution.
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    2/32 [0005] In addition, a series of extracellular matrix products for medical use have been derived from human skin (ALLODERM® Regenerative Tissue Matrix (LifeCell); fetal bovine dermis (PRIMATRIX ™ Dermal Repair Scaffold (TEI Biosciences)); pig urinary bladder (MATRISTEM ™ Extracellular Matrix Wound Sheet (Medicine Industries, Inc.)) and pig small intestine submucosa (OASIS® Wound Matrix (Healthpoint Ltd)). However, there is a need for better products and methods to enhance wound healing and tissue repair. The present invention addresses this need.
    SUMMARY [0006] The extracellular matrix (ECM) of vertebrates is a complex structural unit that surrounds and supports cells. ECM is composed of complex mixtures of structural proteins, the most abundant of which, collagen, and other specialized proteins and proteoglycans. The support material described here is an acellular support, largely intact from components of the natural biological ECM in fish skin. The support may also comprise naturally occurring fish skin lipids. The native three-dimensional structure, composition and functions of the dermal ECM are essentially unchanged, providing support to support cell migration, adherence, proliferation and differentiation, thus facilitating tissue repair and / or replacement. The present invention also involves methods of producing and using the support material.
    BRIEF DESCRIPTION OF THE DRAWINGS [0007] Figure 1 illustrates a sample of an ECM product devoid of cell (support material) made from fish skin according to a process described here.
    [0008] Figure 2 illustrates optical images of cross sections of the support material at 100X (2A) and 400X (2B) in magnitude.
    [0009] Figure 3 illustrates the electron microscope scan of images (SEM) of cross sections of the support material to
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    300x (3A) and 600x (3B) in magnitude.
    DESCRIPTION [0010] A support material according to the present invention is obtained from the intact skin of the fish. An illustrative example shows a sample of the support material of figure 1. Any species of fish, including bone fish or cartilaginous fish, can be used as the origin of the fish skin. For example, the source may be rounded fish such as cod, herring, catfish; flatfish, such as sole, sole; salmonids such as salmon and trout; debris, like tuna; or small fish such as herring, anchovies, mackerel and sardines. In some embodiments, fish skin is obtained from oily freshwater fish and / or fish known to contain large amounts of Omega-3 oil. Examples of fish with a high Omega-3 oil content are salmon, sardines, tuna, herring, cod, sardines, mackerel, smelts, white fish, hake and some varieties of trout.
    [0011] The fish skin is removed from the fish before processing. If the fish skin comes from a fish species that has scales, the fish skin should be peeled off so that a substantial part of the scales is removed or at least the hydroxyapatite removed from the scales. The phrase a substantial part of the scales are removed or substantially free of scale means that at least 95%, preferably at least 99%, and more preferably 100% of the fish skin scales are removed. Fish skin Substantially free of scales can also refer to fish skin from a species of fish without scales. The scales are removed or before all processing, with mechanical pressure (via, for example, knife, scraping with abrasives, water pressure, a device for removing scales that uses the same mechanical force as knives or another pressure device , such as polishing with ceramic or plastic) or after some chemical treatment (for example, decellularization) and then with mechanical pressure in order to remove the scales. If the fish skin is first treated
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    4/32 chemically and / or enzymatically (for example, treatment with TRITON® x100), the mechanical pressure in general needs to be mild, since the skin is more vulnerable to tearing after decellularization. The scales can be removed in more than one step, for example, a partial removal before decellularization followed by additional removal during and / or after decellularization. Alternatively, the scales can be removed by chemical treatment only.
    [0012] After the scales have been removed, the fish skin is optionally frozen before decellularization. The fish skin can be frozen quickly by incubating the skin in liquid nitrogen or by using other special freezing equipment that can freeze the skin at -70 ° C or lower, in order to preserve the collagen structure of the support. Alternatively, the fish skin can be frozen in a conventional type of freezer that could be found, typically in a fish product factory. The freezing process can smooth or partially lyse the cells comprising the intact fish skin, helping to facilitate the decellularization of the fish skin. If the fish skin has been frozen, it can be thawed later for further processing.
    [0013] Whether the fish skin is frozen or not, it can be washed with a buffer solution before further processing. For example, the fish skin can be washed 1 to 3 X with a buffer solution optionally containing one or more antioxidants (for example, ascorbic acid (such as 50 mM ascorbic acid), vitamins A, C, E, and beta-carotene), antibiotics (for example, streptomycin and penicillin), proteases (for example, dispase II) and protease inhibitors (for example , antipain, aprotinin, benzamidine, bestatin, DFP, EDTA, EGTA, leupeptin, pepstatin, phosphoramidone and PMSF) to facilitate disinfection and stabilization of fish skin. The buffer solution can be at a pH of at least 5.5 such as 6.0; 6.5; 7.0; 7.5; 8.0; 8.5; 9.0; 9.5; 10.0 or higher. In some embodiments, the pH is between 7.0 and 9.0 for example, between 7.5 and 8.5. The solution
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    5/32 buffer can also be used as a medium in which fish skin can be stored for a few days to a few weeks or more. In some embodiments, the fish skin is stored in the buffer solution at a temperature of about 4 ° C.
    [0014] After freezing and / or washing and / or storage in a buffer solution, fish skin is treated with one or more decellularization solutions to remove cellular material, including antigenic material, from fish skin with minimal damage to no damage to the mechanical and structural integrity and biological activity of the naturally occurring extracellular matrix.
    [0015] The terms extracellular matrix or ECM as used herein, refers to the non-cellular tissue material present within the fish skin that provides structural support for the skin cells in addition to performing several other important functions. The ECM described here does not include matrix material that has been constituted or completely reformed from ECM components extracted, purified or separated (for example, collagen).
    [0016] The terms acellular, decellularized, decellularized fish skin and others, as used herein, refer to a fish skin from which a substantial amount of cellular content and nucleic acid has been removed leaving a complex three-dimensional interstitial structure of ECM . Decellularization agents are the effective agents in removing a substantial amount of cell content and nucleic acid from the ECM. The ECM is decellularized or substantially free of cellular material and nucleic acid (i.e., a substantial amount has been removed) that at least 50% of viable and non-viable nucleic acids and other cellular material have been removed from the ECM. In some modalities, around 55%, 60%, 65%. 70%, 75%, 80%, 855, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% of the acids viable and non-viable nucleic and cellular material are removed. Decellularization can be verified, for example, by testing the treated fish skin for DNA content. Removing acids
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    6/32 nuclei of ECM can be determined, for example, by histological examination of ECM, and / or by a biochemical assay such as the PICOGREEN® assay, diphenylamine assay, or by PCR.
    [0017] Decellularization breaks cell membranes releasing cell content. Decellularization may involve one or more physical treatments or one or more chemical treatments, one or more enzymatic treatments or any combination of those mentioned. Examples of physical treatments are sound treatment, mechanical agitation, mechanical massage, mechanical pressure, and freezing / thawing. Examples of chemical decellularizing agents are ionic salts (eg, sodium azide) bases, acids, detergents (eg, non-ionic and ionic detergents), oxidizing agents (eg, hydrogen peroxide and peroxyacids), hypotonic solutions, hypertonic solutions, chelating agents (for example, EDTA and EGTA), organic solvents (for example, tri (n-butyl) phosphate, ascorbic acid, methionine, cysteine maleic acid, and DNA-binding polymers (for example, poly- L-lysine, polyethylimine (PEI), and polyaminoamine (PAMAM). Non-ionic detergents include 4- (1,1,3,3-tetramethylbutyl) phenylpolyethylene glycol, tert-octylphenoxypolyethoxyethanol, tert-octylphenyl polyethylene glycol (TRITON® X -100) (Dow Chemical Co.) Ionic detergents include sodium dodecyl sulfate (SDS), sodium deoxycholate, TRITON® X-200, and zwitterionic detergents (eg CHAPS). Other suitable decellularization detergents include polyoxyethi leno (20) sorbitan monooleate and polyoxyethylene (80) sorbitan monooleate (Tween 20 and 80), 3 - [(3-chloramidopropyl) -dimethylamino] -1-porpanosulfonate, octyl glycoside and sodium dodecyl sulfate. Examples of enzymatic decellularizing agents are proteases, endonucleases and exonucleases. Proteases include serine proteases, (eg, trypsin, threonine proteases, cysteine proteases, aspartate proteases, metalloproteases (eg, thermolysin) and glutamic acid proteases. Decellularization is generally done at a pH of at least 5.5 as 6.0; 6.5; 7.0; 7.5; 8.0; 8.5; 9.0; 9.5; 10.0; or more. In some embodiments, the pH is between
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    7.0 and 9.0; for example, between 7.5 and 8.5.
    [0018] As an example of a decellularization step is the incubation of fish skin in a solution composed of 1M NaCl, 2% deoxycholic acid, 0.02% sodium azide and 500 ppm of streptomycin. In another example, the fish skin is incubated with a first decellularization solution composed of a protease (for example, 2.5 U / ml of dispase II) and other components (for example, 0.02% sodium azide). sodium). The first decellularization solution is discarded and the fish skin is then treated with a second decellularization solution as a solution comprising a detergent (eg 0.5% TRITON® X-100) and other components (eg 0.02% sodium azide). In another example, the fish skin is first treated with a decellularization solution comprising detergent (for example, 0.5% TRITON® X-100) with other components (for example, 0.02% EDTA, sodium, and / or deoxy-holic acid) and then incubated in a second decellularization solution comprising a detergent such as SDS.
    [0019] The fish skin may or may not be incubated under agitation. Decellularization steps can be repeated as needed by discarding any remaining decellularization solution, optionally washing the fish skin with a buffer solution (eg Hank's Balanced Salt Solution) and then treating the fish skin again with another stage of decellularization. Once a sufficient amount of cellular material has been removed, the decellularization solution can be removed (for example, by aspiration or by gently discarding the solution).
    [0020] After decellularization, the fish skin can be optionally washed with water, buffer solution and / or saline solution. Examples of suitable washing solutions include Dulbecco's phosphate buffered saline (DPBS), Hank's balanced salt solution (HBSS), Medium 199 (M199, SAFC Biosciences Inc) and / or L-glutamine. The washing steps are
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    8/32 generally made at a pH of at least 5.5, such as 6.0; 6.5; 7.0; 7.5; 8.0; 8.5; 9.0; 9.5; 10.0 or higher. In some embodiments, the pH is between 7.0 and 9.0, for example, between 7.5 and 8.5.
    [0021] Fish skin can optionally be targeted to improve the appearance of the final product. The bleaching can be done before, after and; or at the same time as the decellularization step. For example, one or more bleaching agents can be incorporated into one or more decellularization solutions and / or buffer solutions. Examples of bleaching agents include sodium sulfite, hydrogen peroxide, ammonium persulfate, potassium persulfate, and sodium persulfate. In some embodiments, if a strong bleaching agent such as persulfate is used, bleaching and decellularization can be combined in a single step comprising incubating the fish skin in a mixture of one or more bleaching agents, thickeners and sources of peroxide. For example, a dry bleach mixture can be prepared (see for example, the bleach mixtures described in Example 5), followed by the addition of water, hydrogen peroxide, or a combination thereof, to the dry mixture forming a bleach solution that can still be sufficient for decellularization. Bleaching agents (eg, sodium sulfite, hydrogen peroxide, ammonium persulfate, potassium persulfate, and sodium persulfate) should be about 40-60% w / w of the dry mixture. A combination of EDTA and persulfates can be added to the mixture in order to accelerate bleaching and decellularization. In some embodiments, the concentration of EDTA in the dry mix is about 0.25-5% w / w. Hydrogen peroxide can be approximately 15-25% of the mixture; the source of peroxide can be sodium percarbonate and potassium percarbonate. Sodium phosphate perhydrate and sodium carbonate or magnesium metasilicate and silicon silicate can also be used as a source of peroxide. The dry mixture can also include silica and hydrated silica, for example, 1 -10% w / w, and optionally one or more stearate (for example, ammonium stearate, sodium stearate,
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    9/32 and / or magnesium stearate). In addition, the dry blend may optionally include thickeners, such as hydroxypropyl methylcellulose, hydroxyethylcellulose, alginates, organic gums (eg, cellulose, xanthan gum), sodium metasilicate and combinations of the above in order to increase the viscosity of the bleach solution / decellularization and protect protein fibers from damage. Targeting and / or targeting plus decellularization is generally done at a pH of at least 5.5 such as 6.0; 6.5; 7.0; 7.5; 8.0; 8.5; 9.0; 9.5; 10.0 or higher. In some embodiments, the pH is between 7.0 and 9.0, for example, between 7.5 and 8.5. After bleaching and / or bleaching and decellularization, the fish skin can optionally be washed with a solution composed of L-glutamine under the pH conditions described above.
    [0022] In some modalities, fish skin is treated with a digestion enzyme. Similar to bleaching, digestion can be done before, after, and / or at the same time as decellularization. Suitable enzymes include proteases, for example, serine proteases, threonine proteases, cysteine proteases, aspartate proteases, metalloproteases and glutamic acid proteases. In some embodiments, the digestion enzyme is a serine protease like trypsin. The digestion enzyme can be an enzyme that works in an alkaline environment, limits crosslinking within the ECM, and softens the fish scale. Digestion is generally at a pH of at least 5.5, such as 6.0; 6.5; 7.0; 7.5; 8.0; 8.5; 9.0; 9.5; 10.0 or higher. In some embodiments, the pH is between 7.0 and 9.0, for example, between 7.5 and 8.5.
    [0023] Decellularized fish skin can be optionally cryo-preserved. Cryogenic preservation may involve immersing the fish skin in a cryoprotective solution before freezing. The cryoprotective solution in general comprises a suitable buffering agent, one or more cryoprotectants, and optionally, a solvent, for example, an organic solvent which, in combination with water, undergoes minimal expansion and contraction. Examples of cryoprotectants include, sucrose, raffinose, dextran, trehalose, dimethylacetamide, dimethyl sulfoxide, ethylene glycol, glycerol, propylene
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    10/32 glycol, 2-methyl-2,4-pantandial, some antifreeze proteins and peptides, and their combinations. Alternatively, if the decellularized fish skin is frozen quickly (quick freezing) before sublimation in order to minimize ice crystals formed during the freezing step, the skins can be optionally frozen in a buffer solution that does not include cryoprotectants. Breeding-preservation is generally done at a pH of at least 5.5, such as 6.0; 6.5; 7.0; 7.5; 8.0; 8.5; 9.0; 9.5; 10.0 or higher. In some embodiments, the pH is between 7.0 and 9.0, for example, between 7.5 and 8.5.
    [0024] Decellularized fish skin can be packaged in a sterile container, such as a jar or glass bag. In one embodiment, a TYVEK® bag is used. For example, fish skin can be incubated in a cryoprotective solution, packaged in a TYVEK® bag, then placed in a freeze dryer and frozen at a speed that is compatible with the cryoprotectant.
    [0025] Decellularized fish skin can be lyophilized, that is, frozen at a low temperature under vacuum conditions so that water is removed sequentially from each ice crystal phase without recrystallizing the ice. During lyophilization, water is usually removed first via sublimation and then via desorption, if necessary. Another method of removing excess water after processing and before sterilization is vacuum pressure.
    [0026] In some modalities, the decellularized bundle skin is sterilized before and / or after being frozen. Sterilization methods are known in the art. For example, decellularized fish skin can be placed in an ethylene oxide chamber and treated with suitable ethylene oxide cycles. Other methods of sterilization include sterilization with ozone, carbon dioxide, gaseous formaldehyde or radiation (for example, gamma radiation, X-rays, electron beam processing, and subatomic particles).
    [0027] As an alternative or beyond freezing,
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    11/32 lyophilization and / or vacuum water pressure, decellularized fish skin can be preserved in a non-aqueous solution such as alcohol.
    [0028] The resulting product (support material) is a sterile collagen-based matrix, endowed with properties that facilitate tissue regeneration, repair and / or replacement (for example, repair, regeneration, and / or growth of endogenous tissue ). The term support material refers to a material comprising fish skin that has been decellularized and, optionally bleached, digested, lyophilized, etc., as set out above. The support material can obtain a stable support to support endothelial and / or epithelial cells, can be integrated by the host, being biocompatible, does not significantly calcify, and can be stored and transported at room temperatures. The phrase integrated by the host means at present that the cells and tissues of the patient being treated with the support material can grow on the support material and that the support material is actually integrated / absorbed into the patient's body. The term biocompatible refers to a material that is substantially non-toxic in the in vivo environment of its intended use, and is not substantially rejected by the patient's physiological system (that is, it is not antigenic). This can be scaled by the ability of a material to pass the biocompatibility tests seen in International Standards Organization (ISO) Standard No. 10993 and / or in US Pharmacopeia (USP) 23 and / or US Food and Drug Administration (FDA) memorandum of blue book n ° G95-1, entitled Use of International Standard ISO-10993, Biological Evaluation of Medical Devices Particularly 1: Evaluation and Testing. Typically, these tests measure the toxicity, infectivity, pyrogenicity, irritation potential, reactivity, hemolytic activity, carcinogenicity and / or immunogenicity of a material. A biocompatible or material structure, when introduced in most patients, will not cause a significantly adverse reaction or response, of long life, or on a biological scale, distinguished by a mild, transient inflammation, which typically accompanies surgery or implant of
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    12/32 foreign objects in a living organism.
    [0029] Constantly, high levels of metalloprotease matrices (MMPs) can contribute to the chronic state of injuries. The support material described here can absorb the metalloprotease matrix (MMPs), thus promoting wound healing and transition from a chronic to acute injury.
    [0030] The support material contains extracellular matrix (ECM) proteins from fish skin. The ECM components in the support material can include, for example, structural protein, adhesive glycoproteins, proteoglycans, polysaccharides other than proteoglycans, and proteins from the extracellular matrix. Examples of structural proteins include collagen (the most abundant protein in ECM) such as fibrillar collagens (types, I, II, III, V and Xi), facit collagens (types IX, XII, and XIV), short-chain collagen (types VIII and X), basement membrane collagen (type Iv) and other collagens (types VI, VII and XIII), elastin and laminin. Examples of adhesive glycoproteins include fibronectin, tenascin, and thrombo-respondin. Examples of proteoglycans include heparin sulfate, chondroitin sulfate, and keratan sulfate. An example of a non-proteoglycan polysaccharide is hyaluronic acid. Matrix cell proteins are a structurally diverse group of extracellular proteins that regulate cell function via interactions with cell surface receptors, cytokines, growth factors, proteases and ECM. Examples include thromboespondins (TSPs) 1 and 2; tenascin, and SPARC (secreted, acidic and cysteine-rich protein).
    [0031] In some embodiments, decellularization (and other optional processing steps) does not remove all naturally occurring lipids from the lipid layer of the fish skin. Thus, the support material may comprise one or more lipids from the fish skin, particularly from the lipid layer of the fish skin. For example, the support material can include up to about 25% w / w lipids) dry weight of the total support material after lyophilization), such as 0.1%, 0.5%, 1%, 2%, 3 %, 4%, 5%, 6%, 7% ,, 8%, 9%,
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    10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 195, 20%, 21%, 22%, 23%, or 24% of lipids w / w. The presence of lipids in the support material can be verified, for example, by extraction with organic solvent followed by chromatography. Examples of suitable organic solvents include acetone and chloroform.
    [0032] The lipids in the support material include, for example, fatty acyls (that is, their conjugated fatty acids and derivatives); glycerolipids, glycerophospholipids (ie phospholipids), sphingolipids, saccharolipids, polyketides, lipids sterols (ie sterols), some fat-soluble vitamins; prenol lipids; and / or polyketides. Examples of fatty acyls include saturated fatty acids, such as polyunsaturated fatty acids, fatty esters, fatty amides and eicosanoids. In some embodiments, fatty acids include Omega-3 fatty acids, such as eicosapentaenoic acid (EPA), and docosahexanoic acid (DHA) found in high concentrations in fish oil). Other fatty acids found in fish oil include arachidic acid, gadoleic acid, arachidonic acid, butyric acid, caprylic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, vaccenic acid , linoleic acid, alpha-linolenic acid, gamma-linolenic acid, behenic acid, erucic acid, and lignoceric acid. Examples of glycerolipids include mono-, di- and triglycerols substituted as monoacylglycerols, diacylglycerols and triacylglycerols (i.e., monoglycerides, diglycerides, and triglycerides). Examples of glycerophospholipids include phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. Examples of sphingolipids include phosphofingolipids and glycosphingolipids. Examples of sterol lipids include cholesterol; steroids, and secosteroids (various forms of Vitamin D). Examples of prenol lipids include isoprenoids, carotenoids, and quinones and hydroquinones such as Vitamins E and K.
    [0033] The support material may contain the addition of one or more active agents (ie, an agent that is added during or after the processing of the support material) such as antibiotics, antiseptics, agents
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    14/32 antimicrobial, antiviral, antifungal, antiparasitic and anti-inflammatory. The active ingredient can be a compound or composition that facilitates the treatment of the wound and / or tissue healing as an antioxidant, or pharmaceutical. It can also be a protein or proteins and / or other biological agents. Antibiotic, antiseptic, and antimicrobial agents can be added in an amount sufficient to obtain effective antimicrobial properties for the support material. In some embodiments, the antimicrobial agent is one or more of metal, antimicrobials, such as silver, gold, platinum, copper, zinc or combinations of these. For example, silver can be added to the support material during processing in ionic, metallic, elemental, and / or colloidal form. Silver can also be in combination with other antimicrobials. Anti-inflammatory agents can be added in an amount sufficient to reduce and / or prevent inflammation in the wound or tissue area where the support material is applied.
    [0034] The support material can be used in dry form. Alternatively, the support material can be rehydrated before use. In some embodiments, one or more support materials are laminated together to form a thicker support material.
    [0035] In general, the support material is about 0.1 to 4.0 mm thick (ie, in the cross section) as 0.5, 1.0; 1.5; 2.0; 2.5; 3.0; or 3.5 mm thick. The thickness can depend on a number of factors, including the species of fish used as the starting material, processing, lyophilization, and / or rehydration (see Example 14). Naturally, the thickness is proportionally greater when the product comprises more than one layer of the support material.
    [0036] The support material described here can be used for a number of applications in medicine. For example, the support material can be used as a wound dressing and / or as a suture material, where the support material is applied over or to a part of the wound or tissue area. The term injury refers to any injury
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    15/32 resulting in tissue damage, tissue penetration, laceration, or injury. Wounds that can be treated with support material include injuries that can be located anywhere, including internal, interfacial, external, interstitial, extracorporeal, and / or intracorporeal. Examples of wounds suitable for covering by support material include cuts, deep cuts, open wounds, tissue rupture, decubitus dermatitis, injuries, chronic wounds, battlefield wounds, necrotic, acute, chronic, traumatic wounds, lacerations, abrasions , bruises, necrotic fasciitis, toxic epidermal necrolysis. pressure wounds, venous insufficiency ulcers, arterial ulcers, diabetic or neuropathic ulcers, pressure ulcers, mixed ulcers, burn wounds, mucormycosis, vasculitic wounds, pyoderma, gangrene and equivalent, and / or combinations of those cited, known to those skilled in the art technical. The treatment of injuries to humans and animals is covered.
    [0037] In some modalities, the support material is used for reconstruction of the abdominal wall, for example, for repair of hernias. For example, when repairing a hernia, a surgeon will make an incision near the site of the hernia. For an inguinal hernia, the incision is made just above the crease where the abdomen meets the thigh. To repair an umbilical hernia, it is done close to the navel. If the hernia occurred at the site of a previous operation, the incision for that surgery is reopened. The surgery proceeds in much the same way, regardless of where the incision was made. The hernia sac is carefully opened and the bowel or other tissue is placed back into the abdomen. The weakened area is repaired and reinforced with a synthetic mesh or a suture that removes the abdominal muscle tissue back together.
    [0038] The support material described herein can be used as a mesh or suture material, or used to reinforce a mesh or suture material. The support material can be used to reinforce or enhance a wound treatment or tissue healing product as a
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    16/32 wound dressing, a mesh material, gauze or a suture. For example, the support material can be used close to or interwoven with a wound dressing, mesh material or a suture. When used as a mesh or suture material, or to reinforce a mesh or suture material, the support material can be treated to increase the crosslinking of the ECM component materials, using, for example, chemical crosslinkers such as glutaraldehyde or chromium. The support material can also be used to replace gums lost due to periodontal diseases, form a bladder tongue, and facilitate the reconstruction of the pelvic floor.
    [0039] As used herein, the singular forms one, one, and o, a, include references in the plural, unless the context clearly indicates otherwise.
    [0040] The publications presented here are given only for presentation before the filing date of this application. Nothing in the present should be construed as an agreement that the present description is not entitled to anticipate such publication by virtue of the preceding description. In addition, the publication dates described may differ from the actual publication dates, which may require independent confirmation. All publications, patents, patent applications and other references cited herein are hereby incorporated by way of reference in their entirety.
    [0041] Although the description was made in detail with reference to some of its modalities, it will be evident to the person skilled in the art, that several changes can be made, and the equivalents employed, without departing from the scope of the description. In addition, the following examples are illustrative only and should not be construed as limiting the description in any way.
    EXAMPLES
    Example 1
    Skin removal
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    17/32 [0042] The skin was removed from a cut of fish, along with as much epidermal fat as possible. The fish skin was peeled off a cutting board, scraping the surface of the fish skin with a knife.
    Disinfection and stabilization [0043] The skin removed from scales was washed twice for 1 hour at 4 ° C, with sterile phosphate buffered saline, containing 50 mM ascorbic acid and 500 ppm streptomycin, respectively.
    Decellularization [0044] a) The fish skin was placed in 1M NaCl for 8 hours followed by incubation in 2% deoxycholic acid containing 0.02% sodium azide and 500 ppm of streptomycin.
    [0045] b) The solution in a) was discarded. The fish skin was placed in Hank's Balanced Saline Solution for 10 minutes at 40 rpm at room temperature. The step was repeated and the fish skin was placed in a container with an open laminar flow hood and the solution was aspirated. The procedure was repeated two more times.
    [0046] c) The fish skin was treated with a decellularization solution containing 0.5% SDS, and the container was placed in a centrifuge at 40 rpm for 1 hour at room temperature.
    [0047] d) The decellularization solution was removed as described in b) by aspiration. The fish skin was washed with 50 mL of Dulbecco's phosphate buffered saline.
    Digestion [0048] The fish skin was incubated for 18 hours at room temperature in a digestion solution consisting of 1M Tris-HCl, trypsin at 0.05 pg / mL, at a pH of 8.5.
    Cryogenic protection [0049] The fish skin was immersed in a pre-freezing solution containing 7% dextran, 6% sucrose, 6% raffinose and 1 mM EDTA in Hank's balanced salt solution.
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    Washing [0050] The fish skin was washed with the pre-freezing solution and placed in a centrifuge at 40 rpm for 120 minutes at room temperature.
    Packaging [0051] The fish skin was placed in a pouch that was closed by a hot seal. The bag consisted of a DuPont medical grade porous TYVEK® membrane, suitable for sterilization with ethylene oxide (Etox)
    Freeze drying [0052] The bag containing the fish skin was placed in a freeze dryer and freeze dried.
    Sterilization [0053] The bag containing the fish skin was placed in an ethylene oxide chamber and treated with 1 to 5 cycles of ethylene oxide to sterilize the fish skin.
    Example 2
    Skin removal [0054] The skin was removed from a cut of fish, along with as much epidermal fat as possible. The fish skin was peeled off a cutting board, scraping the surface of the fish skin with a knife.
    Disinfection and stabilization [0055] The skin removed from scales was washed twice for 1 hour at 4 ° C, with sterile phosphate buffered saline, containing 50 mM ascorbic acid and 500 ppm streptomycin, respectively.
    Decellularization [0056] a) The fish skin was placed in phosphate-buffered saline containing 2.5 U / ml of dispase II (or pronase) and 0.02% sodium acid being incubated at 4 ° C for 8 hours with continuous agitation. THE
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    19/32 solution was discarded and the sample incubated under gentle agitation in a second solution of 0.5% TRITON® X-100 and 0.02% sodium acid for 24 hours.
    [0057] b) The second solution in a) was discarded. The fish skin was placed in Hank's Balanced Saline Solution for 10 minutes at 40 rpm at room temperature. The step was repeated and the fish skin was placed in a container in an open laminar flow hood and the solution was aspirated. The procedure was repeated two more times.
    [0058] c) The fish skin was treated with a decellularization solution containing 0.5% SDS, and the container was placed in a centrifuge at 40 rpm for 1 hour at room temperature.
    [0059] d) The decellularization solution of c) was removed as described in b) by aspiration. The fish skin was washed with 50 mL of Dulbecco's phosphate buffered saline.
    Digestion [0060] The fish skin was incubated for 18 hours at room temperature in a digestion solution consisting of 1M Tris-HCl, trypsin at 0.05 pg / mL, at a pH of 8.5.
    Cryogenic protection [0061] The fish skin was immersed in a pre-freezing solution containing 7% dextran, 6% sucrose, 6% raffinose and 1 mM EDTA in Hank's balanced salt solution.
    Washing [0062] The fish skin was washed with the pre-freezing solution and placed in a centrifuge at 40 rpm for 120 minutes at room temperature.
    Packaging [0063] The fish skin was placed in a TYVEK® porous membrane pouch and closed by heat sealing
    Lyophilization
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    20/32 [0064] The bag containing the fish skin was placed in a freeze dryer and freeze dried.
    Sterilization [0065] The bag containing the fish skin was placed in an ethylene oxide chamber and treated with 1 to 5 cycles of ethylene oxide to sterilize the fish skin.
    Example 3
    Skin removal [0066] The skin was removed from a cut of fish, along with as much epidermal fat as possible. The fish skin was peeled off a cutting board, scraping the surface of the fish skin with a knife.
    Freezing [0067] The fish skins were frozen in a standard fish factory freezer. During the freezing process the cells were lysed. Freezing prevents microbial growth.
    Defrosting [0068] Fish skins were thawed at 4 ° C [0069] Disinfection and stabilization [0070] The skin removed from scales was washed twice for 1 hour at 4 ° C, with sterile phosphate buffered saline, containing 50 mM ascorbic acid and 500 ppm streptomycin, respectively.
    Decellularization [0071] a) The fish skin was treated with a decellularization solution containing 0.5% SDS and the container was placed in a centrifuge at 40 rpm powder 1 hour at room temperature.
    [0072] b) The decellularization solution was removed by aspiration. The fish skin was washed with 50 mL of Dulbecco's phosphate buffered saline.
    Digestion
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    21/32 [0073] The fish skin was incubated for 18 hours at room temperature in a digestion solution consisting of 1M Tris-HCl, trypsin at 0.05 pg / mL, at a pH of 8.5.
    Cryogenic protection [0074] The fish skin was immersed in a pre-freezing solution containing 7% dextran, 6% sucrose, 6% raffinose and 1 mM EDTA in Hank's balanced salt solution.
    Washing [0075] The fish skin was washed with the pre-freezing solution and placed in a centrifuge at 40 rpm for 120 minutes at room temperature.
    Packaging [0076] The fish skin was placed in a TYVEK® porous membrane pouch and closed by heat sealing
    Freeze drying [0077] The bag containing the fish skin was placed in a freeze dryer and freeze dried.
    Sterilization [0078] The bag containing the fish skin was placed in an ethylene oxide chamber and treated with 1 to 5 cycles of ethylene oxide to sterilize the fish skin.
    Example 4 [0079] Fish skins were processed as described in each of examples 1 to 3, except that a bleaching agent (sodium sulfite) was also added to the pre-freezing solution at 0.5% concentration in the washing step, obtaining a targeted support matrix.
    Example 5 [0080] Bleach / decellularization mixtures were prepared as follows:
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    Bleach Mixture A
    Glyceryl stearate 5% Ammonium persulphate 20% EDTA 0.5% Xanthan gum 3.5% Potassium persulfate 46% Silica 2% Hydrated silica 2% SDS 2% Magnesium Percarbonate 19%
    Bleach Blend B
    Potassium persulfate 56% Sodium persulfate 3% Silica 2.7% SDS 1% Hydrated silica 1% EDTA 1% Sodium metasilicate 35.3%
    Bleach Mixture C
    Ammonium persulphate 5% Potassium persulfate 40% Sodium persulfate 10% Guar gum 3% Hydrated silica 7% Silica 3% Glyceryl stearate 2% EDTA 0.5%
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    SDS 1% Magnesium Percarbonate 15% Sodium metasilicate 13.5%
    Bleach Mixture D
    Ammonium persulphate 10% Potassium persulfate 40% Sodium persulfate 10% Hydroxyethylcellulose 3% Hydrated silica 7% Silica 3% Glyceryl stearate 1% EDTA 0.25% SDS 0.5 Sodium Percarbonate 15% Sodium metasilicate 10.25%
    Bleach Mixture E
    Ammonium persulphate 15% Potassium persulfate 35% Sodium persulfate 10% Hydroxyethylcellulose 3% Hydrated silica 7% Silica 3% Glyceryl stearate 1% EDTA 0.25% SDS 0.5 Sodium Percarbonate 10.25% Sodium metasilicate 15%
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    Note: The percentages are by weight of the total (dry) bleach mixture.
    Example 6
    Skin removal [0081] The skin was removed from a cut of fish. The fish skin was peeled off substantially by scraping the surface of the fish skin with a wire brush and knife.
    Freezing [0082] The skin was frozen in a quick-freezer (that is, placed directly in a freezer at -80 ° C). The fish skin was removed from the freezer and thawed at room temperature for about 2 hours
    Decellularization [0083] a) The tissue was incubated in a 0.5% TRITON® X-100 solution and 0.02% sodium azide in Dullbecco's phosphate buffered saline (DPBS), shaken at 40 rpm for 2 hours at room temperature with the solution discarded.
    [0084] b) The peeling process continued with a knife, resulting in a scale-free product (no visible scales).
    [0085] c) Placed the tissue in 2 mM L-glutamine in Hank's Balanced Saline Solution (HBSS) for 10 minutes at 40 rpm at room temperature and the solution was discarded.
    [0086] d) Placed the tissue in a 0.5% solution of Sodium Dodecyl Sulfate (SDS) in HBSS for 1 hour at room temperature, shaking at 40 rpm and the solution discarded.
    Digestion [0087] The fish skin was incubated for 18 hours at room temperature in a digestion solution consisting of 1 mM EDTA and 0.05 pg / mL of trypsin, at a pH of 8.5.
    Bleaching
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    25/32 [0088] The fabric was bleached with bleach mixture C and 2% hydrogen peroxide for 30 minutes
    Washing [0089] The fabric was washed with a continuous stream of water for 48 hours at 6 ° C.
    Cryogenic protection [0090] The fish skin was immersed in a pre-freezing solution containing 7% dextran, 6% sucrose, 6% raffinose and 1 mM EDTA in Dullbecco's Phosphate Buffered Saline (DPBS).
    Packaging [0091] The fish skin was placed in a TYVEK® membrane pouch and closed by heat sealing
    Freeze drying [0092] The bag containing the fish skin was placed in a freeze dryer and freeze dried.
    Sterilization [0093] The bag containing the fish skin was placed in an ethylene oxide chamber and treated with 1 to 5 cycles of ethylene oxide to sterilize the support material.
    Example 7 [0094] The product was made according to Example 6, minus the freezing step after removing the skin. Instead of the freezing step, the tissue was washed twice for 1 hour at 4 ° C with sterile phosphate buffered saline, containing 50 mM ascorbic acid, 500 ppm streptomycin, respectively.
    Example 8
    Skin removal [0095] The skin was removed from a cut of fish. The fish skin was peeled off, scraping the surface of the fish skin with a wire brush and knife.
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    Freezing [0096] The skin was frozen in a fast freezer at -80 ° C). The fish skin was removed from the freezer and thawed at room temperature for about 2 hours.
    Decellularization [0097] a) The tissue was incubated in a solution of 0.5% TRITON® X-100 and 0.02% EDTA in Dullbecco's phosphate buffered saline (DPBS), shaken at 40 rpm for 24 hours at room temperature with the solution discarded.
    [0098] b) The peeling process continued with a knife. The result was a scale-free product (no visible scales).
    [0099] c) The tissue was placed in 0.5% Sodium Dodecyl Sulfate (SDS) in Hank's Balanced Saline Solution (HBSS) for 1 hour at room temperature, shaking at 40 rpm, the solution being discarded .
    [0100] d) The tissue was placed in a 2mM solution of L-glutamine in Hank's Balanced saline solution for 10 minutes at 40 rpm and at room temperature, the solution being discarded.
    Bleaching [0101] The fabric was bleached with bleach C and 2% hydrogen peroxide for 30 minutes
    Washing [0102] The fabric was washed with a continuous stream of water for 48 hours at 6 ° C.
    Cryogenic protection [0103] The fish skin was immersed in a pre-freezing solution containing 7% dextran, 6% sucrose, 6% raffinose and 1 mM EDTA in Dullbecco's Phosphate Buffered Saline (DPBS).
    Packing
    Petition 870190077407, of 10/08/2019, p. 40/63
    27/32 [0104] The fish skin was placed in a TYVEK® membrane pouch and closed by heat sealing
    Freeze drying [0105] The bag containing the fish skin was placed in a freeze dryer and freeze dried.
    Sterilization [0106] The bag containing the fish skin was placed in an ethylene oxide chamber and treated with 1 to 5 cycles of ethylene oxide to sterilize the support material.
    Example 9 [0107] The product was made as in Example 8, with the exception of the freezing step after skin removal. Instead of the freezing step, the tissue was washed twice for 1 hour at 4 ° C with sterile phosphate buffered saline, containing 50 mM ascorbic acid, 500 ppm streptomycin, respectively.
    Example 10
    Skin removal [0108] The skin was removed from a cut of fish. The fish skin was peeled off, scraping the surface of the fish skin with a wire brush and knife.
    Freezing [0109] The skin was frozen in a fast-freezer at -80 ° C). The fish skin was removed from the freezer and thawed at room temperature for about 2 hours
    Decellularization [0110] a) The tissue was incubated in a 0.5% TRITON® X-100 solution and 0.02% EDTA in Dullbecco's phosphate buffered saline (DPBS), shaken at 40 rpm for 48 hours at room temperature and the solution is discarded.
    [0111] b) The tissue was placed in a 2mM solution of
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    L-glutamine in Hank's Balanced saline solution (HBSS) for 10 minutes at 40 rpm and at room temperature, the solution being discarded.
    Bleaching [0112] The fabric was bleached with a Kerecis C bleach mixture and 2% hydrogen peroxide for 30 minutes
    Washing [0113] The fabric was washed with a continuous flow of water for 12 hours at 6 ° C.
    Cryogenic protection [0114] The fish skin was immersed in a pre-freezing solution containing 7% dextran, 6% sucrose, 6% raffinose and 1 mM EDTA in Dullbecco's Phosphate Buffered Saline (DPBS).
    Packaging [0115] The fish skin was placed in a TYVEK® membrane pouch and closed by heat sealing
    Freeze drying [0116] The bag containing the fish skin was placed in a freeze dryer and freeze dried.
    Sterilization [0117] The bag containing the fish skin was placed in an ethylene oxide chamber and treated with 1 to 5 cycles of ethylene oxide to sterilize the support material.
    Example 11 [0118] The product was made according to Example 10, except for the freezing step after skin removal. Instead of the freezing step, the tissue was washed twice for 1 hour at 4 ° C with sterile phosphate buffered saline containing 50 mM ascorbic acid, and 500 ppm streptomycin, respectively.
    Example 12
    Skin removal
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    29/32 [0119] The skin was removed from a cut of fish. The fish skin was peeled, scraping the surface of the fish skin with a wire brush and knife, resulting in a scale-free product.
    Freezing [0120] The skin was frozen in a fast freezer at -80 ° C). The fish scale was removed from the freezer and thawed at room temperature for about 2 hours.
    Decellularization and Bleaching [0121] a) The tissue was incubated in a mixture of bleach C and 25 of hydrogen peroxide for 30 minutes.
    [0122] b) The tissue was placed in 0.5% Sodium Dodecyl Sulfate (SDS) in Hank's Balanced Saline Solution (HBSS) for 1 hour at room temperature, stirring at 40 rpm, the solution being discarded .
    [0123] c) The tissue was placed in a 2mM solution of L-glutamine in Hank's Balanced saline solution for 10 minutes at 40 rpm and at room temperature, the solution being discarded.
    Washing [0124] The fabric was washed with a continuous flow of water for 12 hours at 6 ° C.
    Cryogenic protection [0125] The fish skin was immersed in a pre-freezing solution containing 7% dextran, 6% sucrose, 6% raffinose and 1 mM EDTA in Dullbecco's Phosphate Buffered Saline (DPBS).
    Packaging [0126] The fish skin was placed in a TYVEK® membrane pouch and closed by heat sealing
    Freeze drying [0127] The bag containing the fish skin was placed in a freeze dryer and freeze dried.
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    30/32
    Sterilization [0128] The bag containing the fish skin was placed in an ethylene oxide chamber and treated with 1 to 5 cycles of ethylene oxide to sterilize the support material.
    Example 13 [0129] The product was made according to example 12, except the freezing step after removing the skin. Instead of the freezing step, the tissue was washed twice for 1 hour at 4 ° C with sterile phosphate buffered saline containing 50 mM ascorbic acid, and 500 ppm streptomycin, respectively.
    Example 14 [0130] The thickness of the product was measured before and after lyophilization. Thicknesses were measured with a digital calibrator. The average thickness before and after lyophilization was 0.29 mm and 0.46 mm, respectively. The product was rehydrated (for example, in an aqueous saline solution) after lyophilization and before use on a wound. When lyophilized and rehydrated the product was tested, with an average thickness of 0.30 mm. Water loss (based on the differential weight before and after lyophilization) was estimated to be approximately 80-82%.
    Example 15 [0131] The permeability of the product was measured under atmospheric pressure and under vacuum (0.2 atm). The average permeability under atmospheric pressure was 3.73 mg of AGU after mm 2 of the sample / 24 hours. The average permeability under vacuum was 4.93 mg of water per mm 2 of sample / 180 seconds.
    Example 16 [0132] Preliminary biodegradability tests were performed by incubating the product samples in either isotonic saline or Dullbecco's Modified Eagle's Medium (DMEM) (Gibco) at 32 ° C. After about 6-10 days, the samples degraded by more than 50% (by weight).
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    Example 17 [0133] Preliminary cytotoxicity tests were performed by incubating the product samples with human foreskin fibroblast cells (cell line Hs27, ATCC CRL-1634) in DMEM, glucose and 10% fetal bovine serum. The samples did not cause any change in the visible phenotype, decrease or cell death.
    Example 18 [0134] The product was tested to determine the production force, tensile strength and elongation. Uniform samples measuring 33 mm by 6 mm were rehydrated and clamped at 0.4 x 105 MPa (0.4 bar) in a 500 N load cell. The samples were kept moist for the duration of the test. The results can be seen in Table 1 below:
    Table 1
    sample number Sample thickness [mm] ForceMax. [N] Maximum elongation at effort [mm] Maximum stretch [%] Effort at maximum force [Mpa] 1 0.48 18.10 11.68 35.4% 6.5 2 0.63 34.52 10.67 32.3% 9.2 3 0.35 26.66 13.53 41.0% 112.0 4 0.45 8.70 11.32 34.3% 3.1 5 0.56 75.59 20.52 62.2% 22.7 6 6 0.76 86.00 21.85 66.2% 19.0 77 0.76 64.80 27.32 82.8% 14.3 88 0.77 69.75 18.71 57.0% 15.0 99 0.33 23.67 11.66 35.3% 11.74 110 0.29 6.91 17.02 51.6% 4.51
    Note: 1 Mpa (mega pascal) = 1 N / mm 2 [0135] The support material described here is more robust
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    32/32 and stretches considerably more than grafts of acellular matrices produced from urinary bladders of rats, pigs and humans (see Dahms ET al., Compositin and biomechanical properties of the acellular matrix graft bladder: comparative analysis in rat, pig and human. British Journal of Urology, 1998; 82 (3): 411-419) and the FDA-approved ALLODERMDERM® (see Bottino ET AL., Freeze-dried acellular dermal matrix graft: Effects of rehydration on physical, chemical and mechanical properties. Dental Materials, 2009; 25 (9): 1109-1115).
    [0136] Attempts were made to test the tensile strength of OASIS® (pig's small intestine submucosa), and MATRISTEM® (pig's urinary bladder), but samples of both products disintegrated before any measurements could be made. .
    权利要求:
    Claims (11)
    [1]
    1. Support material characterized by comprising:
    fish skin substantially free of scales, decellularized, and lyophilized or vacuum-pressed in a dry form because it has a three-dimensional interstitial structure of components of the natural biological extracellular matrix derived from fish skin.
    the three-dimensional interstitial structure that includes up to about 25% (weight / weight) of natural fish skin lipids.
    [2]
    Support material according to claim 1, characterized in that the support material is in the form of a wound dressing, bandages, suture material and / or mesh material.
    [3]
    Support material according to claim 1, characterized in that the support material is biocompatible.
    [4]
    Support material according to claim 1, characterized in that the support material further comprises the addition of one or more active agents.
    [5]
    5. Support material according to claim 4, characterized in that the addition of the active agent is selected from the group consisting of antibiotics, antiseptics, antimicrobial, antiviral, antifungal, antiparasitic agents, anti-inflammatory agents, antioxidants, drugs, proteins , peptides and combinations thereof.
    [6]
    6. Method for producing the support material according to claim 1, the method comprising:
    a) obtain a fish skin; and
    b) removing substantially any fish scales present on the fish skin; and
    c) decellularize and lyophilize or vacuum fish skin pressure.
    [7]
    Method according to claim 6, characterized in that it also comprises peeling, washing, freezing, bleaching,
    Petition 870190077407, of 10/08/2019, p. 47/63
    2/2 digestion and / or cryogenic preservation of fish skin.
    [8]
    Method according to claim 6, characterized in that the decellularization comprises one or more physical treatments, one or more chemical treatments, one or more enzymatic treatments, or any combinations thereof.
    [9]
    9. Method, according to claim 8, characterized in that the chemical treatment comprises the treatment of fish skin with one or more decellularization agents selected from the group consisting of ionic salts, bases, acids, detergents, oxidizing agents, hypertonic solutions, chelating agents, organic solvents, methionine, maleic acid cysteine, polymers that bind to DNA, and combination thereof.
    [10]
    Method according to claim 8, characterized in that the enzymatic treatment comprises the treatment of fish skin with one or more enzymes selected from the group consisting of proteases, endonucleases and exonucleases.
    [11]
    Support material according to claim 2, characterized in that the support material is used to replace the gum lost due to periodontal disease, to form a bladder tongue, or to facilitate the reconstruction of the pelvic floor and / or wall .
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    同族专利:
    公开号 | 公开日
    KR101755740B1|2017-07-07|
    RU2012118397A|2013-11-20|
    PE20121798A1|2013-01-02|
    JP5893563B2|2016-03-23|
    ZA201203175B|2013-01-30|
    IL218945A|2015-05-31|
    MY160388A|2017-03-15|
    US8613957B2|2013-12-24|
    RU2568595C2|2015-11-20|
    EP2485779B8|2018-04-04|
    CN102781485A|2012-11-14|
    US20110244054A1|2011-10-06|
    BR112012007597A2|2019-05-07|
    IL218945D0|2012-07-31|
    CA2778090A1|2011-04-14|
    JP2013507164A|2013-03-04|
    PT2485779T|2018-04-26|
    AU2010304827A1|2012-05-10|
    EP2485779A2|2012-08-15|
    WO2011042794A2|2011-04-14|
    HUE038789T2|2018-11-28|
    PL2485779T3|2018-08-31|
    DK2485779T3|2018-05-07|
    KR20120134096A|2012-12-11|
    NZ599399A|2014-05-30|
    AU2010304827B2|2015-02-12|
    ES2664293T3|2018-04-19|
    CA2778090C|2018-03-13|
    EP2485779B1|2018-02-21|
    CL2012000891A1|2012-08-31|
    WO2011042794A3|2011-06-09|
    MX2012004037A|2012-10-15|
    NO2485779T3|2018-07-21|
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    法律状态:
    2017-09-12| B11Z| Dismissal: petition dismissal - article 216, par 2 of industrial property law|
    2017-11-28| B12F| Appeal: other appeals|
    2019-05-14| B07A| Technical examination (opinion): publication of technical examination (opinion) [chapter 7.1 patent gazette]|
    2019-05-21| B11M| Dismissal: decision cancelled|Free format text: ANULADA A PUBLICACAO CODIGO 11.6.1 NA RPI NO 2436 DE 12/09/2017 POR TER SIDO INDEVIDA. |
    2019-08-20| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2019-10-15| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 06/10/2010, OBSERVADAS AS CONDICOES LEGAIS. (CO) 20 (VINTE) ANOS CONTADOS A PARTIR DE 06/10/2010, OBSERVADAS AS CONDICOES LEGAIS |
    优先权:
    申请号 | 申请日 | 专利标题
    US24934109P| true| 2009-10-07|2009-10-07|
    US61/249,341|2009-10-07|
    US35332010P| true| 2010-06-10|2010-06-10|
    US61/353,320|2010-06-10|
    PCT/IB2010/002528|WO2011042794A2|2009-10-07|2010-10-06|A scaffold material for wound care and/or other tissue healing applications|
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