• 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 Preparing a Fabric Matrix Composition, Fabric Matrix, Fabric Filling These are methods for producing fabric fills. In certain embodiments, the fabric is similar to flake and has regenerative properties.
    公开号:BR112013026200A2
    申请号:R112013026200
    申请日:2012-04-13
    公开日:2019-08-27
    发明作者:Richter Bowley Melissa；Goel Raghav；Sun Wenquan
    申请人:Lifecell Corp；
    IPC主号:
    专利说明:

    METHOD FOR PREPARING A COMPOSITION OF TISSUE MATRIX AND TISSUE MATRIX
    This order claims priority under 35 U.S.C. $ 119 to Provisional Order No. 61 / 475,378, which was filed on April 14, 2011.
    The present disclosure relates to fabric fills and, more particularly, to methods for preparing fabric fills that have a flake-like shape and fabric fills prepared according to those methods.
    Various types of wound dressings and soft tissue fillers are used to regenerate, repair or otherwise treat diseased or damaged tissues or organs. Such materials include hyaluronan-based gels, solubilized and cross-linked collagen compositions, micronized tissue matrices and synthetic polymer compositions in hydrogel or other forms. Each of these materials has potential disadvantages if used as bulk soft tissue fillers or deep wound dressings, including limited suitability for deep injuries, inability to regenerate, tendency to increase the inflammatory response or tendency to degradation upon radiation exposure.
    According to certain embodiments, a method for preparing a tissue matrix composition is provided. The method comprises selecting a collagen-based tissue matrix, placing the matrix in contact with a cryoprotectant solution, freezing the matrix and cutting the matrix, where the tissue matrix temperature is in the range of -10 ° C to -40 ° C for the cutting step.
    In certain embodiments, a fabric padding is provided. The filling comprises a collagen-based tissue matrix, in which the matrix has been placed in contact with
    2/35 a cryoprotective solution and then frozen and in which the matrix was cut at a temperature between -10 ° C and -40 ° C after freezing.
    In certain embodiments, a method for preparing a tissue matrix composition is provided. The method comprises selecting a collagen-based matrix, placing the matrix in contact with a cryoprotectant solution, freezing the matrix, adjusting the tissue matrix and cryoprotectant temperature between -10 ° C to -40 ° C, cutting the matrix of frozen tissue, place the cut tissue matrix in a liquid to form a suspension and freeze dry the suspension.
    DESCRIPTION OF THE DRAWINGS
    Figure 1 is a diagram of a grating device for carrying out methods of the present disclosure, according to certain modalities.
    Figure 2 is a diagram of an infusion column for producing tissue fillings of the present disclosure, according to certain modalities.
    Figure 3 is a flow chart that summarizes the various steps that can be used to produce fabric fillings, according to certain modalities of the revealed method.
    Figures 4A and 4B are photographs that demonstrate the morphology of various pieces of tissue under light and SEM microscope respectively, according to certain modalities, as described in Example 1.
    Figure 5 is a graph showing the fabric flake size distribution according to certain modalities, as described in Example 1.
    Figure 6 is a graph showing the ice mass and amorphous tissue domain fractions as a function of cryoprotectant concentration, according to certain modalities, as described in Example 2.
    3/35
    Figure 7 is a graph showing tissue flakes thermograms according to certain modalities, as described in Example 4.
    Figures 8A and 8B are graphs showing the resistance of processed tissue material, according to certain modalities, to the digestion of collagenase and trypsin, respectively, as described in Example 4.
    Figure 9A is a photograph of rehydrated tissue flakes, according to certain modalities, and Figure 9B is a schematic of a pressure testing pad, as described in Example 5.
    Figures 10A and 10B are graphs showing the width and length size distribution, respectively, of processed fabric material, according to certain modalities, as described in Example 6.
    Figures 11A to 11D are photographs of a soft tissue foam, prepared according to certain modalities, as described in Example 6.
    Figures 12A to 12D show several histological sections of various tissue matrices after implantation in an animal, according to certain modalities, as described in Example 6.
    DESCRIPTION OF CERTAIN EXEMPLIFICATIVE MODALITIES
    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.
    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 stated. In addition, the use of the term
    4/35 includes, as well as other forms, such as included and included, is not limiting. Any range described in this document will be understood to include endpoints and all values between endpoints. It should be understood that the benefits and advantages described above may refer to one modality or may refer to several modalities. Where appropriate, aspects of any of the examples and modalities described above can be combined with aspects of any of the other examples described to form additional examples that have comparable or different properties and address the same or different problems.
    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.
    As used herein, tissue matrix will refer to material derived from animal tissue that includes a matrix that contains collagen. Such tissue arrays may include intact tissue, tissues that have been partially or completely decellularized or synthetic collagen hues (for example, 3D arrays formed from suspended or otherwise processed fabrics). As further described below, suitable tissue matrices can be acellular. Any suitable tissue matrix can be used, depending on the intended implantation site, as long as the tissue is susceptible to use with the methods described in this document.
    The flake-like tissue matrix revealed in the
    5/35 this document has several properties that make it suitable for use as a soft tissue filler or a deep wound dressing. The flake-like tissue matrix is regenerative, allowing for revascularization and restocking of host cells. The large size of the individual tissue flakes, in relation to the micronized tissue particles, prevents the migration of the flakes in the surrounding areas. The flake-like tissue matrix disclosed herein is found to be more stable and resistant to enzymatic degradation compared to micronized tissue particles. The geometry of the fabric flakes allows them to form a stable suspension without freezing or phase separation, which makes the fabric fills suitable for filling large gaps (tens to hundreds of milliliters). When used to form a suspension that can be freeze-dried, the fabric flake suspension can contain relatively large interconnected channels that allow fluids to flow freely. When used as wound dressings and / or negative pressure injury therapy systems, the channels can help support host cell restocking revascularization. These interconnected channels can convert pressure, making the flake-like tissue matrix more susceptible to the treatment of deep wounds, while also being compatible with negative pressure therapy.
    According to certain embodiments, a method for preparing a tissue matrix composition is provided. The method comprises selecting a collagen-based tissue matrix, placing the matrix in contact with a cryoprotectant solution, freezing the matrix and cutting the matrix, where the tissue matrix temperature is in the range of -10 ° C to -40 ° C for the cutting step. In certain embodiments, a fabric padding is provided. Filling
    6/35 comprises a collagen-based tissue matrix, in which the matrix was placed in contact with a cryoprotective solution and then frozen and in which the matrix was cut after freezing at a temperature between -10 ° C and -40 ° C. In certain embodiments, an additional method for preparing a tissue matrix composition is provided. The method comprises selecting a collagen-based matrix, placing the matrix in contact with a cryoprotectant solution, freezing the matrix, adjusting the tissue matrix and cryoprotectant temperature between -10 ° C to -40 ° C, cutting the matrix of frozen tissue, place the cut tissue matrix in a liquid to form a suspension and freeze dry the suspension. In certain embodiments, a tissue matrix is provided. In addition, in various embodiments, fabric matrices produced according to the methods described in this document are provided.
    After a tissue matrix has been selected, a cryoprotective solution can be used to treat the tissue matrix before freezing. The cryoprotective solution can prevent tissue damage from damage as a result of freezing and / or melting, can reduce the amount of water frozen in the tissue through osmotic dehydration, and can guarantee the formation of a glassy matrix of sub-zero high temperature in the tissue. The use of a cryoprotectant can also help to maintain a balance between ice content and unfrozen tissue after freezing. A frozen tissue matrix that contains a lot of ice can be fragile and difficult to cut. Conversely, the tissue matrix frozen with insufficient ice is very soft and heats up quickly during cutting, making it difficult to cut too. Thus, the concentration of the cryoprotectant in the cryoprotectant solution can be used to control the ice content and hardness of the frozen matrix. In some embodiments, the ice content of the matrix
    Frozen 7/35 is in the range of 40 to 60%.
    Any suitable cryoprotectant can be used in the cryoprotectant solution. In certain embodiments, suitable cryoprotectants may include maltodextrin, sucrose, polyethylene glycol (PEG) and polyvinylpyrrolidone (PVP) or combinations thereof. In some embodiments, the cryoprotectant comprises maltodextrin. In some embodiments, the cryoprotective solution contains 5 to 50% (w / v) of maltodextrin. In some embodiments, the solution contains 15 to 25% (w / v) of maltodextrin.
    After treatment with the cryoprotective solution, the tissue matrix is then frozen. In certain embodiments, the tissue matrix is frozen at -80 ° C. Freezing can be performed using a -80 ° C freezer. Before cutting, the temperature of the tissue matrix can be adjusted if necessary to a temperature in the range of -10 ° C to -40 ° C for the cutting step. This temperature range can help maintain the proper balance between ice content and unfrozen tissue, which facilitates cutting.
    After adjusting the temperature, the fabric can be cut. In some embodiments, the fabric is cut into pieces of irregular shape and size. In some embodiments, the irregular size and shape give the fabric a flake-like appearance. In some embodiments, the cut fabric has a selected size distribution. In certain embodiments, the cut fabric has a size distribution in the range of 0.2 to 5 mm in length, 0.2 to 3 mm in width and 0.2 to 0.3 mm in thickness. When hydrated, most of the tissue cut within that size distribution weighs between 0.5 and 2.0 mg. The irregular size and shape distribution of the cut tissue facilitates the formation of large interconnected channels that allow body fluid to flow freely when the cut tissue is placed
    8/35 in suspension and thus helps revascularization and restocking of host cells. In some uses, the large size of the cut tissue, relative to the smaller micronized tissue particles, prevents the migration of the cut tissue to surrounding anatomical sites when implanted or placed on or over a wound. Cut tissue that is larger than the revealed size distribution may also be undesirable. If the cut fabric is too large, the individual pieces can take up a lot of space when suspended and also prevent the formation of sufficiently large channels.
    Various methods can be used to cut the fabric and still conform to the revealed method, as long as the fabric is cut into pieces of irregular size and shape. For example, the fabric matrix can be cut manually, using scissors. In some embodiments, cutting the tissue matrix comprises grating the tissue matrix. In some embodiments, a grating device is used to perform the grating step. Any grating device can be used in accordance with the disclosed method, as long as it cuts the fabric matrix into pieces of irregular shape and size. In some embodiments, the grating device is a grater, such as a MICROPLANE® grater. In other embodiments, the grating device is a grating wheel.
    In certain embodiments, the cutting step can be automatic. Automation reduces the amount of time needed to prepare the cut fabric and makes it easier to cut larger amounts of fabric. In several embodiments, one or more aspects of the cutting process can be automatic. For example, the grating device itself can be automatic so that manual effort is no longer required to cut the tissue matrix. It is also possible to automate
    9/35 delivery of the tissue matrix to the cutting device. In addition, it is possible to automate the removal of the tissue matrix once it has been cut. An example of an automatic cutting apparatus is illustrated in Figure 1. As shown, the apparatus comprises a grating wheel 101 which is fitted with blades 102 to cut a die 107. Below the grating wheel is a collection tray 103. The grating wheel 101 and the collecting tray are confined to a housing unit 104, which protects the operator from direct contact with the blades 102. The housing unit 104 is fitted with a loading platform 106. In this example, the loading platform 106 is tilted so that any samples loaded in it will move towards the grating wheel 101 by gravity. The fabric matrix 107 is placed on the loading platform 106 and moves towards the grating wheel 101. The blades 102 of the grating wheel 101 cut the fabric matrix 107 into flakes 108 which fall into the collection tray 103 below.
    In various embodiments, the disclosed methods may further comprise further processing before or after cutting. For example, in various embodiments, the methods disclosed comprise the use of a pre-processed acellular tissue matrix, which is described in more detail below. In other embodiments, an intact cell tissue can be used, which can be further processed to produce a suitable acellular tissue matrix. Further processing may comprise decellularization, removal of DNA and removal of epitopes from a-gal or other antigens. Decellularization, DNA removal and a-gal epitope removal are described in more detail below. Further processing of the cut tissue may include disinfecting the tissue matrix. In some embodiments, the flake-like tissue matrix is disinfected with isopropyl alcohol (IPA) (for example, the
    10/35 about 70% IPA).
    The processing of fabric flakes can also be susceptible to automation. Automation can include any process that does not require manual delivery and removal of solutions that facilitate processing. Automating fabric processing reduces the time spent manually replacing processing solutions and minimizes the handling of fabric flakes by the operator. In some modalities, the automation of tissue processing includes the use of a closed, low pressure perfusion column. Decellularization, DNA removal, a-gal antigen removal and disinfection can all be performed inside the column, which allows high-throughput processing of the cut tissue. The cut tissue matrix is loaded onto the column and air is purged from the column. Decellularization, DNA removal, agal antigen removal and disinfection are achieved by stepwise perfusion of the appropriate solutions at or near atmospheric pressure. Solutions of α-galactosidase, DNAse and detergent are described in more detail below.
    An example of an automatic processing system is shown in Figure 2. The processing system comprises a solution pumping system 201 in which various processing solutions are placed. The pumping system 201 sends an appropriate solution through piping 202 into a closed perfusion column 204, which has been fitted to an inlet 203 and outlet 205 of process solution. The appropriate solution then contacts the tissue flakes 108 within the perfusion column 204 and then leaves the column 204 through tubing 202 connected to the process solution outlet 205 at one end and the solution pumping system 201 at the other far end.
    The revealed fabric fills can be
    11/35 still treated to produce aseptic or sterile materials. In this way, in various modalities, the tissue fillers can be sterilized after preparation. As used in this document, a sterilization process can include any process that reduces the biocharge in a sample, but it is not necessary to make the sample absolutely sterile.
    Certain exemplary processes include, but are not limited to, a gamma irradiation process, an electron beam irradiation process, ethylene oxide treatment and propylene oxide treatment. Suitable sterilization processes include, without limitation, those described, for example, in Patent Publication η Ω US 2006 / 0073592A1, Sun et al .; Patent η Ω US 5,460,962 to Kemp; Patent Publication η Ω US 2008 / 0171092A1, Cook et al. In some modalities, sterilization is performed in conjunction with the packaging of the flakes, while in other modalities, sterilization can occur after packaging.
    After the tissue flakes are prepared by the disclosed methods, they can be stored for some time before implantation in or on a patient. In certain embodiments, the fabric filler can be packed in a Tyvek pouch for storage purposes. The fabric filling can also be stored in different states. In some embodiments, the filling of flake-like fabric is freeze-dried after preparation. In certain embodiments, freeze drying of the flake-like fabric filling is carried out before or during packaging. In certain embodiments, the tissue flakes are stored in a hydrated state. The tissue flakes can be hydrated in various solutions, for example, an aqueous preservation solution.
    The various steps described above can be
    12/35 combined, added, deleted or otherwise modified as needed. For example, if the starting material is porcine skin, removing the epidermis and subcutaneous fat may be necessary before cutting. However, removal of these components is not required if the starting material is an acellular tissue matrix. In addition, if porcine skin is used as the starting material instead of an acellular tissue matrix, further processing may be necessary after cutting.
    The same protocol in accordance with the revealed methods is provided in Figure 3, using porcine skins as the starting material. Specific details regarding each step are provided throughout the present disclosure. Fresh porcine skins are collected with the hairs subsequently removed 301. The epidermis and subcutaneous fat layers are then removed, leaving the dermal tissue 302. The dermal tissue is then incubated in a cryoprotective solution before freezing to maintain the appropriate balance between ice and amorphous tissue during cutting 303. After incubation in the cryoprotectant solution, the tissue is frozen, placed at a temperature between -10 ° C and -40 ° C and cut to a desired size and shape. The flakes are then incubated in an appropriate solution to decellularize the tissue 305. The tissue is then treated with enzymes to remove DNA and epitopes from a-gal 30 6. After the enzyme treatment, the flakes are disinfected using a solution of IPA 307 and washed in a 308 buffer.
    After washing, the fabric flakes are packed for storage. The packaging can be carried out in conjunction with freeze drying 309 or sterilization 312. If the flakes are freeze dried together with packaging 309, they are then sterilized 310, resulting in flakes dried by
    13/35 sterile freezing 311. Alternatively, if the packaging and sterilization are carried out together 312, the flakes can be freeze dried afterwards 314, which also results in sterile freeze-dried flakes 311. The flakes can also be hydrated after the step of packaging and sterilization 312, resulting in sterile hydrated tissue flakes 313.
    The developed flakes can form a stable, non-gelled, low-density suspension that can be used in a variety of ways. In certain embodiments, the flake-like tissue filler can be used as a bulk tissue filler for tissue regeneration and repair. Treatment methods that use tissue filling include selecting an anatomical site for treatment and implanting tissue filling at the treatment site. Examples include direct application of flake-like tissue material to deep wounds and large soft tissue spans that may occur during certain types of surgery, such as lumpectomies. Tissue filling can also be used to treat pressure ulcers, diabetic foot ulcers or periosteal bone defects. Tissue filling can also be used to reconstruct facial features as well as correct facial defects, including the treatment of wrinkles, skin loss or skin atrophy. In other embodiments, the flake-like fabric material can be used as a carrier for the controlled delivery of other bioactive substances. Examples of bioactive substances include, but are not limited to, microbicidal agents, cytokines, growth factors and drugs. Bioactive substances can also include non-collagen tissue, such as adipose tissue or cells, including stem cells. In certain embodiments, flakes of tissue that have been subsequently freeze dried can be
    14/35 hydrated in solutions containing bioactive substances and then applied to sites as needed. In other embodiments, the flake-like fabric filler can be applied as a slurry. If an aqueous suspension of tissue flakes is mixed briefly (30 to 300 seconds, for example), it becomes a loosely interwoven fibrillar slurry that is flowable for convenient application.
    In certain circumstances, a fabric foam may be desired. Depending on surgical procedures and particular tissue repair circumstances, the use of a tissue foam may be appropriate. Fabric foams can be used to treat wounds or damaged tissues that are not defined by gaps with a particular limit. For example, surgical adhesives or sutures could be used to attach tissue foam to tissues or organs. In other cases, fabric flakes may be more desirable when there is a need to fill in gaps of any shape, which are defined by a particular boundary. For example, tissue flakes are suitable when tumors are removed using laparoscopic procedures or cryosurgery, due to the small openings that result. Fabric foams can also be made to have specific sizes and shapes, such as sheets, spheres and cubes, for certain well-defined surgeries. For example, tissue foam sheets can be used to partially or completely cover surgical implants to reduce the dramatic effect of initial implant / body interactions and potentially slow capsule formation. In addition, tissue foams can be used as components of negative pressure injury therapy systems, such as the VAC® system, which is produced by Kinetic Concepts, Inc. Such systems can be used to treat a
    15/35 variety of tissue sites and include, for example, a source of negative pressure, such as a pump and one or more treatment materials, which normally include a porous foam or collector. General examples of such systems are described in U.S. Patent Publication Number 2010/0040687 A1, which was filed on August 13, 2009.
    The use of the revealed fabric flakes facilitates the preparation of fabric foam in several ways. The flakes of fabric have a small mass, but a large surface area, which facilitates further processing and the flakes are susceptible to the preparation of a uniform fiber suspension. In contrast to using micronized or crushed fibers or tissue particles, tissue flakes are unlikely to become gel or cake during decellularization. Finally, the process for preparing the fabric flakes first adds an additional layer of size reduction, which assists in preparing a uniform suspension.
    Accordingly, the methods described in this document can also be used to prepare a regenerative foam using the cut fabric described above as the starting material. As discussed above, a tissue matrix is selected and a cryoprotectant is used to treat the tissue matrix before freezing. The tissue matrix is then frozen and cut at a temperature in the range of -10 ° C to -40 ° C after freezing. Like the previous one, a pre-processed acellular tissue matrix can be used in conjunction with the disclosed method. In other modalities, the
    fabric matrix Can be decellularized after the cut as described above. The matrix of fabric cut is then placed on a
    liquid to form a suspension. Any suitable liquid can be used, as long as it does not interfere with the regenerative properties of the tissue matrix. In some
    16/35 modalities, the cut tissue matrix is placed in an aqueous solution. After being placed in the solution, the tissue matrix can be mixed into the solution. In various embodiments, the solution is mixed until a stable tissue suspension is formed and / or until the tissue size distribution reaches a desired level. The mixing can be carried out by any suitable means that achieve these ends, such as shaking, shaking or centrifuging the tissue matrix, once it is in the solution. Blending can also be used to mix the tissue matrix in the solution. In certain embodiments, a mixer is used to mix the matrix. The mixture can also be achieved with the use of a pressure jet, an ultrasonic device or a combination of the two. For example, after decellularization, the tissue flakes in solution can pass through a pressurized nozzle, where the tissue flakes are broken into fibers. You can also place the fabric flakes in solution in an ultrasonic field to break the fabric flakes into a fiber suspension. A sonic mouthpiece can also be used, which combines ultrasound and pressure to break the tissue flakes into a fibrous suspension. The consistency of the suspension can be controlled by how much cut tissue matrix is added to the liquid. In some embodiments, the amount of tissue matrix cut in the liquid is in the range of 20 to 40% (w / v). In certain embodiments, the amount of tissue matrix cut in the liquid is 25% (w / v).
    After a suspension has formed, the tissue suspension is then freeze-dried. Freeze drying can be performed under aseptic conditions to prevent contamination of the tissue matrix. The fabric suspension can also be aliquoted in an appropriate container, so that upon freeze drying, the fabric matrix is molded into the mold
    Desired 17/35.
    0 mixing process and freeze drying results in a composition of fabric in which filaments small in fabric of various dimensions are interwoven and
    integrated with each other. In some embodiments, the fabric composition has a foam-like appearance. Due to the interwoven pieces of small tissue, the tissue composition contains interconnected macropores, which allow free flow of fluid and help support the revascularization and restocking of host cells.
    The fabric foam disclosed in this document can be further processed as needed. The fabric foam can be disinfected or sterilized as described above. The freeze-dried material can also be further treated to increase the strength of the fabric foam with the use of heat and vacuum conditions. Without sticking to a theory, tissue foam reinforcement can occur through physical incorporation, biochemical cross-linking or a combination of the two. Physically, final dehydration results in surface tension, which propels closer tissue fragments and forms hydrogen bonds between the hydroxyl groups of adjacent collagen fibers. Chemically, the treatment results in the formation of amide between the carboxyl and amino groups, as well as esterification and glycation of collagen and other amino groups of extracellular matrix proteins. The heat applied to the tissue matrix cannot be so great as to cause denaturation of the dried proteins contained in the tissue matrix. The dried proteins in the tissue matrix typically denature between 130 ° C and 170 ° C. In one embodiment, the strength of the fabric foam can be increased by treating the freeze-dried material at a temperature above 30 ° C, but below temperatures
    18/35 denaturation listed above under vacuum conditions. In some embodiments, the strength of the fabric foam can be increased by treating the freeze-dried material at approximately 100 ° C under vacuum for 24 hours.
    After processing, tissue foam can be stored for some time before implantation in or on a patient. In certain embodiments, the fabric foam may be packaged in a Tyvek pouch for storage purposes. The fabric foam itself can also be stored in different states. In some embodiments, the fabric foam is stored in its freeze-dried state. In other embodiments, the fabric foam is stored in a hydrated state.
    Fabric Matrices
    As noted above, the methods described in this document can be used to produce flake-like fabric fillings using a variety of different types of fabric, as long as the fabric includes a matrix containing collagen susceptible to use with the methods described above. Such tissue arrays may include intact tissue, tissues that have been partially or completely decellularized or synthetic collagen hues (for example, 3D arrays formed from suspended or otherwise processed fabrics).
    The tissue matrix can be produced from a range of tissue types. For example, the tissue matrix can be derived from fascia, pericardial tissue, dura, umbilical tissue, placental tissue, heart valve tissue, ligament tissue, tendon tissue, arterial tissue, venous tissue, neural connective tissue, tissue of the urinary bladder, ureter tissue and intestinal tissue. In other embodiments, the tissue matrix comprises a dermal tissue matrix. In certain embodiments, the tissue matrix
    19/35 comprises a porcine dermal matrix.
    In certain embodiments, the tissues may include a mammalian soft tissue. For example, in certain embodiments, the tissue may include mammalian dermis. In certain embodiments, the dermis can be separated from the surrounding epidermis and / or other tissues, such as subcutaneous fat. In certain embodiments, the tissue sample may include small intestinal submucosa. In certain embodiments, tissue samples may include human or non-human sources. Suitable and exemplary non-human tissue sources include, but are not limited to, pigs, sheep, goats, rabbits, monkeys and / or other non-human mammals.
    Tissue matrices can be implanted in a variety of different anatomical sites. For example, tissue arrays can be implanted around breast implants; around or replacing vascular structures; surrounding or replacing luminal structures (for example, ureters, nerves, lymphatic tissues, gastrointestinal structures); over or replacing heart valves, pericardium or other cardiac structures; in or on bone or cartilaginous materials (for example, ears, noses, joint surfaces, around dental structures or along any short or long bone); and / or surrounding, coating, supporting or replacing any body cavity (eg bladder, stomach).
    Fabric matrices can be selected to provide a variety of different biological and mechanical properties. For example, an acellular tissue matrix can be selected to allow internal tissue growth and remodeling to assist with tissue regeneration normally found at the site where the matrix is implanted. For example, an acellular tissue matrix, when implanted
    20/35 over or within the band, can be selected to allow regeneration of the band without scarring or excessive fibrosis. In certain embodiments, the tissue matrix can be formed from ALLODERM® or STRATTICE ™, which are acellular dermal matrices of humans or pigs, respectively. Alternatively, other suitable acellular dermal matrices can be used, as further described below.
    In some embodiments, the collagen-based material comprises an acellular tissue matrix. In certain embodiments, these matrices can be completely decellularized to yield matrices of acellular tissue to be used for patients. For example, various tissues, such as skin, intestine, bone, cartilage, nerve tissue (e.g., nerve or hard fibers), tendons, ligaments or other tissues can be completely decellularized to produce tissue matrices useful for patients. Suitable processes for producing acellular tissue arrays are described below.
    In general, the steps involved in producing an acellular tissue matrix include harvesting tissue from a donor (for example, a human corpse or animal source) and removing cells under conditions that preserve biological and structural function. In certain embodiments, the process includes chemical treatment to stabilize the tissue and prevent biochemical and structural degradation together with or before removal of cells. In several embodiments, the stabilization solution stops and prevents osmotic, hypoxic, autolytic and proteolytic degradation, protects against microbial contamination and reduces the mechanical damage that can occur with tissues containing, for example, smooth muscle components (for example , blood vessels). The stabilization solution may contain a buffer
    21/35 appropriate, one or more antioxidants, one or more oncotic agents, one or more antibiotics, one or more protease inhibitors and / or one or more smooth muscle relaxants.
    The tissue is then placed in a decellularization solution to remove viable cells (for example, epithelial cells, endothelial cells, smooth muscle cells and fibroblasts) from the structural matrix without damaging the biological and structural integrity of the collagen matrix. The decellularization solution may contain a suitable buffer, salt, an antibiotic, one or more detergents (for example, TRITON X-100 ™, sodium deoxycholate, polyoxyethylene (20) sorbitan monooleate), one or more agents to prevent crosslinking , one or more protease inhibitors and / or one or more enzymes. In some embodiments, the decellularization solution comprises TRITON X-100 ™ at 1% in RPMI medium with Gentamicin and EDTA at 25 mM (ethylenediaminetetraacetic acid). In some embodiments, the tissue is incubated in a decellularization solution overnight at 37 2 C with gentle shaking at 90 rpm. For example, in some embodiments, 2% sodium deoxycholate is added to the decellularization solution.
    After the decellularization process, the tissue sample is washed carefully with saline. In some exemplary embodiments, for example, when xenogenic material is used, the decellularized tissue is then treated overnight at room temperature with a deoxyribonuclease solution (DNase). In some embodiments, the tissue sample is treated with a DNase solution prepared in the DNase buffer (20 mM HEPES (4 (2-hydroxyethyl) -1-piperazine ethanesulfonic acid), 20 mM CaCl 2 and 20 mM MgCl 2 ). Optionally, an antibiotic solution (for example, Gentamicin) can be added to the DNase solution. Any suitable buffer can be used as long as the
    22/35 buffer provides adequate DNase activity.
    Although an acellular tissue matrix can be made from the same species as the acellular tissue matrix graft receptor, different species can also serve as tissue sources. Thus, for example, an acellular tissue matrix can be made from pig tissue and implanted in a human patient. Species that serve as receptors for the acellular tissue matrix and donors of tissues or organs for the production of the acellular tissue matrix include, without limitation, mammals, such as humans, non-human primates (eg monkeys, baboons or chimpanzees), pigs, cows, horses, goats, sheep, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats or mice.
    The elimination of a-gal epitopes from collagen-containing material may decrease the immune response against collagen-containing material. The epitope of a-gal is expressed in non-primate mammals and New World monkeys (South American monkeys) as well as macromolecules such as proteoglycans from extracellular components. U. Galili et al., J. Biol. Chem. 263: 17755 (1988). This epitope is absent in Old World primates (monkeys from Asia and Africa and apes) and humans, however. Id. Anti-gal antibodies are produced in humans and primates as a result of an immune response to a-gal epitope carbohydrate structures in gastrointestinal bacteria. U. Galili et al., Infect. Immun. 56: 1730 (1988); R. M. Hamadeh et al., J. Clin. Invest. 89: 1223 (1992).
    Since non-primate mammals (for example, pigs) produce epitopes of a-gal, xenotransplantation of a material containing collagen from these primate mammals often results in immune activation because of anti-Gal primate antibodies that bind to these epitopes
    23/35 in the material that contains collagen. U. Galili et al., Immunology Today 14: 480 (1993); M. Sandrin et al. , Proc. Natl. Acad. Know. USA 90: 11391 (1993); H. Good et al., Transplant. Proc. 24: 559 (1992); Β. H. Collins et al. , J. Immunol. 154: 5500 (1995). Furthermore, a xenotransplant results in a greater activation of the immune system to produce increased amounts of anti-gal antibodies with high affinity. Consequently, in some embodiments, when animals that produce a-gal epitopes are used as the tissue source, the substantial elimination of a-gal epitopes from cells and extracellular components of collagen-containing material, and the prevention of expression of cellular a-gal epitopes can decrease the immune response against collagen-containing material associated with an anti-gal antibody that binds to a-gal epitopes.
    To remove epitopes from a-gal, after washing the tissue completely with saline to remove the DNase solution, the tissue sample can be subjected to one or more enzyme treatments to remove certain immunogenic antigens, if present in the sample. In some embodiments, the tissue sample can be treated with an α-galactosidase enzyme to eliminate epitopes of a-gal if present in the tissue. In some embodiments, the tissue sample is treated with α-galactosidase at a concentration of 300 U / L prepared in 100 mM phosphate buffer at pH 6.0. In other embodiments, the α-galactosidase concentration is increased to 400 U / L for adequate removal of a-gal epitopes from the harvested tissue. Any suitable buffer and enzyme concentration can be used as long as sufficient removal of antigens is achieved.
    Alternatively, instead of treating the tissue with enzymes, animals that have been genetically modified to lack one or more antigenic epitopes can be selected
    24/35 as the tissue source. For example, animals (for example, pigs) that have been genetically engineered to lack the terminal α-galactose part can be selected as the tissue source. For descriptions of appropriate animals, see the U.S. patent copending serial application in U.S. 10 / 896,594 and the patent document in U.S. 6,166,288, the disclosures of which are incorporated herein by reference in their entirety for reference. In addition, certain exemplary methods for processing tissues to produce acellular matrices with or without reduced amounts of alpha-1,3-galactose parts or without them, are described in 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 as a reference in its entirety.
    After the acellular tissue matrix is formed, viable histocompatible cells can optionally be seeded into the acellular tissue matrix to produce a graft that can be further remodeled by the host. In some embodiments, viable histocompatible cells can be added to the matrices using standard in vitro cell co-culture techniques prior to transplantation or through in vivo repopulation after transplantation. In vivo repopulation can occur by the recipient's own cells that migrate to the acellular tissue matrix or by infusing or injecting cells obtained from the recipient or histocompatible cells from another donor into the acellular tissue matrix in situ. Various types of cells can be used, including embryonic stem cells, adult stem cells (for example, mesenchymal stem cells) and / or neuronal cells. In various modalities, cells can be directly applied to the
    25/35 inner portion of the acellular tissue matrix just before or after an implant. In certain embodiments, cells can be placed within the matrix of acellular tissue that will be implanted and cultured before an implant.
    Although general process parameters for producing acellular tissue arrays are described, a variety of acellular materials that contain collagen are available and methods for processing such materials to produce flake-like tissue fillers can be used with any of these materials . For example, a number of biological framework materials are described by Badylak et al. and the methods of the present disclosure can be used to produce flake-like fabric fillings using any of those materials or any other similar materials. Badylak et al., Extracellular Matrix as a Biological Scaffold Material: Structure and Function, Acta Biomaterialia (2008), doi: 10.1016 / j.actbio.2008.09.013.
    Example 1: Cryopreservation and cryo-cutting of divided porcine dermis
    Fresh porcine skins were collected and hair was removed. A dermis tissue was obtained from the skins dividing the epidermis layer and the subcutaneous fat layer (hypodermis). The porcine dermis was cleaned with Dulbecco's phosphate buffered saline (PBS) and incubated for 4 to 6 hours in the cryoprotective solution containing 50 mM sodium phosphate, 10 mM ethylenediaminetetraacetic acid (EDTA) and 35% (w / v ) of maltodextrin (pH 7.0). The cryoprotectant treated dermis slides were then frozen in an 80 ° C freezer. The frozen dermis tissue was grated into tissue flakes using a medium sized MICROPLANE® grater (the grater openings are approximately 2.2 to 3.2 mm wide and 0.2 mm thick).
    26/35
    The tissue flakes were then washed for 10 to 15 minutes in PBS to remove the cryoprotectant and then fixed in 2% glutaraldehyde solution. The morphology of the tissue flakes was then observed using light microscopy and scanning electron microscopy (SEM). For an examination under light microscopy, the tissue samples were stained with Sirius red. For SEM examination, the tissue samples were dehydrated in stages in ethanol solutions of increasing concentration (25%, 50%, 75%, 90%, 95%, 98% and 100%), dried with supercritical CO 2 in a critical point dryer and coated with gold crackle. The tissue specimens were seen under high vacuum and low electronic voltage (5 kV). As shown in Figures 4A and 4B, the morphology of the cut pieces was observed to be flake-like under both types of microscopy.
    To determine the size (mass) distribution of the tissue flakes, the glutaraldehyde-fixed tissue pieces were washed in distilled water to remove a glutaraldehyde residue. The net weight of more than 200 pieces of tissue was determined after staining a surface water with surgical gauze. Figure 5 shows the size distribution based on mass of the fabric flakes. More than 70% of the tissue fragments weighed between 0.5 and 2.0 mg, with an average weight of 1.3 mg. Few pieces of tissue weighed over 4 mg. The size of the tissue fragment was in the expected range after using a
    medium microplane (2 to 3 mm wide and 0.2 mm thick, nominal tissue volume from 0.8 to 1.8 mm 3 ). Example 2: Content control dermis ice frozen pigThe reason between ice and dermis fabric frozen phase amorphous facilitate cutting dermis blades into a tec matrix flake-like acid. At dermis blades
    Frozen 27/35 that were not treated in cryoprotectant solution contained a lot of ice, were very brittle and were difficult to cut. On the other hand, the frozen dermis blades with low ice content were soft and heated up quickly during a cut, which also made them difficult to cut. The ice content and hardness of the frozen dermis slides were controlled by pre-treating the dermis slides in cryoprotective solutions that reduced ice formation while facilitating the formation of an amorphous tissue matrix with a high glass transition temperature below zero.
    The divided porcine dermis was prepared as described in example 1. Maltodextrin solutions containing 0% to 35% (w / v) maltodextrin were made with a phosphate buffer solution (50 mM sodium phosphate, 10 mM EDTA , pH 7.0). The samples of divided porcine dermis were incubated in cryoprotective solutions overnight. After an incubation, a water and cryoprotectant content in dermis tissue samples was determined. The ice contents of frozen tissue samples were determined using differential scanning calorimetry according to their ice melting enthalpies. Figure 6 shows the ice mass fractions and the amorphous tissue domain in the pre-treated frozen tissue in different cryoprotective solutions. In the absence of maltodextrin (PBS buffer alone), the porcine dermis had 75% (w / v) water and 25% dry weight. Upon freezing, -93% of dermis water crystallized as ice, resulting in a mass fraction of 70% ice and a mass fraction of 30% freeze-concentrated amorphous domain (not frozen). The corresponding volume fractions were 77% and 23% for ice and non-frozen domain, respectively. With increasing concentrations of maltodextrin, the ice fraction of dermis tissue
    28/35 incubated porcine decreased and the fraction of the non-frozen domain increased. For porcine dermis incubated in 35% maltodextrin solution, the ice mass and non-frozen domain fractions were reduced to 34% and increased to 66%, respectively (corresponding to volume fractions of 42% and 58%, respectively ). A reduced ice formation was due to both osmotic tissue dehydration and a penetration of maltodextrin into the extracellular matrix of the dermis tissue. As a result of a penetration of maltodextrin into the extracellular dermis matrix, the glass transition temperature of the amorphous domain of the frozen dermis increased to -17 ° C. A porcine tissue treated with 15 to 35% maltodextrin solutions can be easily grated into tissue flakes.
    Example 3: Processing of dermal tissue flakes
    The tissue flakes prepared as described in example 1 were further processed to remove cellular components and epitopes from α-galactosyl. The process included the following steps: (i) cleaning the cryoprotectants, (ii) decellularization, (iü) enzyme treatment, (iv) disinfection, (v) lyophilization, (vi) sterilization and (vii) secondary packaging.
    (i) Removing cryoprotectants. The tissue flakes were placed in 225 ml (~ 30 g / bottle) cuneiform centrifuge bottles. The material was washed with 150 ml of sterile PBS for about 10 minutes. The tissue suspension was centrifuged at 500 rpm for 3 minutes and the supernatant was discarded. The tissue pellet was resuspended with 150 ml of sterile distilled water and centrifuged again at 500 rpm for 3 minutes to collect the tissue flakes.
    (ii) Decellularization. The tissue material underwent decellularization at room temperature with agitation at 150
    29/35 ml of 2% (w / v) sodium deoxycholate dissolved in a 10 mM HEPES buffer with 10 mM EDTA (pH 7.8). The decellularization solution was changed after 1 hour by centrifugation at 500 rpm for 3 minutes. After a fresh solution was added, the material tissue was allowed to incubate for another 4 hours. The decellularization solution was drained after another round of centrifugation.
    (iii) Enzyme treatment. The decellularized tissue flakes were washed twice for 30 minutes at a time with a 10 mM HEPES buffer containing 5 mM EDTA (pH 7.3). The enzyme treatment was performed for 4 hours in 150 ml of HEPES buffer with 2 mM MgCfo, 2 mM CaCÍ2, 1 mg / 1 dornase alfa and 1 mg / 1 α-galactosidase. The enzyme solution was drained after centrifugation at 500 rpm for 3 minutes.
    (iv) Disinfection. The enzyme-treated tissue flakes were resuspended and washed twice with a 10 mM HEPES buffer with 5 mM EDTA. The first wash was for 60 minutes and the second wash was carried out overnight. After centrifugation, the tissue material was cleaned with 100 ml of sterile distilled water for 30 minutes. For some bottles, 100 ml of isopropyl alcohol (IPA) solution (70% w / v) was added to a tissue suspension. After about 10 minutes, the suspension was centrifuged at 500 rpm for 3 minutes. The old IPA solution was drained and fresh 70% IPA solution was added to resuspend the material. The material was treated with IPA for at least 4 to 6 hours before being packed in Tyvek bags for lyophilization. For other bottles, the fabric flakes were packaged directly in Tyvek bags for freeze drying without IPA disinfection.
    (v) Lyophilization. The processed tissue material was freeze-dried. Freeze drying
    30/35 consisted of 3 stages. (a) cool the material from room temperature to -35 ° C to approximately 1 ° C / minute, then maintenance at -35 ° C for 10 minutes; (b) raise to -10 ° C to approximately 1 ° C / minute under 40 mT, then maintenance for 16 hours; and (c) rise to 20 ° C at approximately 1 ° C / minute under 20 mT and then maintained for 8 hours.
    (vi) Sterilization. Freeze-dried tissue samples that were not disinfected with IPA were sterilized with ethylene oxide (EO). Sterilization with EO included (a) conditioning at 52 ° C to 63 ° C and 55 to 75% Relative Humidity for 30 to 45 minutes, (b) exposure of EO with a gas concentration of 600 ± 50 mg / 1 per 4 hours and (c) aeration at 38 to 54 ° C for at least 12 hours.
    (vii) Secondary packaging. The samples treated by IPA were immediately packaged in foil bags after freeze drying. The samples treated with EO were packed in foil bags after treatment with EO.
    The processed tissue flakes were acellular.
    Both materials treated with IPA how much the treaties eat the tested must beExample sterile.4: Stability in Flakes Fabric ProcessedThe stability thermal From flakes of fabric processed was tested with the use of scanning calorimetry
    differential (DSC). Both materials treated by IP and those treated by EO were rehydrated in saline solution PBS (pH 7.5). The samples were scanned at a heating rate of 3 ° C / min from 2 to 125 ° C (DSC Q200, TA Instruments). Both IPA-treated and EO-treated tissues had a small low temperature peak (approximately 30 to 32 ° C). As shown in the Figures
    31/35
    7, the initial temperature of the main collagen denaturation peaks was 53.3 ± 0.2 ° C and 58.0 ± 0.2 ° C (N = 4) for EO treated tissue flakes and IPA, respectively. The initial temperature of the tissue flakes treated with IPA was similar to the fresh dermal samples. The data indicated that sterilization with EO destabilizes the tissue matrix compared to treatment with IPA.
    The susceptibility of freeze-dried tissue flakes processed to enzyme degradation was tested with collagenase and trypsin assays. Figures 8A and 8B show the resistance of processed tissue material to the digestion of collagenase and trypsin, respectively. The flakes of tissue disinfected with IPA resisted the digestion of collagenase and trypsin reasonably well. The material sterilized with EO had an increased susceptibility to proteolysis compared to the material disinfected with IPA.
    Example 5: Suitability of Tissue Flakes to Enable Fluid Flow and Pressure Conversion
    Upon rehydration, the tissue flakes form a stable suspension without freezing or phase separation, allowing the tissue flakes to be used to fill gaps or large defects (tens to hundreds of milliliters). The suspension of tissue flakes contains large interconnected channels that allow fluid to flow freely and thus assist in revascularization and restocking of cells when the flakes are used as tissue fillers. The ability of the suspension of tissue flakes to allow fluid flow and the conversion of pressure differentials was investigated. The rehydrated fabric flakes were placed in Organza mesh and spread over a 7.62 cm x 7.62 cm (3 x 3) pressure distribution pad with 36 sensor ports, as shown
    32/35 in Figures 9A and 9B. A piece of GranuFoam ™ (7.62 cm x 7.62 cm (3 x 3) and 3.81 cm (1.5) thick) was placed on top of the fabric flake material and a VAC® of 15, 24 cm x 15.24 cm (6 x 6) was fixed to the dressing assembly together with the TRAC® pad. The assembly was connected to the V.A.C.® ATS therapy unit to produce a continuous pressure at 16.67 KPa (125 mmHg). After 5 minutes of equilibrium, the negative pressure detected in the 36 sizes was mentioned. Subsequently, 4 of the ports were disconnected from the pressure sensors and connected to a reservoir of 0.9% colored saline solution for infusion at a rate of 500 ml per day using a peristaltic pump. The pressure on the remaining 32 ports was then monitored over time. The average pressure detected was approximately 14.67 to 14.93 KPa (110 to 112 mmHg). The negative pressure was 14.85 ± 0.11 (111.4 ± 0.9) (mean ± SD), 14.68 ± 0.18 (110.1 ± 1.4) and 14.75 ± 0, 17 KPa (110.6 ± 1.3 mmHg) after 1, 2 and 3 hours respectively. The consistency indicated that the pressure was evenly distributed across the entire dressing assembly and the interconnected channels were able to convert pressure differentials well.
    Example 6: Reconstructive fabric foam made from flake-like fabric material
    An acellular tissue matrix derived from porcine dermis was frozen at -80 ° C and used to produce tissue flakes aseptically with a medium sized MICROPLANE® cheese grater. Approximately 50 g of tissue sample was suspended in 200 ml of sterile water and then mixed using a RETSCH® mixer at 4,000 rpm at one minute intervals for a total of 5 cycles. As shown in Figures 10A and 10B, the blend reduced the size of the fabric flake material. Merging the fabric flakes also resulted in a stable and consistent fabric suspension. The suspension
    33/35 tissue was distributed in plastic petri dishes of 80 cm 2 in 25 ml of suspension per plate and freeze-dried aseptically. The freeze-drying process included controlled cooling of a tissue suspension from room temperature to -30 ° C within 60 minutes and drying at a chamber pressure of 100 mT and a shelf temperature of 20 ° C for 24 hours.
    The blending and freeze drying process resulted in small pieces of tissue of various dimensions intertwined and integrated with each other, forming a soft tissue foam with interconnected macropores and a preserved extra matrix of acellular tissue structures, as shown in Figures 11A to 11D. Figure 11A shows a thick appearance of the fabric foam. Figure 11B shows the interconnected macropores of the soft tissue foam under the SEM microscope. Figure 11C is a histogram of the extracellular matrix structure of the foam using a hematoxylin and eosin stain. Figure 11D is an enlarged view of a section found within Figure 11C. As shown in Figures 11C and 11D, the foam tissue matrix has a filamentous, fibrous nature after mixing and freeze drying. The aseptically freeze-dried foam had a dry tissue mass of 9.9 ± 0.3% (w / v, N = 5).
    Part of the freeze-dried tissue material was further treated at approximately 100 ° C under vacuum for 24 hours to increase the strength of the tissue foam. The calorimetric measurement detected an initial denaturation temperature of 62.2 ± 0.1 ° C and a denaturation enthalpy of 60.5 J / g of tissue mass, indicating that there is no denaturation of tissue collagen.
    In a separate experiment using the freeze-dried material, the in vivo response of reconstructive tissue foam made of flake-like tissue material was
    34/35 investigated using an athematic rat model (Rattus norvegicus, nude rat). Tissue specimens (10 mm x 10 mm and approximately 3 mm thick) were prepared from the fabric foam and rehydrated in 0.9% saline. After rehydration, tissue specimens were then implanted subcutaneously in athymic rats. For each rat, four separate incisions were made on the right and left side of the back through the skin and parallel to the lumbar region of the spine. Pockets were formed by blind dissection in the subcutaneous tissue into which the tissue material was introduced. Two specimens implanted on the right side of the spine and two specimens were implanted on the right, with the skin closed afterwards. The animals were euthanized either four or eight weeks after implantation. The specimens implanted with attached soft tissue were excised and the excised tissue samples were fixed in 10% formalin and processed for histological evaluation using hematoxylin and eosin stains. Histological slides were evaluated by microscope for evidence of revascularization and restocking of host cells. As shown in Figure 12, revascularization and restocking of significant cells were observed in 4-week implants (Figures 12A and 12B), while implanted tissue foams were completely repopulated in 8 weeks (Figures 12C and D). Inflammation was observed to be moderate in 4-week explants and mild in 8-week explants.
    Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification or a practice of the invention disclosed herein. It is intended that the specification and examples are considered as exemplary only, with a true scope and spirit of the invention being indicated
    35/35 for the following claims.
    权利要求:
    Claims (3)
    [1]
    characterized by the cryoprotective solution comprising a maltodextrin solution.
    Method according to either of Claims 7 and 8, characterized in that the solution comprises 5 to 50% (w / v) of maltodextrin.
    METHOD, according to claim 7 or 78, characterized in that the solution comprises 15 to 25% (w / v) of maltodextrin.
    METHOD according to claim 1, characterized in that cutting the tissue matrix comprises grating the tissue matrix.
    12. METHOD, according to claim 1, characterized in that the cut fabric has a size distribution in the range of 0,
    [2]
    2 to 5 mm in length, 0.2 to 3 mm in width and 0.02 to 0.3 mm in thickness.
    METHOD, according to any one of claims 1 to 11, characterized in that it further comprises exposing the tissue matrix to a disinfectant.
    14. METHOD, according to claims 1 to 13, characterized in that the disinfectant is isopropyl alcohol.
    15. METHOD, according to claim 1, characterized in that it further comprises sterilizing the tissue matrix.
    16. METHOD according to claim 15, characterized in that sterilizing the tissue matrix comprises the application of ethylene oxide, propylene oxide, gamma irradiation or electron beam irradiation to the tissue matrix.
    17. METHOD according to claim 1, characterized in that it further comprises mixing the tissue matrix suspension prior to freeze drying.
    18. METHOD, according to claim 17, characterized by the mixing step also comprising the
    [3]
    3/3 reduction in the size of the tissue matrix.
    19. METHOD according to claim 1, characterized in that the fabric matrix forms interlaced filaments during mixing and freeze drying.
    20. TISSUE MATRIX, characterized by being prepared by the process, as defined in any one of claims 1 to 19.
    21. TISSUE MATRIX according to claim 20, characterized in that it is for use in the treatment of a tissue defect.
    22. FABRIC MATRIX, 21, characterized by defect
    23. FABRIC MATRIX, 21, characterized by the pressure defect.
    24. TISSUE MATRIX, 21, characterized by the diabetic defect.
    according to the tissue claim it is a lumpectomy.
    according to the claim to be a foot ulcer according to the claim and to be a foot ulcer
    25. TISSUE MATRIX according to claim 21, characterized in that the tissue defect is a defect of the periosteal bone.
    26. TISSUE MATRIX according to claim 21, characterized in that the tissue defect is a facial defect.
    27. TISSUE MATRIX, according to claim 26, characterized by the facial defect being selected from a wrinkle, skin loss or skin atrophy.
    28. TISSUE MATRIX according to any one of claims 21 to 27, characterized in that it further comprises applying negative pressure to the collagen-based matrix during treatment.
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    同族专利:
    公开号 | 公开日
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    AU2012242694A1|2013-10-17|
    US20160271295A1|2016-09-22|
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    法律状态:
    2019-09-03| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|
    2019-12-17| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|
    2020-04-14| B09B| Patent application refused [chapter 9.2 patent gazette]|
    2020-06-30| B12B| Appeal against refusal [chapter 12.2 patent gazette]|
    2021-10-05| B350| Update of information on the portal [chapter 15.35 patent gazette]|
    优先权:
    申请号 | 申请日 | 专利标题
    US201161475378P| true| 2011-04-14|2011-04-14|
    PCT/US2012/033533|WO2012142419A1|2011-04-14|2012-04-13|Regenerative materials|
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