Adipose tissue matrix containing tropoelastin

Abstract

This invention provides tissue products manufactured from adipose tissue and methods for manufacturing the same. [Solution] A method for producing a tissue product is provided, comprising the steps of: selecting adipose tissue; processing the tissue to remove substantially all cellular material from the tissue; suspending the tissue in a liquid to form a suspension containing 2-4% by weight of solids; and freezing and drying the suspension to form a porous sponge.

Description

Technical Field

[0001] The present disclosure relates to tissue products, and more particularly, to an extracellular tissue matrix made from adipose tissue and containing tropoelastin.

[0002] This application claims priority to U.S. Provisional Application No. 63 / 004,794, filed on April 3, 2020, under 35 U.S.C. § 119, the entire content of which is hereby incorporated by reference.

Summary of the Invention

[0003] Various tissue-derived products are used to regenerate, repair or otherwise treat diseased or damaged tissues and organs. Such products can include tissue grafts and / or processed tissues (e.g., acellular tissue matrices from skin, intestine or other tissues, with or without cell seeding). Such products generally have properties determined by the tissue source (i.e., tissue type and animal from which it is derived) and the processing parameters used to manufacture the tissue product. Since tissue products are often used for surgical applications and / or tissue replacement or augmentation, the product needs to support tissue growth and regeneration as desired at the selected implantation site. The present disclosure provides adipose tissue products that can improve tissue growth and regeneration for various applications such as breast implants. The tissue product can further contain tropoelastin to improve its biological or mechanical properties. The product can be crosslinked to maintain the desired shape when implanted.

[0004] According to certain embodiments, a method for producing a tissue product is provided. This method may include the steps of: selecting adipose tissue; mechanically processing the adipose tissue to reduce its size; processing the mechanically processed tissue to remove substantially all cellular material from it; suspending the tissue in a liquid to form a suspension; and drying the suspension in a mold to form a porous sponge. In some embodiments, the suspension is dried and subjected to a treatment such as dehydrothermal crosslinking. The tissue product may contain tropoelastin.

[0005] In various embodiments, adipose tissue is treated to control specific mechanical properties. For example, the treated tissue can be crosslinked to provide a stable three-dimensional structure. Additionally or alternatively, the solid content of the sponge or suspension can be controlled, as will be described in more detail below. Furthermore, in some embodiments, tropoelastin can be added to the composition before forming the suspension.

[0006] Furthermore, this specification also provides organizational products created through the disclosure process.

[0007] In some embodiments, the tissue product comprises a decellularized extraadipocyte tissue matrix, the tissue matrix being formed into a predetermined three-dimensional shape, and the tissue matrix being partially crosslinked to maintain the three-dimensional shape. The product may contain a desired amount of tropoelastin.

[0008] Furthermore, this specification also provides tissue products including breast implants. These implants may include adipose tissue matrix formed to have a desired set of mechanical properties controlled by crosslinking and / or solid content. The product may further contain tropoelastin.

[0009] Furthermore, this specification also provides tissue products containing an injectable fat matrix or particulate fat matrix. This implant may be at least partially crosslinked solids. The product may further contain tropoelastin.

[0010] Furthermore, this specification provides a therapeutic method comprising the steps of selecting a tissue site and implanting a tissue product disclosed herein into the tissue site. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 is a flowchart outlining the process for manufacturing an adipose tissue matrix sponge according to a specific embodiment. [Figure 2] Figure 2 is a side view of a biological breast implant according to a specific embodiment. [Figure 3] Figure 3A is a perspective view of the configuration of a breast implant according to a particular embodiment. Figure 3B is a perspective view of another configuration of the breast implant according to a particular embodiment. Figure 3C is a perspective view of another configuration of the breast implant according to a particular embodiment. [Figure 4] Figure 4 shows the implantation of a system for surgical breast surgery according to a specific embodiment. [Figure 5] Figures 5A to 5G are tissue images showing the effect of EDC crosslinking on lipid synthesis. [Figure 6] Figure 6A is a bar graph showing the effect of fat matrix solids on compressive strength. Figure 6B is a bar graph showing the effect of fat matrix solids on recovery rate. Figure 6C is a bar graph showing the effect of fat matrix solids on elasticity. Figure 6D is a bar graph showing the effect of fat matrix solids on elastic modulus. [Figure 7]Figure 7A is a bar graph showing the effect of changes in crosslinking agent content or tropoelastin content on the compressive strength of the adipose matrix product. Figure 7B is a bar graph showing the effect of changes in crosslinking agent content or tropoelastin content on the recovery rate of the adipose matrix product. Figure 7C is a bar graph showing the effect of changes in crosslinking agent content or tropoelastin content on the elastic modulus of the adipose matrix product. [Figure 8] Figures 8A to 8D are representative histological images showing the effect of tropoelastin addition on adipogenesis in a rat model 8 weeks post-transplantation, with and without EDC crosslinking of the adipose matrix product. [Figure 9] Figure 9 shows graphs of fat increase in the adipose matrix at 8 weeks post-transplantation, with and without EDC crosslinking, and with and without tropoelastin supplementation. [Figure 10] Figures 10A to 10D are representative histological images showing the effect of tropoelastin addition on adipogenesis in a rat model 16 weeks post-transplantation, with and without EDC crosslinking of the adipose matrix product. [Figure 11] Figure 11 shows graphs of fat increase in the adipose matrix at 16 weeks post-transplantation, with and without EDC crosslinking, and with and without tropoelastin supplementation. [Figure 12] Figures 12A and 12B are Masson's trichrome stained sections of explants at week 16, demonstrating the persistence of TE. [Modes for carrying out the invention]

[0012] Hereinafter, specific exemplary embodiments relating to this disclosure will be referenced in detail. These specific examples are shown in the accompanying drawings. Wherever possible, the same reference numerals will be used throughout the drawings to refer to identical or similar parts.

[0013] In this application, the use of the singular form includes the plural form unless otherwise specified. In this application, the use of "or" means "and / or" unless otherwise specified. Furthermore, the use of the term "includes," as well as other forms such as "includes" and "contains," is not limiting. Any range described herein should be understood to include both endpoints and all values ​​between those endpoints.

[0014] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described herein. All documents or parts of documents cited herein, including but not limited to patents, patent applications, articles, books, and papers, are expressly incorporated herein by reference in whole for any purpose.

[0015] As used herein, “tissue product” refers to any human or animal tissue containing extracellular matrix proteins. “Tissue product” includes cell-free tissue matrix or partially decellularized tissue matrix, as well as decellularized tissue matrix rearranged with exogenous cells.

[0016] As used herein, the term “cell-free tissue matrix” refers to an extracellular matrix derived from human or animal tissue that holds a substantial amount of natural collagen, other proteins, proteoglycans, and / or glycoproteins necessary to serve as a scaffold supporting tissue regeneration. “Cell-free tissue matrix” is distinct from purified collagen materials such as acid-extracted purified collagen, which substantially lack other matrix proteins and do not retain the natural microstructural properties of the tissue matrix due to the purification process. While referred to as “cell-free tissue matrix,” it should be understood that such tissue matrix may be bound to exogenous cells, such as stem cells or cells from the patient to whom the “cell-free tissue matrix” is transplanted. “Decellularized adipose tissue matrix” will be understood to refer to adipose tissue from which all cells have been removed to produce an adipose extracellular matrix. “Decellularized adipose tissue matrix” may include further processed or intact matrices, as discussed herein, including mechanical processing, sponge formation, and / or further processing to produce a particle matrix. As used herein, AAM refers to “cell-free adipose matrix” from any source, and pAAM refers to “porcine-derived adipose matrix.”

[0017] It should be understood that "cell-free" or "decellularized" tissue matrix refers to a tissue matrix in which no cells are visible even when using a light microscope.

[0018] Products for treating patients can be manufactured using various human and animal tissues. For example, various tissue products are manufactured for the regeneration, repair, augmentation, reinforcement and / or treatment of human tissue damaged or lost due to various diseases and / or structural damage (e.g., trauma, surgery, atrophy, and / or long-term wear and degeneration). Such products may include, for example, cell-free tissue matrices, tissue allografts or xenografts, and / or reconstituted tissues (i.e., at least partially decellularized tissue from which cells have been seeded to provide material that can grow).

[0019] A variety of tissue products are manufactured for treating soft and hard tissues. For example, ALLODERM® and STRATTICE™ (LIFECELL CORPORATION, Branchburg, New Jersey) are two acellular dermal tissue matrices made from human and porcine dermis, respectively. Such materials are very useful for treating certain types of diseases, but materials with different biological and mechanical properties may be desirable for certain applications. For example, ALLODERM® and STRATTICE™ are used to assist in the treatment of structural defects and / or to provide tissue support (e.g., for the abdominal wall or in breast reconstruction), and their strength and biological properties make them suitable for such uses. However, such materials may not be ideal for the regeneration, repair, replacement, and / or augmentation of adipose-containing tissue when the production of adipose tissue containing viable adipocytes is a desired outcome. Accordingly, the present disclosure provides tissue products useful for treating tissue defects / deficiencies associated with adipose-containing tissue. The present disclosure also provides methods for manufacturing such tissue products.

[0020] Tissue products may include adipose tissue that has been processed to remove at least some of its cellular components. In some cases, all or substantially all of the cellular material is removed, leaving behind adipose extracellular matrix proteins. Furthermore, the product may be processed to remove some or all of the extracellular and / or intracellular lipids. However, in some cases, the complete removal of extracellular and / or intracellular lipids may damage the structure and function of the adipose matrix. For example, adipose tissue that has been chemically or enzymatically processed for a long period of time may have denatured or otherwise damaged collagen, or depleted proteins necessary for fat regeneration. For this reason, in some cases, the product will contain a certain level of residual lipids. The residual lipid content may be, for example, about 5%, 6%, 7%, 8%, 9%, or 10% by weight of the product. Further processing of extracellular matrix proteins may be used to produce three-dimensional porous or spongy materials, and further processing of porous or spongy materials may be used to produce injectable products.

[0021] As described above, the tissue product of the present disclosure is formed at least in part from adipose tissue. The adipose tissue is derived from a human or animal source. For example, human adipose tissue may be obtained from cadavers. Additionally, human adipose tissue can also be obtained from a living donor (e.g., autologous tissue). Adipose tissue can also be obtained from animals such as pigs, monkeys or other sources. When using a source of animals other than primates, the tissue can be further processed to remove antigenic components such as the 1,3-alpha-galactose moiety that is present in pigs and other mammals but not in humans and primates. See Xu, Hui, et al., "A Porcine-Derived Acellular Dermal Scaffold that Supports Soft Tissue Regeneration: Removal of Terminal Galactose-α-(1,3)-Galactose and Retention of matrix Structure", Tissue Engineering, Vol. 15, 1-13 (2009). This document is hereby incorporated by reference in its entirety. Further, the adipose tissue may be obtained from an animal that has been genetically modified to remove antigenic portions.

[0022] Figure 1 shows an exemplary process for manufacturing the tissue product of the present disclosure. Figure 1 provides a flowchart showing the basic steps and can be used to produce a suitable adipose tissue sponge. As shown, the process can include several steps, but it should be understood that additional or alternative steps may be added or substituted depending on the particular tissue used, the desired application or other factors.

[0023] As shown in the figure, process 100 as a whole can begin with step 110 receiving the tissue. The tissue may include various types of adipose tissue, including, for example, human or animal adipose tissue. Suitable tissue sources include allograft tissue, autograft tissue, or xenograft tissue. If xenografts are used, the tissue may include fat from animal, livestock or wild sources, including pigs, cattle, dogs, and cats, and / or any other suitable mammalian or non-mammalian fat sources.

[0024] Tissue can be collected from the animal source using any preferred method, but it may be preferred to collect it using sterile or sterilization techniques if possible. Tissue may be stored at low temperatures or frozen, or processed immediately to prevent undesirable changes due to long-term storage.

[0025] After receiving the tissue, it first undergoes a mechanical sizing process in step 120 and / or a mechanical degreasing process in step 130. Mechanical sizing includes visible or large cuts of the tissue using a manual blade or any other suitable grinding process.

[0026] The mechanical degreasing in step 130 is crucial in tissue preparation. Specifically, the fat is subjected to various mechanical processing conditions to aid in lipid removal. For example, mechanical processing may include grinding, mixing, shredding, grating, or other processing of the tissue. Mechanical processing may also be carried out under conditions that allow for some degree of heating, which can aid in the liberation or removal of lipids. For example, mechanical processing may be carried out at temperatures of 42-45°C for porcine fat and slightly lower for human fat, under conditions that the adipose tissue is heated up to 122°F (50°C). The application of external heat may be insufficient to release lipids, and therefore, the heat generated during mechanical destruction may be preferable to aid in lipid removal. In some examples, the heating during mechanical processing may be a short-duration pulse of temperature rise. This thermal pulse may cause liquefaction of lipids released from adipocytes destroyed by mechanical destruction, which can then lead to efficient phase separation for bulk lipid removal. For example, when processing porcine adipose tissue, the temperature reached during the process will exceed 100°F but will not exceed 122°F (50°F). The temperature range can be adjusted depending on the origin of the adipose tissue. For example, when processing tissue with less saturated fat, such as primate tissue, the temperature can be further reduced to approximately 80°F, 90°F, 100°F, 110°F, or 120°F. Alternatively, the process can be selected so that the fat reaches a minimum temperature, such as 80°F, 90°F, 100°F, 110°F, or 120°F.

[0027] In some cases, mechanical degreasing can be performed by mechanically treating the tissue with little or no washing solution added. For example, the tissue may be mechanically treated by grinding or mixing without the use of a solvent. Alternatively, water may be used for tissue disruption if moisture is needed, for example, to increase fluidity or decrease viscosity, and this water may include pure water, saline, or other buffers including saline or phosphate-buffered saline. In some cases, the tissue is treated by adding a specific amount of a biocompatible solvent, such as saline (e.g., plain saline, phosphate-buffered saline, or a solution containing salts and / or surfactants). Other solutions that promote cell lysis, including salts and / or surfactants, may also be appropriate.

[0028] In step 140, fat can be washed after mechanical processing and lipid removal. For example, tissue can be washed with one or more rinses in various biocompatible buffers. Suitable washing solutions include, for example, saline, phosphate-buffered saline, or other suitable biocompatible materials or physiological solutions. In one embodiment, water can be used as a rinse agent to further disrupt the cells, and then phosphate-buffered saline or other suitable saline can be introduced to return the matrix proteins to the biocompatible buffer.

[0029] Washing can be performed in conjunction with centrifugation or other processes to separate lipids from the tissue. For example, in some embodiments, the material is diluted with water or another solvent. The diluted material is then centrifuged, with free lipids flowing to the top and extracellular matrix proteins settling as a pellet. The protein pellet is then resuspended, and washing and centrifugation are repeated until a sufficient amount of lipids are removed.

[0030] After washing, the fat can be treated to remove some or all of the cells from the adipose tissue, as shown in step 150. The cell removal process can include several suitable processes. For example, a suitable method for removing cells from adipose tissue includes treatment with a surfactant such as deoxycholic acid, polyethylene glycol, or other surfactants at a concentration and time sufficient to destroy the cells and / or remove the cellular components.

[0031] After cell removal, additional processing and / or washing steps can be incorporated, depending on the tissue to be used or the desired final structure, as shown in step 160. For example, additional washing or processing can be performed to remove antigenic substances, such as antigenic components, including alpha-1,3-galactose moieties that may be present in non-primate animal tissues. Furthermore, the material may be treated with additional solutions or reagents during, before, and / or after the washing steps. For example, enzymes, surfactants, and / or other agents can be used in one or more steps to further remove cellular material or lipids, remove antigenic substances, and / or reduce bacteria or other bioburden in the material. For example, one or more washing steps may include the use of surfactants such as sodium dodecyl sulfate or Triton to assist in the removal of cells and lipids. Furthermore, enzymes, such as lipases, DNAses, RNAses, alpha-galactosidases, or other enzymes, can be used to reliably destroy nuclear material, antigens from heterologous sources, residual cellular components, and / or viruses. Furthermore, acidic solutions and / or peroxides can be used to further remove cellular material and help destroy bacteria and / or viruses, or other potentially infectious pathogens.

[0032] The material can be formed into a porous or spongy material after the removal of lipid and cellular components. As shown in step 170, the extracellular matrix is ​​usually first resuspended in an aqueous solvent to form a slurry-like material of AAM (cell-free lipid matrix). By using a sufficient amount of solvent, the material can be formed into a liquid substance that can be poured into a mold having the size and shape of the desired tissue product. The amount of water or solvent added may vary based on the desired porosity of the final material. In some cases, the slurry-like material can have a solid content concentration of about 2% to about 10% by weight, preferably about 2% to about 5% by weight. Weight should be understood as the dry weight of AAM or TE (if added). For example, a 3% AAM slurry contains 3g of AAM in 100mL of liquid. After freeze-drying, the weight of the final material will be 3g. Alternatively, if the slurry has 1% TE and 3% AAM, then 1g of TE and 3g of AAM will be contained per 100mL of slurry. In some cases, the resuspended extracellular matrix may be further mechanically processed one or more times by grinding, cutting, mixing or other processes, and the processed material may be centrifuged and resuspended one or more times to further remove cellular material or lipids (if necessary) and / or control the viscosity of the extracellular matrix.

[0033] In some cases, tropoelastin can be added to the material before sponge formation, as shown in step 180. Various suitable tropoelastins can be selected. For example, tropoelastin can be provided in a form suitable for mixing into AAM slurry. For example, tropoelastin can be formed into elastic materials, viscoelastic materials, or hydrogels by treating a tropoelastin solution with chemical processes and / or heat. After processing to form elastic materials, viscoelastic materials, or hydrogels, tropoelastin can be mixed with a pre-prepared cell-free adipose matrix by cutting or other methods.

[0034] An exemplary process for producing tropoelastin will be described further in a separate section.

[0035] Tropoelastin can be added to the AAM slurry within an appropriate concentration range. For example, the amount of tropoelastin can be selected to impart the desired mechanical and / or biological properties. For instance, tropoelastin can be mixed with AAM in weight percentages of 0.1–50%, or 0.5–3%, 1–5%, 0.5–2%, 10–75%, or other appropriate values.

[0036] Tropoelastin can be produced and mixed with the AAM sponge slurry in step 170 in many forms, including, for example, mixing tropoelastin into the AAM slurry while the tropoelastin is in the form of a dry powder, solution, slurry, or hydrogel. For example, a TE hydrogel can be incorporated into the AAM slurry to a desired weight. Exemplary TE hydrogels can be produced by crosslinking human TE using EDC, BS3, or other crosslinking agents. Alternatively, TE can be dried by freeze-drying, pulverized to produce a dry powder, and this powder can be incorporated into the AAM slurry.

[0037] The amount of TE can be varied based on many factors, including desired mechanical or biological properties. For example, TE can be incorporated into a slurry to form a slurry containing 0.1 to 10% by weight of AAM, with 0.1 to 10% AAM in the slurry. In some cases, TE and AAM may be included in a desired ratio (by weight) of TE:AAM in the slurry, such as 1:3, 1:4, or 1:5, or in other preferred ranges.

[0038] After suspending the material by mixing tropoelastin with AAM, the material is placed in a container or mold as shown in step 190 to form a porous, spongy product. Typically, porous or spongy materials are formed by drying the material to leave a three-dimensional matrix of porous structure. In some embodiments, the material is freeze-dried. Freeze-drying allows for the production of a three-dimensional structure that roughly conforms to the shape of the mold, as shown in Figure 3. Specific freeze-drying protocols can be modified to optimize the solvent used, sample size, and / or processing time. One suitable freeze-drying process involves cooling the material; holding the sample at the cooled temperature and further cooling the sample to freeze it completely; applying a vacuum; and raising the temperature in one or more steps. The freeze-dried sample can then be removed from the freeze-dryer and packaged in a foil pouch under nitrogen.

[0039] After the formation of a solid or sponge, the material can optionally be stabilized as shown in step 195. In some cases, stabilization may include additional processes such as crosslinking, treatment in a dehydrothermal (DHT) process, or other suitable stabilization methods. For example, mechanically processed tissues, when formed into a porous matrix, generally form a more putty- or paste-like material when embedded in the body, wetted, or placed in a solution. This can lead to a loss of desired shape and size. Furthermore, the porous structure, which is important for supporting cell adhesion, tissue growth, blood vessel formation, and tissue regeneration, may be lost. For this reason, the material may be further processed to stabilize its size, shape, and structure.

[0040] In some embodiments, the TE-AAM material is crosslinked for stabilization. An exemplary crosslinking process may include contacting the freeze-dried material prepared as described above with glutaraldehyde or EDC.

[0041] In some embodiments, the material is crosslinked after freeze-drying. However, the material can also be crosslinked before or during the freeze-drying process. Crosslinking can be carried out in various ways. In one embodiment, the material can be crosslinked by contacting it with a crosslinking agent such as glutaraldehyde, genine, carbodiimide (e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)), and diisocyanate.

[0042] Furthermore, crosslinking may be performed by heating the material in a vacuum (DHT). For example, in some embodiments, the material may be heated under reduced pressure or vacuum to 70°C to 120°C, or 80°C to 110°C, or to about 100°C, or any value within a specific range. Chemical crosslinking may also be performed following the DHT treatment (e.g., EDC or other chemical crosslinking following DHT) to obtain the desired mechanical properties. Furthermore, other crosslinking processes, or combinations of processes, including ultraviolet irradiation, gamma irradiation and / or electron beam irradiation, may be used to produce any of the disclosed products. Furthermore, although vacuum is not essential, it can shorten the crosslinking time. Furthermore, lower or higher temperatures can be used as long as the matrix protein does not melt and / or sufficient time is provided for crosslinking.

[0043] In various embodiments, the crosslinking process can be controlled to produce tissue products with desired mechanical, biological, and / or structural characteristics. For example, crosslinking can affect the overall strength of the material, and the process can be controlled to produce the desired strength. Furthermore, the amount of crosslinking can affect the product's ability to maintain a desired shape and structure (e.g., porosity) upon implantation. For this reason, the amount of crosslinking can be selected to produce a stable three-dimensional shape when implanted in the body, when in contact with an aqueous environment, and / or when compressed (e.g., by surrounding tissue or material).

[0044] 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 the tissue product is placed in adipose tissue sites or other anatomical locations. Furthermore, excessive crosslinking can make the material brittle or weak. For this reason, the amount of crosslinking is controlled to provide the desired level of stability while maintaining the desired biological, mechanical, and / or structural characteristics.

[0045] In some cases, instead of providing the disclosed tissue product as a sponge, the product may be formed into a particulate or fluid injectable material. Such compositions may be used for applications such as bulking (for example, to smooth wrinkles, to increase the tissue size of lips or other structures, or to improve the shape or features of other anatomical structures).

[0046] Injectable compositions can be formed in many suitable ways. In particular, tropoelastin components can be mixed with adipose tissue matrix products to produce injectable compositions in many ways. For example, in some cases, an adipose tissue matrix sponge containing tropoelastin is produced up to step 195 as shown in Figure 1. After stabilization, the sponge can be micronized by mechanical means such as cutting or grinding, and the microparticles can be sorted or size-selected as needed to obtain a desired size distribution range.

[0047] Alternatively, in some cases, AAM can be formed into a sponge and then atomized into fine particles. The particulate AAM can then be mixed with particulate tropoelastin, and the composition can be used as an injectable agent. The tropoelastin can be added in an appropriate proportion (e.g., 10-75%).

[0048] Devices manufactured using the method described above can have a variety of configurations. For example, Figure 2 is a side view of a biological breast implant 30 formed of tropoelastin and adipose tissue matrix. This implant can include a variety of appropriate breast implant shapes, contours, or protrusions. Furthermore, it should be understood that a variety of shapes can be used, including rounded shapes, irregular shapes, concentric elliptical shapes, or concentric irregular 3D shapes, or custom-molded implants. For example, Figures 3A–3C show exemplary shapes of implants manufactured using the method disclosed, including a teardrop-shaped implant 36 (Figure 3A), an irregular implant 37 (Figure 3B), and / or a spherical implant 38 (Figure 3C).

[0049] Devices 30, 36-38 can have various sizes. However, as stated above, the method provided herein can offer advantages by enabling the manufacture of fat implants having larger sizes that can be matched with conventional breast implants or tissue expanders. For example, using the method described herein, implants with at least one dimension of 5 cm or more can be manufactured. In other cases, the devices may have dimensions of at least 6 cm, at least 7 cm, at least 8 cm, at least 10 cm or more.

[0050] This specification also discloses a method for treating the breast by transplanting a tissue product. Figure 4 shows the transplantation of a system for surgical breast surgery. This method may first include the step of identifying an anatomical site within the breast 60. (As used herein, “intra-breast” is understood to mean within the mammary gland tissue, or within or near the tissue surrounding the breast, such as tissue immediately below, laterally or internally to the breast, or below surrounding tissue, such as below the chest muscles (pectoralis muscles), and also includes transplantation to a site where part or all of the breast has already been surgically removed). The site may include, for example, any suitable site that requires reconstruction, repair, augmentation or treatment. Such sites may include sites where surgical tumor removal (mastectomy, mammary gland tumor removal) has been performed, sites where cosmetic surgery (breast augmentation or revision breast augmentation) has been performed, or sites that require treatment due to disease or trauma.

[0051] Furthermore, this specification provides a therapeutic method comprising the steps of selecting a tissue site and implanting a tissue product disclosed herein into the tissue site. This method may include the steps of implanting a therapeutic device into or near a wound or surgical site and fixing at least a portion of the therapeutic device to tissue in or near the treatment site. The tissue product is implanted behind the tissue site, thereby reinforcing, repositioning, or projecting outward the natural tissue.

[0052] Furthermore, this specification also provides a therapeutic method comprising the steps of selecting a tissue site within the breast, implanting a device into the tissue site, and growing the tissue within a cell-free adipose tissue matrix. In one embodiment, the device comprises a synthetic breast implant or tissue expander and a cell-free adipose tissue matrix surrounding the breast implant or tissue expander. The method may further include the steps of removing the breast implant or tissue expander and implanting additional cell-free adipose tissue matrix into the space created by the removal of the breast implant or tissue expander.

[0053] The tissue products described herein can be used to treat a variety of different anatomical sites. For example, as described herein, the tissue products of this disclosure are produced from adipose tissue matrix and can be used to treat the breast. In some cases, the tissue product may be implanted in other sites, including tissue sites that are primarily or substantially composed of adipose tissue. In some cases, the tissue site may include the breast (for example, for breast augmentation, replacement of excised tissue, or placement around an implant). Furthermore, any other site containing adipose tissue can be selected. For example, the tissue product can be used for reconstructive or cosmetic purposes in the breast, face, buttocks, abdomen, waist, thigh, or any other site where additional adipose tissue with a structure and feel similar to the patient's own fat is desired. In any of those sites, the tissue can be used to reduce or eliminate wrinkles, sagging, or undesirable shapes.

[0054] The process of the present invention and the elastic materials formed thereby are particularly useful in tissue augmentation applications, such as applications where there is a need to cosmetically enhance or improve appearance (e.g., lip plumping, nasolabial fold filling, wrinkle reduction, or other tissue strengthening), or in medical applications where there is a need to support congenital defects or defects resulting from disease or surgical excision.

[0055] In some cases, the product comprises an injectable composition containing particulate AAM and TE in a carrier solution. In certain forms, the material can be configured to allow for desired fluidity or injectability. For example, various carriers can be added in specific proportions to enable fluidity. Suitable carriers include solutions (e.g., PBS), hydrogels, hyaluronic acid or hyaluronic acid derivatives, gelatin, or other biocompatible fluid materials.

[0056] Exemplary methods for producing tropoelastin: As mentioned above, tropoelastin can be provided in many forms, including elastic materials, viscoelastic materials, hydrogels, or other materials. Generally, “elastic materials” are not free-flowing liquids. They can be gels, pastes, solids, or other phases that significantly lack fluid properties. “Elastic materials” generally return to a specific shape or form when a force, such as compression or stretching, applied to them is removed. “Elastic materials” are also called elastically compressible and stretchable, mechanically durable, or flexible materials, with relatively low hysteresis. Such materials may also be referred to as stretchable materials, tensile materials, elastic materials, or resilient materials.

[0057] It will be understood that "tropoelastin" generally refers to peptides that contain, or consist of, sequences identical or similar to the hydrophilic domain of tropoelastin. The hydrophilic domain typically has sequences rich in lysine and alanine residues. These domains often consist of stretches of lysine separated by two or three alanine residues, such as AAAKAAKM (SEQ ID NO: 1). Other hydrophilic domains do not contain a polyalanine tract but instead have lysine near proline. In contrast, the hydrophobic domain of tropoelastin is rich in nonpolar amino acids, particularly glycine, valine, proline, and alanine, and often arises from repeats of 3-6 peptides, such as GVGVP (SEQ ID NO: 2), GGVP (SEQ ID NO: 3), and GVGVAP (SEQ ID NO: 4).

[0058] Examples of tropoelastins that may be used with this AAM include those consisting of a hydrophilic domain or its homolog, and those containing a hydrophilic domain or homolog and some or all of a hydrophobic domain. Several examples are given below.

[0059] GGVPGAIPGGVPGGVFYP,(Sequence ID: 5)

[0060] GVGLPGVYP, (Sequence ID: 6)

[0061] GVPLGYP, (Sequence ID: 7)

[0062] PYTTGKLPYGYGP, (Sequence ID: 8)

[0063] GGVAGAAGKAGYP,(Sequence ID: 9)

[0064] TYGVGAGGFP;(Sequence ID: 10)

[0065] KPLKP, (Sequence ID: 11)

[0066] ADAAAAYKAAKA,(Sequence ID: 12)

[0067] GAGVKPGKV, (Sequence ID: 13)

[0068] GAGVKPGKV, (Sequence ID: 14)

[0069] TGAGVKPKA,(Sequence ID: 15)

[0070] QIKAPKL, (Sequence ID: 16)

[0071] AAAAAAAKAAAK,(Sequence ID: 17)

[0072] AAAAAAAAAAKAAKYGAAAGLV,(Sequence ID: 18)

[0073] EAAAKAAAKAAKYGAR, (Sequence ID: 19)

[0074] EAQAAAAAKAAKYGVGT,(Sequence ID: 20)

[0075] AAAAAKAAAKAAQFGLV,(Sequence ID: 21)

[0076] GGVAAAAKSAAKVAAKAQLRAAAGLGAGI,(Sequence ID: 22)

[0077] GALAAAKAAKYGAAV, (Sequence ID: 23)

[0078] AAAAAAAKAAAKAA,(Sequence number: 24)

[0079] AAAAKAAKYGAA,(Sequence ID: 25)

[0080] CLGKACGRKRK.(Sequence ID: 26)

[0081] "Tropoelastin" may have the same sequence as the entry shown in GenBank entry AAC98394. Other tropoelastin sequences containing a hydrophilic domain are known in the art and include, but are not limited to, CAA33627 (Homo sapiens), P15502 (Homo sapiens), AM42271 (Norway rat), AAA42272 (Norway rat), AAA42268 (Norway rat), AAA42269 (Norway rat), AAA80155 (House mouse), AAA49082 (Red Junglefowl), P04985 (Cattle), ABF82224 (Zebrafish), ABF82222 (Xenopas tropicalis), and P11547 (Sheep).

[0082] "Tropoelastin" may also be fragments of these sequences, provided that the fragment contains at least a portion of the hydrophilic domains described above. An example is amino acids 27-724 of AAC98394. The tropoelastin used in this disclosure may include human tropoelastin or selected domains of human tropoelastin.

[0083] Tropoelastin may include a peptide having the sequence described above, particularly a homolog of AAC98394, or a homolog of a peptide having the sequence described above, or a fragment of a homolog of a peptide having the sequence described above. Here, "homolog" means a protein that has a similar sequence to, but is not identical to, the reference sequence. It also has the same function as the reference sequence, for example, the ability to form an elastic material when the alkalinity, temperature, or salt concentration of a homolog solution is adjusted by manipulating it, as described herein.

[0084] In certain embodiments, the homolog has at least 60% homology to the peptide described above, particularly AAC98394, or a fragment of the peptide described above that includes at least a portion of the hydrophilic domain.

[0085] It should be understood that "tropoelastin" may be natural or recombinant.

[0086] There are subsets of temperature, alkalinity, and salt concentration conditions under which a solution of tropoelastin forms an elastic material. In one embodiment, a process is provided for producing an elastic material from tropoelastin, comprising the step of heating a solution of tropoelastin having an alkaline pH to form an elastic material from the tropoelastin in the solution.

[0087] In another embodiment, a process is provided for producing an elastic material from tropoelastin, comprising the step of providing an alkaline pH to a solution of tropoelastin having a temperature of about 37°C to form an elastic material from the tropoelastin in the solution.

[0088] In another embodiment, a process is provided for producing an elastic material from tropoelastin, comprising the steps of providing an alkaline pH to a solution of tropoelastin and raising the temperature of the solution to about 37°C to form an elastic material from the tropoelastin in the solution.

[0089] In another embodiment, a process is provided for producing an elastic material from tropoelastin, comprising the step of adding tropoelastin to a solution having an alkaline pH and a temperature of about 37°C to form an elastic material from the tropoelastin in the solution.

[0090] In another embodiment, a process for producing an elastic material from tropoelastin is provided, comprising the steps of adding tropoelastin to a solution having an alkaline pH and raising the temperature of the solution to about 37°C to form an elastic material from the tropoelastin in the solution.

[0091] In another embodiment, a process is provided for producing an elastic material from tropoelastin, comprising the step of adjusting the salt concentration of a solution of tropoelastin having an alkaline pH and a temperature of about 37°C to form an elastic material from the tropoelastin in the solution.

[0092] Typically, solutions with a tropoelastin concentration exceeding approximately 1.5 mg / mL can form elastic materials with desirable integrity (although lower concentrations are also useful). In most applications, the solution concentration is less than approximately 300 mg / mL. Therefore, tropoelastin solutions with concentrations between approximately 1.5 mg / mL and approximately 300 mg / mL are preferred. More preferably, tropoelastin solutions with concentrations between approximately 10 mg / mL and approximately 300 mg / mL are used. Most preferably, tropoelastin solutions with concentrations between approximately 10 mg / mL and approximately 200 mg / mL are used.

[0093] It has been found that a pH of approximately 7.5 or higher is sufficient to form an elastic material from tropoelastin in solution. Beyond this, sufficient elastic material formation is not achieved, so the pH is usually maintained below approximately 13. More preferably, a pH of approximately 9 to 13 is desirable. However, most preferably, a pH of approximately 10 to 11 is used. Other usable pH ranges include 8.0, 8.5, 9.5, 10, 10.5, and 11.5.

[0094] Alkalinity can be adjusted by several approaches, including 1) directly adding a pH-increasing agent to the tropoelastin solution, and 2) mixing a solution containing a sufficient amount of a pH-increasing agent with the tropoelastin solution to make it alkaline. pH-increasing agents can be bases, buffers, or proton adsorbents. Examples including Tris bases, NH4OH, and NaOH have been found to be useful as pH-increasing or controlling agents.

[0095] When the pH is alkaline and less than about 9.5, salt may be required to form the elastic tropoelastin material useful for forming the sponge of the present invention. When salt is used, its concentration is usually above 25 mM and may reach up to 200 mM. Preferably, the salt concentration is about 100 mM to 150 mM. More preferably, the salt concentration is about 150 mM. In particular, the inventors have found that when the pH drops below 10 (while remaining alkaline), salt is required to induce the formation of the elastic material, and the amount of salt required increases as the pH decreases. For example, at a pH of about 9 to 10, salt is required, and it is necessary to provide the solution with a salt concentration equivalent to, for example, about 60 mM. In some embodiments, the solution needs to have an osmotic pressure equivalent to or less than that of mammalian isotonic saline (150 mM). In other embodiments, the solution needs to have an osmotic pressure greater than 150 mM. The salt concentration may be 0 mM.

[0096] The salt concentration of a solution can be controlled by adding a salt containing any ionic compound, monovalent or divalent ion, or low molecular weight species that can affect the osmotic pressure of the solution. For example, NaCl, KCl, MgSO4, Na2CO3, or glucose can be used. NaCl is a preferred salt.

[0097] A method for forming an elastic material from a tropoelastin solution is: (1) A step of providing a solution of tropoelastin, (2) The step of adjusting the pH of the solution to form an alkaline solution and precipitating the tropoelastin in the solution, (3) A step of removing the precipitate, (4) Adding the removed precipitate to a solution having a substantially non-alkaline pH and / or a substantially low temperature to disperse the precipitate in the solution, (5) The step of raising the temperature of the solution to approximately 37°C to form an elastic material from the tropoelastin in the solution. It can include...

[0098] In one embodiment, the temperature of the solution is preferably about 4°C to about 37°C in step (2) and less than about 4°C in step (4). Furthermore, in one embodiment, the pH of the solution is preferably at least about pH 9 in step (2) and less than about pH 9 in step (4). The pH may be as low as about pH 7.5 in step (4). Furthermore, in one embodiment, the salt concentration of the solution is preferably about 0 mM to 200 mM.

[0099] In other embodiments, a process for producing an elastic material from tropoelastin includes the step of heating a solution of tropoelastin having an alkaline pH of less than 10 and a salt concentration of 150 mM or less to form an elastic material from the tropoelastin in the solution. These embodiments are particularly preferred for in vivo applications because the pH of the solution of tropoelastin and elastic material is close to the pH of mammals.

[0100] In further embodiments, a process for producing an elastic material from tropoelastin includes the steps of adjusting the salt concentration of a tropoelastin solution having an alkaline pH, and raising the temperature of the solution to about 37°C to form an elastic material from the tropoelastin in the solution.

[0101] It has been found that a temperature of approximately 37°C is preferable for forming elastic materials from tropoelastin in solution. However, in certain embodiments, temperatures below 37°C may be used. Typically, this temperature is 4°C higher. It is usually below 42°C.

[0102] A solution can be heated by providing it in or on mammalian tissue and raising the temperature of the solution by heat transfer from the tissue, or by irradiating the tissue with radiation.

[0103] Alternatively, the solution can be heated by bringing it into contact with a non-living surface and heating that surface. The non-living surface may be provided to a mold or casting device for providing the elastic material formed by this method in a predetermined shape or form.

[0104] If heating of the solution is provided to induce the formation of an elastic material (for example, if appropriate pH and / or salt conditions are provided), the solution is typically stored at a temperature of less than 30°C, preferably about 4°C, until it is required to form the elastic material used in the method for forming the sponge of the present invention.

[0105] In certain embodiments, the elastic material formed from a tropoelastin solution by the process described above can be crosslinked with an agent capable of crosslinking the side chains of tropoelastin residues (e.g., lysine residues). In certain applications described later, crosslinking the side chains during the formation of the elastic material is useful because it can impart further properties to the elastic material. Specifically, compared to elastic materials formed in the absence of a crosslinking agent, using a crosslinking agent such as glutaraldehyde can yield an elastic material that is harder, denser, stronger, and therefore considered to have higher biostability in vivo. Crosslinked materials may be preferable to uncrosslinked elastic materials for more demanding tissue repair applications or when compatibility with surrounding natural tissues is not essential.

[0106] Any crosslinking agent that can be used to form elastin, whether natural or artificial, is considered usable. Examples include lysyl oxidase, transglutaminase, carbodiimides (e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), glutaraldehyde, genepine, and bis(sulfosuccinimidyl)sverate (BS3) and other amine-reactive crosslinking agents. In one embodiment, the crosslinking agent is glutaraldehyde, used at a concentration of about 0.001 w / v% to about 0.5 w / v%, or BS3 is used at 1 mM to 100 mM.

[0107] In some cases, tropoelastin (also referred to herein as TE) can be crosslinked by heating, for example, by heating at 120-180°C for 10-24 hours, which may enable suitable crosslinking.

[0108] Example: Effect of crosslinking on lipogenesis 3D cell-free adipose matrix (AAM) sponges can reduce serumomas, hematomas, and scar formation, while also promoting lipogenesis. The mechanical properties of the sponge must be sufficient to withstand compressive forces within the body. To improve the mechanical strength and elasticity of 3D AAM sponges, the sponges were modified by chemical crosslinking (e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; EDC). However, there is often a trade-off between the mechanical strength achieved by crosslinking and the biological response. Therefore, the biological response to crosslinked sponges was evaluated using a subcutaneous nude rat model.

[0109] Porcine cell-free adipose matrix (pAAM) slurry was prepared, lyophilized, and crosslinked with DHT at 80°C for 24 hours. Several samples were also crosslinked with 0.016% or 0.125% EDC. N-hydroxysuccinimide (NHS) was added in an EDC:NHS ratio of 5:3. The sponges were then sterilized to the final stage using an electron beam. Sponges approximately 5 mm thick were cut with a 10 mm biopsy punch and transplanted subcutaneously into nude rats (n=5). After 4 weeks, the explants were cut in half, one half fixed in 10% formalin for Masson's trichrome staining, and the other half fixed in sucrose.

[0110] By week 4, uncrosslinked sponges showed cell endoproliferation, angiogenesis, and adipogenesis (Figures 5A and 5B). In contrast, sponges crosslinked with 0.125% EDC showed no adipocytes on oil red O staining (Figures 5E and 5F). Sponges with an intermediate crosslinking level (0.016%) showed intermediate levels of adipocytes, similar to those seen in the 0.125% and uncrosslinked sponges (Figures 5C and 5D). However, trichrome staining revealed widespread cell endoproliferation and angiogenesis in all types of sponges (Figures 5A, 5C, 5E, and 5G). This suggests that adipogenesis is merely delayed by EDC crosslinking, not completely prevented.

[0111] Overall, increasing EDC crosslinking simultaneously reduced adipogenesis, as evidenced by trichrome staining and oil red O staining. All three types of sponges promoted cell endoplasty and angiogenesis, regardless of the crosslinking conditions.

[0112] Example: Effect of treatment on mechanical properties AAM sponges typically need to possess mechanical properties that adequately withstand the compressive forces within the body. To improve the mechanical strength and elasticity of 3D AAM sponges, the sponges were modified by (1) changing the solid content of the AAM, or (2) chemical crosslinking (e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; EDC).

[0113] AAM slurry was prepared in 20% PBS with a solid content of 3% or 4%. The slurry was then lyophilized to form a sponge, followed by DHT crosslinking at 80°C for 24 hours. When sponges were formed from 3% or 4% solid slurry and crosslinked with EDC, they were incubated at room temperature for 4 hours in either 0.03% or 0.1% EDC in MES buffer. N-hydroxysuccinimide (NHS) was then added to the buffer in an EDC:NHS = 5:3 ratio. After crosslinking, the sponges were washed twice with PBS. The solid content and EDC amounts of the samples were as follows: TIFF2026076164000002.tif49170

[0114] Compression tests were performed on sponges hydrated with PBS to evaluate the compressive strength at 50% strain, the shape recovery rate after compression, and the modulus of elasticity. Here, the modulus of elasticity is defined as the slope of the linear region of the force-displacement curve. To evaluate elasticity, tensile tests were performed on sponge strips that had been hydrated with PBS and then gently squeezed to remove excess liquid.

[0115] As the proportion of EDC increased, a linear trend was observed overall in compressive strength, elasticity, and modulus of elasticity (Figures 6A, 6C, and 6D). For each EDC crosslinking condition, the 4% AAM sponge was stronger than the 3% control.

[0116] A 4% AAM sponge using 0.1% EDC (Sample 6) showed the highest strength for these parameters. Figure 6B shows that both the 0.03% and 0.1% EDC crosslinking conditions similarly improved the shape recovery rate by an average of 7.2% compared to the non-crosslinked version.

[0117] Increasing the solid content from 3% to 4% increased the mechanical strength of the sponge.

[0118] Crosslinking with EDC further increased the mechanical strength of the sponge, resulting in a stronger sponge than when the EDC concentration was low (0.03%) compared to when it was high (0.1%).

[0119] Examples: Effects of treatment on lipogenesis and mechanical properties In a different sponge composition, 10 mg / ml of tropoelastin in PBS was crosslinked with 10 mM bis(sulfosuccinimidyl)sverate (BS3) at 37°C for 18 hours to form a hydrogel. The tropoelastin hydrogel was then washed with PBS, cleaved, and incorporated into an AAM slurry to a final concentration of 1% w / v (1% of a slurry with 3% AAM content). Alternatively, the tropoelastin hydrogel was lyophilized, cleaved, and freeze-ground to form a powder, which was then incorporated into an AAM slurry to a final concentration of 1%. Subsequently, the tropoelastin:AAM sponge was lyophilized and crosslinked (with DHT or EDC) as described above.

[0120] Compression tests were performed on PBS-hydrated sponges to evaluate compressive strength at 50% strain, shape recovery rate after compression, and elastic modulus. The biological response of tropoelastin / AAM sponges was also tested in vivo. The sponges were sterilized using an electron beam at 15–25 kGy. Sponges approximately 5 mm thick were cut with a 10 mm biopsy punch, washed with saline, and then transplanted subcutaneously into nude rats (n=5). At weeks 8 and 16, the explants were fixed with 10% formalin and stained with Masson's trichrome.

[0121] As the percentage of EDC increased, there was an overall linear trend in compressive strength and modulus (Figures 7A and 7C). The addition of tropoelastin hydrogel increased compressive strength by approximately twofold compared to the AAM control, while tropoelastin powder was intermediate. The 3% AAM sponge and tropoelastin hydrogel with 0.1% EDC showed the best strength for their respective parameters. Figure 7B shows that both the 0.03% and 0.1% EDC crosslinking conditions similarly improved shape recovery by approximately 10% on average compared to the uncrosslinked version.

[0122] Evaluation of Masson's trichrome-stained explants revealed evidence of extensive cell regrowth and angiogenesis in nude rats at both 8 and 16 weeks (Figures 8A–8D (8 weeks) and 10A–10D (16 weeks)). Inflammatory cell infiltration was higher in the crosslinked group compared to the uncrosslinked control at both time points. 0.03% EDC crosslinking significantly reduced the mean adipose tissue percentage at both time points compared to the uncrosslinked control (ANOVA; Tukey post hoc; p<0.05). Figure 9 shows that the addition of tropoelastin hydrogel significantly increased the mean adipose tissue percentage at 8 weeks compared to all groups (74±8.9; ANOVA; Tukey post hoc; p<0.05). Similarly, when tropoelastin was added to AAM sponges crosslinked with 0.03% EDC, the average percentage of adipose tissue increased significantly compared to the crosslinked group, but it was still lower than that of the uncrosslinked counterpart (ANOVA; Tukey post hoc; p<0.05).

[0123] By week 16, the overall percentage of adipose tissue increased in all groups. However, adipocyte coverage for the 3% pAAM crosslinked group remained below 10%, indicating that the adipogenic capacity of pAAM is impaired by crosslinking at least 0.03% EDC concentration. Addition of tropoelastin significantly improved the percentage of fat at week 16 compared to the corresponding uncrosslinked 3% pAAM sample (ANOVA; Tukey post hoc; p<0.05) (Figure 11). The tropoelastin hydrogel remained within the sponge, with some persisting for 16 weeks (Figures 12A and 12B). Crosslinked pAAM containing tropoelastin did not show a significant difference compared to the other crosslinked groups at week 16 (ANOVA; Tukey post hoc; p<0.05).

[0124] Tropoelastin hydrogel improved both the mechanical and biological properties of AAM sponges. Tropoelastin powder showed intermediate improvements in compressive strength and modulus of elasticity. EDC crosslinking improved the mechanical strength of the sponges, with higher EDC concentrations (0.1%) resulting in stronger sponges than lower EDC concentrations (0.03%). EDC crosslinking at 0.03% significantly reduced the internal growth of adipose tissue. Incorporation of TE hydrogel significantly increased the percentage of adipose tissue in explants at 8 weeks by 1.6 times and 4 times, respectively, in uncrosslinked and 3% AAM sponges crosslinked with 0.03% EDC (p<0.05).

Claims

1. A method for manufacturing tissue products, The step of selecting adipose tissue, The steps include processing the tissue to remove virtually all cellular material from it, The steps include suspending the tissue in a liquid to form a suspension containing 2-4% by weight of solids, A method characterized by comprising the steps of freezing and drying a suspension to form a porous sponge.

2. In the method according to claim 1, A method characterized by further comprising the step of crosslinking a porous sponge.

3. In the method of claim 2, A method characterized in that crosslinking is performed using a dehydrothermal process.

4. In the method according to claim 3, A method characterized by further comprising a step of performing a chemical crosslinking process.

5. In the method according to claim 1, A method characterized in that a porous sponge has a desired thickness at least in the thickest part of the sponge, and that thickness exceeds 10.0 cm.

6. In the method according to claim 1, A method further comprising the step of adding a suspension to a mold.

7. In the method according to claim 6, A method characterized in that the mold is in the shape of a rounded breast implant or a teardrop-shaped breast implant.

8. In the method according to claim 4, A method characterized in that the chemical crosslinking step includes at least one of glutaraldehyde, genepine, carbodiimide, and diisocyanate.

9. In the method according to claim 4, A method characterized by crosslinking, which includes heating a porous sponge.

10. In the method according to claim 9, A method characterized by heating a porous sponge in a vacuum.

11. In the method according to claim 10, A method characterized by heating a porous sponge to a temperature range of 70°C to 120°C.

12. In the method according to claim 4, A method characterized in that a porous sponge is crosslinked so that the material maintains a stable three-dimensional structure when it comes into contact with an aqueous environment.

13. In the method according to claim 12, A method characterized in that the aquatic environment is the body of a mammal.

14. It is an organizational product, A tissue product comprising a breast implant, wherein the implant comprises a construct of a cell-free adipose tissue matrix that is homogenized, dried, and stabilized to form a suspension, the implant having at least one dimension of at least 5 cm, and comprising tropoelastin.

15. In the tissue product according to claim 14, The implant is a tissue product characterized by having at least one dimension of at least 8 cm.

16. In the tissue product according to claim 14, A tissue product characterized by the rounded shape of the breast implant.

17. In the tissue product according to claim 14, A tissue product characterized by the implant having a teardrop shape as a breast implant.

18. A method for manufacturing tissue products, The step of selecting adipose tissue, The steps include processing the tissue to remove virtually all cellular material from it, The steps include suspending the tissue in a liquid to form a suspension, The step of adding tropoelastin to the suspension, A method characterized by comprising the steps of freezing and drying a suspension to form a porous sponge.

19. In the method according to claim 18, A method characterized by further comprising the step of crosslinking a porous sponge.

20. In the method according to claim 19, A method characterized in that crosslinking is performed using a dehydrothermal process.

21. In the method of claim 20, A method characterized by further comprising a step of performing a chemical crosslinking process.

22. In the method according to claim 18, A method characterized in that a porous sponge has a desired thickness at least in the thickest part of the sponge, and that thickness exceeds 10.0 cm.

23. In the method according to claim 18, A method further comprising the step of adding a suspension to a mold.

24. In the method according to claim 23, A method characterized in that the mold is in the shape of a rounded breast implant or a teardrop-shaped breast implant.

25. In the method according to claim 21, A method characterized in that the chemical crosslinking step includes at least one of glutaraldehyde, genepine, carbodiimide, and diisocyanate.

26. In the method according to claim 19, A method characterized by crosslinking, which includes heating a porous sponge.

27. In the method according to claim 26, A method characterized by heating a porous sponge in a vacuum.

28. In the method according to claim 26, A method characterized by heating a porous sponge to a temperature range of 70°C to 120°C.

29. In the method according to claim 19, A method characterized in that a porous sponge is crosslinked so that the material maintains a stable three-dimensional structure when it comes into contact with an aqueous environment.

30. In the method of claim 29, A method characterized in that the aquatic environment is the body of a mammal.

31. In the method according to any one of claims 18 to 30, A method further comprising the step of forming fine particles from a sponge.

32. It is an organizational product, The first component contains cell-free adipose matrix (AAM), A tissue product characterized by containing a second component containing tropoelastin (TE).

33. In the tissue product according to claim 32, A structured product characterized by having a sponge-like form.

34. In the tissue product according to claim 33, A structured product characterized by a sponge AAM to TE ratio of approximately 3:1 to approximately 1:

3.

35. In the tissue product according to any one of claims 32 to 34, A textured product characterized by its particulate nature.

36. In the tissue product according to any one of claims 32 to 35, A tissue product characterized by the fact that TE is human TE.

37. In the tissue product according to any one of claims 32 to 36, A tissue product characterized by AAM being derived from porcine fat.

38. In the tissue product according to any one of claims 32 to 37, A tissue product characterized by being at least partially cross-linked.

39. In the tissue product according to claim 38, A tissue product characterized by crosslinking using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC).

40. It is an organizational product, The step of selecting adipose tissue, The steps include processing the tissue to remove virtually all cellular material from it, The steps include suspending the tissue in a liquid to form a suspension, The step of adding tropoelastin to the suspension, A tissue product characterized by being manufactured by a process comprising the steps of freezing and drying a suspension to form a porous sponge.

41. In the tissue product according to claim 40, A tissue product characterized by further comprising the step of crosslinking porous sponges.

42. In the tissue product according to claim 41, A tissue product characterized by crosslinking being performed using a dehydrothermal process.

43. In the tissue product according to claim 42, A tissue product characterized by further including a chemical crosslinking process.

44. In the tissue product according to claim 43, A tissue product characterized in that the chemical crosslinking step includes at least one of glutaraldehyde, genepine, carbodiimide, and diisocyanate.

45. In the tissue product according to claim 41, A tissue product characterized by crosslinking, which includes heating a porous sponge.

46. In the tissue product according to claim 41, A tissue product characterized by the heating of a porous sponge in a vacuum.

47. In the tissue product according to claim 46, A structured product characterized by including heating of a porous sponge in the range of 70°C to 120°C.

48. In the tissue product according to claim 19, A structured product characterized by a porous sponge that is cross-linked so that the material maintains a stable three-dimensional structure when in contact with an aqueous environment.

49. In the tissue product according to any one of claims 18 to 30, A tissue product characterized by further comprising the step of forming fine particles from a sponge.

50. It is a treatment method, A method characterized by comprising the step of transplanting a tissue product containing a cell-free adipose tissue matrix and tropoelastin into or on an anatomical site.

51. In the method according to claim 50, A method characterized in that the product is implanted in or around the breast.

52. In the method according to claim 50, A method characterized in that the anatomical area is a cavity left behind by surgical procedures.

53. In the method according to claim 50, A method characterized in that the anatomical site is at least one of the hand, waist, buttocks, or facial structure.

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