• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
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    • 专利摘要:
    Summary Method for Producing a Tissue Product, Tissue Product Produced by a Process, Method of Treatment, and Treatment System The present disclosure provides tissue products made from adipose tissues, as well as methods for producing such tissue products. tissue products can include extracellular acellular matrices. in addition, the present disclosure provides systems and methods for using such products.
    公开号:BR112013030492B1
    申请号:R112013030492-8
    申请日:2012-05-30
    公开日:2020-03-17
    发明作者:Jerome Connor
    申请人:Lifecell Corporation；
    IPC主号:
    专利说明:

    METHOD TO PRODUCE A FABRIC PRODUCT, FABRIC PRODUCT PRODUCED BY A PROCESS
    [001] This application claims priority under 35 U.S.C. § 119 to provisional application No. U.S. 61 / 491,787, filed on March 31, 2011, which is incorporated herein by reference in its entirety.
    [002] The present disclosure relates to tissue products and, more particularly, to extracellular tissue arrays made of adipose tissue.
    [003] Various tissue products are used to regenerate, repair or otherwise treat diseased or damaged tissues and organs. Such products may include tissue grafts and / or processed tissues (for example, matrices of acellular tissue from the skin, intestine or other tissues with or without cell seeding). Such products in general have properties determined by the tissue source (i.e., type of tissue and animal from which it originated) and the processing parameters used to produce tissue products. Such tissue products are normally used for surgical applications and / or tissue replacement or augmentation, the products must support tissue growth and regeneration as desired for the selected implant site. The present disclosure provides adipose tissue products that can enable improved tissue growth and regeneration for various applications.
    SUMMARY
    [004] According to certain modalities, methods for producing fabric products are provided. Methods include selecting an adipose tissue; mechanically process adipose tissue to reduce tissue size; and treating the mechanically processed tissue to remove substantially all cellular material from the tissue. The processed tissue is suspended in a solution and cross-linked to produce a stable three-dimensional structure.
    [005] Additionally, fabric products produced by the disclosed processes are provided. Processes may include selecting an adipose tissue; mechanically process adipose tissue to reduce tissue size; and treating the mechanically processed tissue to remove substantially all of the cellular material from the processed tissue. In addition, processes may include suspending the processed tissue in a solution and cross-linking the tissue to produce a stable three-dimensional structure.
    [006] Additionally, adipose tissue products are supplied. Products may include a decellularized adipose extracellular tissue matrix, in which the tissue matrix has been formed in a predetermined three-dimensional shape and in which the tissue matrix is particularly reticulated to maintain the three-dimensional shape.
    [007] In addition, treatment methods are provided. The methods may comprise placing a negative pressure collector on a wound, wherein the collector comprises a matrix of decellularized adipose extracellular tissue. The tissue matrix is formed in a predetermined three-dimensional shape and is partially cross-linked to maintain the three-dimensional shape. A field is placed over the collector to seal the wound and negative pressure is applied to drain fluid through the collector.
    DESCRIPTION OF THE DRAWINGS
    [008] Figure 1 is a flow chart that illustrates a process for producing adipose tissue products, according to certain modalities.
    [009] Figures 2A to 2D are macroscopic images of adipose tissue products produced according to various modalities.
    [010] Figures 3A to 3D are sections stained with hematoxylin and eosin from adipose tissue products produced according to various modalities.
    [011] Figures 4A to 4D are electron micrographs for transmitting adipose tissue products produced according to various modalities.
    [012] Figures 5A to 5D are scanning electron microscope images of adipose tissue products produced according to various modalities.
    [013] Figures 6A to 6C are sections stained with hematoxylin and eosin from adipose tissue products produced according to various modalities after implantation in nude rats.
    [014] Figures 7A to 7C are sections stained with hematoxylin and eosin from adipose tissue products produced according to various modalities after implantation in nude mice.
    [015] Figures 8A to 8C are sections stained with hematoxylin and eosin from adipose tissue products produced according to various modalities after implantation in nude mice.
    [016] Figures 9A to 9C are sections stained with hematoxylin and eosin from adipose tissue products produced according to various modalities after implantation in nude mice.
    [017] Figure 10 shows two examples of drain collectors based on adipose tissue. Exemplary collectors contain grooves (left) and holes (right).
    DESCRIPTION OF CERTAIN EXEMPLIFICATIVE MODALITIES
    [018] Reference will now be made in detail to certain exemplary modalities, in accordance with the present disclosure, certain examples of which are illustrated in the accompanying drawings. Whenever possible, the same reference numbers will be used by all drawings to refer to the same or similar parts.
    [019] In this application, the use of singular includes the plural unless specifically stated otherwise. In this application, the use of "or" means "and / or" unless otherwise specified. In addition, the use of the term "which includes", as well as other forms, such as "includes" and "included", is not limiting. Any range described in this document will be understood to include endpoints and all values between endpoints.
    [020] The section headings used in this document are for organizational purposes only and should not be construed as limiting the material described. All documents or portions of documents mentioned in this application, including, but not limited to, patents, patent applications, articles, books and research, are expressly incorporated herein by reference in their entirety for any purpose.
    [021] Various human and animal tissues can be used to produce products to treat patients. For example, various tissue products for regeneration, repair, augmentation, strengthening and / or treatment of human tissues that have been damaged or lost due to various diseases and / or structural damage (for example, trauma, surgery, atrophy and / or wear and long-term degeneration) have been produced. Such products may include, for example, arrays of acellular tissue, allografts or xenografts of tissue and / or reconstituted tissues (i.e., at least partially decellularized tissues that have been seeded with cells to produce viable materials).
    [022] A variety of fabric products have been produced to treat soft and hard fabrics. For example, ALLODERM® and STRATTICE ™ are two matrices of acellular dermal tissue produced from human and porcine dermis, respectively. Although such materials are very useful for treating certain types of conditions, materials that have different biological and mechanical properties may be desired for certain applications. For example, ALLODERM® and STRATTICE ™ have been used to assist in the treatment of structural defects and / or to provide support for tissues (for example, for abdominal walls or in breast reconstruction) and their biological and resistance properties make them suitable for such uses. However, such materials may not be ideal for regeneration, repair, replacement and / or augmentation of tissues that contain adipose tissue. Accordingly, the present disclosure provides tissue products that are useful for treating tissues containing adipose tissue as well as other tissue sites. The present disclosure also provides methods for producing such fabric products.
    [023] Tissue products can include adipose tissues that have been processed to remove at least part of the cellular components. In some cases, all, or substantially all, of the cellular material is removed, thus leading to extracellular adipose matrix proteins. Additionally, the products can be processed to remove part or all of the extracellular and / or intracellular lipids. As described below, extracellular matrix proteins can be further treated to produce a three-dimensional porous, or sponge-like material. In addition, to allow treatment of a selected tissue site, the material can be further processed (for example, by crosslinking) to form a stable structure.
    [024] As perceived, the tissue products of the present disclosure are formed from adipose tissues. Adipose tissues can be derived from human or animal sources. For example, human adipose tissue can be obtained from cadavers. In addition, human adipose tissue could be obtained from living donors (for example, with an autologous tissue). Adipose tissue can also be obtained from animals, such as pigs, monkeys or other sources. If animal sources are used, the tissues can be further treated to remove antigenic components such as chemical portions of 1,3-alpha-galactose, which are present in pigs, but not in humans or primates. Additionally, adipose tissue can be obtained from animals that have been genetically modified to remove antigenic chemical moieties. See Xu, Hui. et al., "A Porcine-Derived Acellular Dermal Scaffold that Supports Soft Tissue Regeneration: Removal of Terminal Galactose-a- (1,3) -Galactose and Retention of Matrix Structure," Tissue Engineering, Volume 15, 1 to 13 (2009 ), which is incorporated by reference in its entirety.
    [025] An exemplary process for producing the tissue products of the present disclosure is illustrated in Figure 1. As shown, the process in general includes obtaining adipose tissue (Step 10); mechanically process adipose tissue to produce small pieces (Step 12); further processing the tissue to remove substantially all cellular material and / or lipids from the tissue (Step 14); resuspend the tissue in a solution to form a porous matrix or sponge (Step 16); and crosslink the tissue to produce a stable three-dimensional structure (Step 18). Each of these steps is explained in more detail below.
    [026] To assist in the removal of cellular components and to produce a fluid mass, the tissue is first processed to produce small pieces. In various embodiments, the material is cut, crushed, mixed or otherwise mechanically treated to reduce the size of the fabric and / or to form a fluid or solid material. Adipose tissue can be treated using any repetitive cutting, crushing or blending process. For example, in one embodiment, the fabric is first cut into relatively small pieces (for example, about 2 cm x 2 cm). These pieces can then be placed in an aqueous solution, which is treated with a blade crusher or similar instrument.
    [027] After processing, the tissue is then treated to remove cell contents and lipids. The cellular material can be removed by washing the material. For example, in some embodiments, the material is further diluted with water or another solvent. The diluted material is then centrifuged and free lipids and cell debris will flow to the top, while extracellular matrix proteins are deposited like a pellet. The protein pellet can then be resuspended and the washing and centrifugation can be repeated until a sufficient amount of the lipids and cell materials are removed. In some cases, the process is repeated until substantially all of the cellular material and / or lipids are removed.
    [028] During, before and / or after the washing steps, additional solutions or reagents can be used to process the material. For example, enzymes, detergents and / or other agents can be used in one or more steps to remove cellular materials or lipids, remove antigenic materials and / or to reduce bacteria or other biocharge of the material. For example, one or more washing steps can be included using detergents, such as sodium dodecyl sulfate or TRIS, to assist in the removal of cells and lipid. In addition, enzymes, such as lipases, DNAses, RNAses, alpha-galactosidase or other enzymes, can be used to ensure the destruction of nuclear materials, antigens from xenogenic sources and / or viruses. In addition, solutions of peracetic acid and / or peroxides can be used to help remove cellular materials and destroy bacteria or other potentially infectious agents.
    [029] After removing cellular components, the material can also be formed into a porous or sponge-like material. In general, the extracellular matrix is first resuspended in an aqueous solvent. A sufficient amount of solvent is used to allow the material to form a liquid mass that can be poured into a mold that is the size and shape of the desired fabric product. The amount of water added can be varied based on the desired porosity of the final material. In some cases, the resuspended cell matrix can be mechanically treated by crushing, cutting, blending or other processes one or more additional times and the treated material can be centrifuged and resuspended one or more times to further remove cell material or lipids (if necessary) and / or to control the viscosity of the extracellular matrix.
    [030] Once any additional washing and crushing steps are completed, the suspended material is again placed in a container or mold to form the porous, sponge-like product. In general, the porous or sponge-like material is formed by drying the material to leave a three-dimensional matrix with a porous structure. In some embodiments, the material is freeze-dried. Freeze-drying can allow the production of a three-dimensional structure that generally conforms to the shape of the mold.
    [031] The specific freeze drying protocol can be varied based on the solvent used, sample size and / or to optimize processing time. A suitable freeze-drying process may include freezing the material at -35 ° C for a period of 45 minutes; keep the samples at -35 ° C for 90 minutes to ensure complete freezing; apply vacuum; raise the temperature to -10 ° C and keep for 24 hours; raise the temperature to 0 ° C and keep for 24 hours; and raise the temperature to 20 ° C and keep for 12 hours. The freeze-dried samples can then be removed from the dryer by freezing and packed in foil bags under nitrogen.
    [032] After forming the porous material, similar to a sponge, the fabric can be treated so that it forms a stable three-dimensional shape. For example, in general, a mechanically processed tissue, when formed in a pore matrix, can form a material similar to mass or paste when it is implanted in a body, becomes moist or is placed in a solution. Therefore, the desired shape and size may be lost. Additionally, the porous structure, which may be important to support cell fixation, tissue growth, vascular formation and tissue regeneration, may be lost. In this way, the material could be further processed to stabilize the material's size, shape and structure.
    [033] In some embodiments, the material is cross-linked. In some embodiments, the material is cross-linked after freeze-drying. However, the material could also be cross-linked before or during the freeze drying process. Crosslinking can be performed in a variety of ways. In one embodiment, crosslinking is carried out by contacting the material with a crosslinking agent such as glutaraldehyde, genipin, carbodiimides and diisocyanates. In addition, crosslinking can be carried out by heating the material. For example, in some embodiments, the material can be heated between 70 ° C to 120 ° C, or between 80 ° C and 110 ° C, or about 100 ° C, or any values between specific ranges at reduced pressure or vacuum. In addition, other crosslinking processes can be used to produce any of the disclosed products, including ultraviolet irradiation, gamma irradiation and / or electron beam irradiation. Additionally, vacuum is not necessary, but it can reduce the crosslinking time. In addition, lower or higher temperatures could be used as long as the fusion of the matrix proteins does not occur and / or sufficient time is provided for crosslinking.
    [034] In various modalities, the crosslinking process can be controlled to produce a tissue product with the desired mechanical, biological and / or structural resources. For example, crosslinking can influence the overall strength of the material and the process can be controlled to produce a desired strength. In addition, the amount of crosslinking can affect the product's ability to maintain a desired shape and structure (eg, porosity) when implanted. In this way, the amount of crosslinking can be selected to produce a stable three-dimensional shape when implanted in a body, when placed in contact with an aqueous environment and / or when compressed (for example, by surrounding fabrics or materials).
    [035] Excessive crosslinking can alter extracellular matrix materials. For example, excessive crosslinking can damage collagen or other extracellular matrix proteins. Damaged proteins may not support tissue regeneration when tissue products are placed in an adipose tissue site or other anatomical site. Additionally, excessive crosslinking causes the material to become delicate or fragile. In this way, the amount of crosslinking can be controlled to produce a desired level of stability, while maintaining the desired biological, mechanical and / or structural resources.
    [036] Exemplary crosslinking processes may include bringing into contact a freeze-dried material, produced as discussed above, with glutaraldehyde. For example, a 0.1% glutaraldehyde solution can be used and the tissue can be submerged in the solution for about 18 hours followed by extensive rinsing in water to remove the solution. Alternatively, or in combination, a dehydrothermal process can be used. For example, an exemplary dehydrothermal process includes treating the material at 100 ° C and approximately 67.73 kPa (20 inches Hg) for 18 hours, followed by submersion in water. The final cross-linked fabric products can be stored in a foil pouch.
    [037] As discussed above, tissue products should have the ability to support internal cell growth and tissue regeneration when implanted in or on a patient. In addition, tissue products should have the ability to act as a carrier for and support the growth of cells, including stem cells, such as stem cells derived from adipose tissue. Thus, the processes discussed above could not alter the extracellular matrix proteins (for example, by damage to the protein structure and / or removal of glycosaminoglycans and / or growth factors). In some modalities, the products will have a normal collagen band as evidenced by transmission electron microscopy.
    [038] In various modalities, tissue products are treated with a process that retains any of either native hyaluronic acid or chondroitin sulfate. Thus, tissue products can include any of or both hyaluronic acid and chondroitin sulfate. Additionally, the process can be selected to maintain native growth factors. For example, tissue products can be produced so that tissue products contain one or more growth factors selected from PECAM-1, HGF, VEGF, PDGF-BB, follistatin, IL-8, and FGF-basic.
    USE OF FABRIC PRODUCTS
    [039] The tissue products described in this document can be used to treat a variety of different anatomical sites. For example, as discussed throughout the present, the tissue products of the present disclosure are produced from adipose tissue matrices. Thus, it is believed that adipose tissue products will provide superior regenerative capabilities when implanted in certain tissue sites, as compared to materials produced from other types of tissue. In some cases, tissue products can be implanted in tissue sites that are predominantly or significantly fatty tissue. In some cases, tissue sites may include the breast (for example, for enlargement, replacement of amputated tissue or placement around an implant). In addition, any other site that contains adipose tissue can be selected. For example, tissue products can be used for reconstructive or cosmetic use on the face, buttocks, abdomen, hips, thighs or any other site where additional adipose tissue that is structured and appears to approach native adipose tissue may be desired. At any of these sites, the fabric can be used to reduce or eliminate wrinkles, sagging or unwanted shapes.
    [040] When used to replace or augment breast tissue, the tissue can provide advantages over other tissue products. For example, although some tissue products allow for internal growth and tissue formation, these products can form significant fibrotic tissue that does not mimic the texture and feel of normal breast and appears abnormal in X-ray imaging. Since the tissue products of the present disclosure are formed from adipose tissue, they can withstand more normal regeneration of adipose tissue.
    [041] The fabric products disclosed in this document can also be formed into thin sheets for use as adhesion barriers, covers, wrappers and the like. For example, a sample of adipose tissue can be placed on a flat surface and a second flat surface can be placed on top of the sample. In some embodiments, force is applied (for example, by applying pressure or adding weight to the second surface) sufficient to flatten the fat tissue sample to a thickness of about 1.5 mm or less (for example, about 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0, 3, 0.2, 0.1 or 0.05 mm, or any value between them).
    Alternatively, a rolling pin (for example, a rolling pin) can be used to apply pressure and flatten the adipose tissue sample. In some embodiments, an absorbent material on the flat surface or roller can be used to remove felt from hydration of the adipose tissue as it is made flat.
    [042] The adipose tissue sheets disclosed in this document may retain some or all of the collagen, growth factors and / or other biological molecules found in the adipose tissue prior to film preparation. The film can also provide a flexible material that has a malleability similar to cellophane. In some embodiments, applying pressure to generate a film material compresses the collagen fibers in the fabric product, thereby increasing the mechanical strength of the fabric product (for example, as a result of bundling, condensing and / or mixing the fibers of collagen in the film). In certain embodiments, after the film is prepared, the film can be cross-linked (for example, with the use of chemical cross-linking agents, dihydrothermal treatment, irradiation and / or any other cross-linking method known to one skilled in the art). The crosslinking of the adipose film can serve to further increase its mechanical strength (for example, by increasing its ability to handle tension, torsion and / or other forces without tearing or breaking).
    [043] Film fabric products can be used as adhesion barriers. In some embodiments, the compactly packaged collagen network of the compressed film can prevent cells or fibrous tissue (e.g., scar tissue) from re-populating or passing through the film. For example, a film of adipose tissue may be wrapped around a temporary implant to prevent the establishment of unwanted cells or fibrous tissue in the implant, thereby preventing subsequent removal of the implant. Similarly, the film can be placed between two or more static fabrics where it is desirable to prevent the fabrics from joining or adhering to each other.
    [044] Film fabric products can also be used as covers or wrappers. For example, a film tissue product can be wrapped around an artery, vein, tendon or ligament to protect the artery, vein, tendon or ligament (for example, as part of a ligament repair or transplant procedure) . In this example, the film could serve to protect the artery, vein, tendon or ligament from abrasion and / or increase the resistance of the artery, vein, tendon or ligament. Similarly, a film fabric product can be placed over and around a suture line to protect the line from damage (for example, to protect suture lines from tearing caused by abrasion or other forces imposed on the sutures). Similarly, film tissue products could be used to protect a surgically prepared anastomosis (eg, an arterial or other blood vessel anastomosis, an anastomosis associated with intestinal resection, etc.). In these embodiments, a thin film product, which comprises biological material, can provide an advantage in allowing the non-bulky wrapping of a surgical site with a bio-absorbable and biocompatible material. In these embodiments, it may also be desirable to cross-link the tissue product in film to increase structural strength and the ability to handle tension, torsion or other forces without tearing or breaking.
    [045] The tissue products described in this document can also be used in conjunction with negative pressure therapy systems, such as the VAC® system, which is produced by Kinetic Concepts, Inc. Such systems can be used to treat a variety of tissue sites and include, for example, a source of negative pressure, such as a pump and an or, but treatment materials, which usually include a porous foam or collector. General examples of such systems are described in US Patent Publication number 2010/0040687 A1, which was filed on August 13, 2009. The products can be used when implanted or placed on or near a wound or other surgical site as tissue products may allow prolonged pressure collection, which is necessary for negative pressure therapy.
    [046] Accordingly, the present disclosure also provides systems for treating tissue comprising one or more tissue products and a negative pressure therapy system. The negative pressure therapy system can include a negative pressure source, for example, a pump, and a fluid connection source (e.g., tube or conduit for connecting the negative pressure source to the tissue product). In addition, the systems may include a flexible sheet or other mechanism to seal a wound or other surgical site to be treated. For example, in some embodiments, the system may include a thin flexible sheet of material that is placed on a treatment site. The sheet can be configured to seal the treatment site using an adhesive or other connection system and the fluid connection can pass at or near the treatment site to produce negative pressure within the treatment site.
    [047] Furthermore, tissue products can be used as carriers for cells. For example, the products can be implanted at any of the sites or used as discussed above, but they can also be seeded with cells. In some cases, the cells may include stem cells such as stem cells derived from adipose tissue. In addition, other pluripotent cells can be used, as well as cells from any tissue source (for example, blood, bone marrow, fetal stem cells, umbilical cord blood cells, etc.). The cells can be seeded into the tissue after implantation or before implantation. In addition, cells can be cultured in the tissue product prior to implantation and then implanted in or on a body.
    [048] The adipose tissue products of the present disclosure can also be used as drains and / or drain collectors. For example, adipose tissue products can be cut and / or otherwise shaped to form a tube, column, sheet or any other shape desired for use as a drain or drain collector. In some embodiments, a fabric product is shaped to have a cross section that is at least up to about 5 mm in size (for example, about 5, 4, 3, 2 or 1 mm in size, or any value between them). In certain embodiments, the fabric product has a length of up to about 20 cm (for example, about 1, 5, 10, 15 or 20 cm, or any length in between). In some embodiments, the fabric product has grooves, channels and / or holes added to it. In some embodiments, the grooves, channels and / or holes constitute up to about 70% (for example, about 70, 60, 50, 40, 30, 20 or 10% or any percentage between them) of the surface area of the product of fabric and allow the passage of fluids and / or pressure through the drain or collector. Examples of this type of drain manifold are shown in Figure 10.
    [049] Alternatively, in various modalities, an adipose tissue product can be cut into thin strips and laminated to form a drain in the shape of a tube or other desired shape. In some embodiments, the thin strips have a thickness of up to about 2 mm (for example, about 0.5, 1.0, 1.5 or 2.0 mm or any value between them), a width of up to about 20 mm (for example, about 1, 5, 10, 15, or 20 mm, or any value in between) and a length of about 20 cm (for example, about 1, 2, 3, 4 , 5, 10, 15, or 20 cm, or any value in between). In some embodiments, the laminated adipose tube comprises a hollow tube. In some embodiments, the laminated tube has grooves, channels and / or holes notched in it. In some embodiments, the grooves, channels and / or holes allow the passage of fluids and / or pressure through the collector.
    [050] Drains and / or adipose tissue drain collectors can be implanted in a patient to provide channel (s) to cause negative pressure to an implant site and / or to remove blood or other fluids from the implant site. In some modalities, the drain and / or collector is implanted to prevent the formation of seromas and / or bruises after a surgical procedure. In some embodiments, the collector is implanted to provide adequate drainage, for example, after a surgical procedure such as tumor removal, abdominal surgery, breast implantation or other plastic surgery. In some embodiments, the drain and / or adipose tissue collector is used in conjunction with negative pressure therapy systems, such as the VAC® system, which is produced by Kinetic Concepts, Inc., as described above. In various modalities, adipose tissue products provide biocompatible and bioabsorbable drains and / or drain collectors. In some embodiments, the implanted drain and / or adipose tissue may be repopulated with native tissue cells surrounding an implant site, thus avoiding the need to remove the drain and / or collector at the conclusion of a drainage procedure.
    EXAMPLES
    [051] The following examples serve to illustrate, and in no way to limit, the present disclosure.
    A. ADIPOSE TISSUE PROCESSING
    [052] Human adipose tissue was obtained from cadaveric sources following all the requirements of the American Association of Tissue Banks (AATB). Full-thickness samples containing dermis and fat were obtained from AATB-certified tissue banks. The adipose tissue was mechanically removed from the dermis and was placed in buffered saline and kept at 4 ° C until processing.
    [053] The adipose tissue, of various thicknesses, was cut into small pieces of approximately 2 cm x 2 cm using scissors. The adipose pieces were suspended in water at a rate of 1 g of adipose tissue to 2 ml of water. Then, 150 ml of the suspension was then ground using a blade crusher (RETSCH® GRINDMASTER GM200) for three minutes at 10,000 rpm at room temperature. The grinding process was repeated until all the fat pieces were ground.
    [054] The ground fat suspension was further diluted at a 1: 1 ratio with water and the final mixture was centrifuged at 2 · 500 x g for 5 minutes. The free lipid and cell residue floated to the top and were removed by expression. The extracellular matrix proteins derived from adipose tissue (ECM) formed a pellet and the pellet fraction was retained. Centrifugation and pelletizing were repeated until all materials had been processed. All ECM pellets were combined, resuspended in water and washed by centrifugation three times.
    [055] The washed adipose tissue ECM was resuspended in water at a rate of 50 ml ECM to 100 ml water and was ground using the blade crusher for 2 minutes at 10,000 rpm at room temperature. The suspension was washed using centrifugation as described above. The final ECM suspension was centrifuged at 3,500xg for 10 minutes to generate a final packaged ECM pellet. The washed ECM pellet was slowly resuspended in water at a rate of 1 ml ECM to 2 ml water to generate an adipose ECM slurry.
    B. PRODUCTION OF SPONGE FROM ADIPOSE ECM
    [056] The slurry was dispersed in 8 cm x 8 cm molds. The molds were placed in a standard industrial freeze dryer and subjected to freeze drying according to the following procedure. The ECM slurry was frozen at -35 ° C for a period of 45 minutes. The samples were kept at -35 ° C for 90 minutes to ensure complete freezing. The vacuum was then started in the system. The shelf temperature was raised to -10 ° C and maintained for 24 hours. The temperature was then raised to -0 ° C and maintained for 24 hours. Finally, the temperature was raised to 20 ° C and maintained for 12 hours. The freeze-dried ECM samples were removed from the dryer by freezing and packed in foil bags under nitrogen.
    C. ADHESIVE FABRIC ECM SPONGES HARNESS
    [057] Freeze-dried ECM products were cross-linked by both methods. The first was chemical cross-linking with the use of glutaraldehyde and the second was biophysical cross-linking with dehydrothermal treatment.
    C (1). GLOSSARALDEHYTE crosslinking
    [058] The fatty sponge was removed from the foil pouch and placed in a glass dish that contains an excess of 0.1% glutaraldehyde solution prepared in water. The sponge was kept submerged until the sample was completely hydrated. The sponge was incubated in the 0.1% glutaraldehyde solution for 18 hours followed by extensive rinsing in water to remove the glutaraldehyde solution. The final cross-linked fatty sponge was stored in a foil pouch C (2). Crosslinking with DHT
    [059] The fatty sponge was removed from the foil pouch and placed in a vacuum oven and subjected to a temperature of 100 ° C and approximately 67.73 kPa (20 inches of Hg) for 18 hours. After dehydrothermal treatment, the sponge was hydrated by submersion in water. The final cross-linked fatty sponge was stored in a foil pouch.
    [060] Figures 2A to 2D are macroscopic images of adipose tissue products produced according to various modalities. Figure 2A is an adipose tissue product that has been cross-linked with glutaraldehyde, as described in C (1) above, in a hydrated state. Figure 2B is an adipose tissue product that has been cross-linked with glutaraldehyde as described in C (2) above and has been compressed to remove most of the water.
    [061] Figures 2C and 2D are sections stained with hematoxylin and eosin (H&E) of adipose tissue products produced according to various modalities. Figure 2C is an adipose tissue product that has been cross-linked with a dehydrothermal process, as described in C (2) above, in a hydrated state. Figure 2D is an adipose tissue product that has been cross-linked with a dehydrothermal process, as described in C (2) above and has been compressed to remove most of the water.
    [062] The tissue products were subjected to a collection test in which the material was placed in a vacuum port and negative pressure was applied to the material to determine whether the material could act as a sink for negative pressure therapy and / or it could maintain a porous structure over time. The collection test demonstrated an extended collection capacity. Thus, it was revealed that the fabric products maintain a porous structure even after contact with aqueous solutions. Therefore, the materials will be suitable for use in negative pressure treatment systems and will maintain a porous structure to ensure internal growth and tissue regeneration when implanted within a body.
    [063] Figures 4A and 4B are electron micrographs for the transmission of adipose tissue products produced with crosslinking with glutaraldehyde, as described in C (1). Figures 5A and 5B are scanning electron microscope images of the adipose tissue products produced with crosslinking with glutaraldehyde, as described in C (1). Figures 4C and 4D are electron micrographs for transmitting adipose tissue products produced with dehydrothermal cross-linking, as described in C (2) above. Figures 5C and 5D are scanning electron microscope images of adipose tissue products produced with dehydrothermal cross-linking, as described in C (2) above. As shown, the cross-linked adipose tissue products maintained a porous structure intact. TEM images from Figures 4A to 4D showed normal collagen band.
    D. ANIMAL IMPLANT STUDY
    [064] The hydrated adipose sponges were subjected to an electron beam irradiation at a dose of 15 to 18 kGy to achieve sterilization. The samples of sterile sponge (n = 4) (GA and DHT cross-linked) were implanted subcutaneously in nude rats for 21 days.
    Biopsies were collected, processed for histology and evaluated by a histopathologist to determine the biological response. Both sponge configurations were observed to have significant infiltration of native fibroblast cells, robust presence of new blood vessels and evidence of newly deposited native collagen in the absence of a significant immune cell presence. The fat sponges showed positive evidence of biocompatibility.
    [065] Figures 6A to 6C are sections stained with hematoxylin and eosin from adipose tissue products produced with crosslinking with glutaraldehyde, as described in C (1) after implantation in nude rats. Figures 7A to 7C are sections stained with additional hematoxylin and eosin from adipose tissue products produced with crosslinking with glutaraldehyde, as described in C (1) above, after implantation in nude rats. Figures 8A to 8C are sections stained with hematoxylin and eosin from adipose tissue products produced with dehydrothermal cross-linking, as described in C (2) above, after implantation in nude rats. Figures 9A to 9C are sections stained with hematoxylin and eosin from adipose tissue products produced with dehydrothermal cross-linking, as described in C (2) above, after implantation in nude rats.
    [066] Each of the crosslinking products with glutaraldehyde and dehydrothermal showed significant cell infiltration, neovascularization and formation of new collagen. Thus, tissue products are believed to be suitable for tissue regeneration when implanted in tissues containing adipose tissue or other tissue sites. The samples showed modest inflammatory infiltrates.
    [067] The previous examples are intended to illustrate and in no way limit the present disclosure. Other modalities of the disclosed devices and methods will be apparent to those skilled in the art from the consideration of the description and practice of the devices and methods disclosed in this document.
    权利要求:
    Claims (11)
    [1]
    1. METHOD TO PRODUCE A TISSUE PRODUCT, characterized by understanding: selecting an adipose tissue; mechanically process adipose tissue to reduce tissue size; treating the tissue to remove substantially all cellular material from the tissue; suspend the tissue in a solution; placing the fabric in a mold to produce a desired shape; freeze and dry the fabric to form a porous sponge; and stabilizing the tissue after freezing and drying the tissue by heating the tissue to produce a stable three-dimensional structure.
    [2]
    2. METHOD according to claim 1, characterized in that the fabric is heated between 70 ° C and 120 ° C.
    [3]
    3. METHOD, according to claim 1, characterized in that the fabric is cross-linked so that the material maintains the stable three-dimensional structure when placed in contact with an aqueous environment.
    [4]
    4. METHOD, according to claim 3, characterized by the material maintaining the three-dimensional structure stable when implanted in a body.
    [5]
    5. METHOD, according to claim 1, characterized by mechanically processing the fabric including at least one among cutting, grinding or mixing to produce small particles.
    [6]
    6. TISSUE PRODUCT PRODUCED BY A PROCESS, characterized by comprising: selecting an adipose tissue; mechanically process adipose tissue to reduce tissue size; treating the tissue to remove substantially all cellular material from the tissue; suspend the tissue in a solution; placing the fabric in a mold to produce a desired shape; freeze and dry the fabric to form a porous sponge; and stabilizing the tissue after freezing and drying the tissue by heating the tissue to produce a stable three-dimensional structure.
    [7]
    7. PRODUCT according to claim 6, characterized in that the fabric is heated between 70 ° C and 120 ° C.
    [8]
    8. PRODUCT according to claim 6, characterized in that the fabric is cross-linked so that the material maintains the stable three-dimensional structure when placed in contact with an aqueous environment.
    [9]
    9. PRODUCT, according to claim 8, characterized by the material maintaining the stable three-dimensional structure when implanted in a body.
    [10]
    10. PRODUCT according to claim 6, characterized in that the fabric contains hyaluronic acid and chondroitin sulfate.
    [11]
    11. PRODUCT, according to claim 6, characterized by mechanically processing the fabric including at least one among cutting, crushing or blending to produce small particles.
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    同族专利:
    公开号 | 公开日
    BR112013030492A2|2016-09-27|
    AU2017216486A1|2017-08-31|
    EP2714111B1|2021-03-17|
    WO2012166784A1|2012-12-06|
    AU2012262311A1|2013-11-28|
    EP3851131A1|2021-07-21|
    AU2016203823A1|2016-06-30|
    AU2021200593A1|2021-03-04|
    AU2012262311B2|2016-04-28|
    EP2714111A1|2014-04-09|
    AU2019210597A1|2019-08-22|
    AU2019210597B2|2020-11-12|
    US20190076582A1|2019-03-14|
    AU2017216486C1|2019-11-14|
    CA2837196C|2020-03-24|
    AU2016203823B2|2017-05-18|
    ES2874487T3|2021-11-05|
    CA2837196A1|2012-12-06|
    AU2017216486B2|2019-05-16|
    US20120310367A1|2012-12-06|
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    法律状态:
    2016-10-04| B15I| Others concerning applications: loss of priority|Free format text: PERDA DA PRIORIDADE US 61/491,787 DE 31/05/2011 REIVINDICADA NO PCT/US2012/039969, CONFORME AS DISPOSICOES PREVISTAS NA LEI 9.279 DE 14/05/1996 (LPI) ART. 167O, ITEM 28 DO ATO NORMATIVO 128/97 E NO ART. 29 DA RESOLUCAO INPI-PR 77/2013. ESTA PERDA SE DEU PELO FATO DE O DEPOSITANTE CONSTANTE DA PETICAO DE REQUERIMENTO DO PEDIDO PCT ("LIFECELL CORPORATION") SER DISTINTO DAQUELE QUE DEPOSITOU A PRIORIDADE REIVINDICADA E NAO APRESENTOU DOCUMENTO COMPROBATORIO DE CESSAO, CONFORME AS DISPOSICOES PREVISTAS NA LEI 9.279 DE 14/05/1996 (LPI) ART. 166O, ITEM 27 DO ATO NORMATIVO 128/97 E NO ART. 28 DA RESOLUCAO INPI-PR 77/2013. |
    2016-11-16| B12F| Other appeals [chapter 12.6 patent gazette]|
    2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2019-02-19| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|
    2019-10-08| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|
    2020-01-28| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2020-03-17| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 30/05/2012, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    US201161491787P| true| 2011-05-31|2011-05-31|
    US61/491,787|2011-05-31|
    PCT/US2012/039969|WO2012166784A1|2011-05-31|2012-05-30|Adipose tissue matrices|
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