Adipose tissue products and manufacturing methods

Abstract

The present invention provides a method for producing compositions for injection, small-volume transplantation, or filling or adding volume to larger voids using adipose tissue products. [Solution] A method for manufacturing a tissue product, comprising 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 the tissue; suspending the tissue in a solution to form a suspension; processing the suspension to generate a stabilized three-dimensional structure having a microporous structure; and mechanically processing the stabilized three-dimensional structure to generate particles.

Description

Technical Field

[0001] The present disclosure relates to tissue matrices, and more particularly, to injection materials for treating or regenerating adipose tissue.

[0002] The present disclosure claims priority to U.S. Provisional Application No. 62 / 573,892, filed on October 18, 2017, under 35 U.S.C. § 119, the entire disclosure of which is hereby incorporated by reference.

Background Art

[0003] Currently, there is a need for improvement in injection materials for tissue treatment. For example, injection materials such as hyaluronic acid-based materials may be used to treat various facial features (e.g., lines, wrinkles, insufficient size, or parts that are not of a desired shape or form). Such materials, however effective they may be, provide only temporary improvement and ultimately are absorbed by the body and cannot induce the regeneration of tissues such as adipose tissue. Furthermore, research has been conducted to develop adipose-based materials for tissue treatment and regeneration, but current materials are not suitable for small-scale procedures or injections or have not been proven to be very effective in adipose regeneration.

Summary of the Invention

[0004] Thus, the present disclosure provides compositions for injection, small grafting, or filling of larger voids or volume augmentation with adipose-based tissue products. The present disclosure also provides methods for manufacturing such compositions.

[0005] This disclosure provides a method for producing an injectable product from an adipose tissue matrix. The 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 solution to form a suspension; processing the suspension to generate a stabilized three-dimensional structure having a microporous structure; and mechanically processing the stabilized three-dimensional structure to generate particles.

[0006] This disclosure also provides tissue product compositions. These compositions may include a particulate tissue matrix, wherein the tissue product composition includes adipose-free tissue matrix formed into particulate matter after being formed into a porous sponge, and the particulate tissue matrix includes particles having a maximum dimension of about 0.05 mm to 3 mm.

[0007] This disclosure also provides a method of treatment using the disclosed product.

[0008] It should be understood that both the general description above and the detailed description below are merely illustrative and descriptive, and do not limit the invention as defined in the claims. [Brief explanation of the drawing]

[0009] The accompanying drawings are incorporated herein and constitute part thereof, and are useful in illustrating exemplary embodiments of the present disclosure and in illustrating the principles of the present disclosure together with the description thereof. [Figure 1] Figure 1 is a flowchart outlining the process for generating an adipose tissue matrix sponge according to an embodiment of the present disclosure. [Figure 2] Figure 2 is a flowchart outlining the process for manufacturing an injectable adipose tissue matrix product according to an embodiment of the present disclosure. [Figure 3] Figure 3 shows bulk pieces of adipose tissue matrix sponge manufactured according to various embodiments. [Figure 4] Figure 4 is a magnified view of particulate tissue matrix with dimensions of 2-3 mm after being produced by crushing adipose tissue matrix sponge. [Figure 5] Figure 5 shows a group of cell-free tissue matrix particles produced by crushing or shredding an adipose tissue matrix sponge to generate particles of 100-300 microns, and the paste / pudding-like injection material after hydration. [Figure 6] Figure 6 shows scanning electron microscope (SEM) and atomic force microscope images of adipose tissue matrix particles produced according to the process of the embodiments of the disclosure. [Figure 7] Figure 7A shows differential scanning calorimetry curves of adipose tissue matrix material produced according to the embodiments of the disclosure. Figure 7B shows Masson trichrome stained sections of adipose tissue matrix material produced according to the embodiments of the disclosure. Figure 7C shows collagenase digestion curves of adipose tissue produced according to the embodiments of the disclosure, with and without electron beam sterilization. [Figure 8] Figure 8A shows hematoxylin and eosin ("H&E") stained sections of adipose tissue matrix material produced according to the examples of disclosure. Figure 8B is a table of DNA and lipid content measurements of adipose tissue produced according to the examples of disclosure. Figure 8C shows immunostained portions of adipose tissue matrix material produced according to the examples of disclosure, which are ineffective against MHC-1&II staining compared to a natural adipose control. [Figure 9] Figure 9 provides a histological image of an adipose tissue matrix produced according to the embodiments of the disclosure, compared to a natural adipose tissue control, and subject to immunohistochemical analysis of major extracellular matrix proteins (e.g., type I, type III, and type IV) collagen. [Figure 10] Figure 10 provides optical microscope images of adipose tissue matrix generated according to the embodiments of the disclosure, supporting the growth of three different cell types (e.g., adipogenic mesenchymal stem cells, endothelial cells, and dermal fibroblasts) in vitro. [Figure 11] Figure 11 provides a whole photograph of an injectable adipose tissue matrix explant after subcutaneous transplantation of nude rats, prepared according to the embodiments of the disclosure. [Figure 12] Figure 12 shows Masson's trichrome staining of the explant shown in Figure 11. [Figure 13] Figure 13 is a bar graph showing the volume of the explant remaining after 4 or 8 weeks of subcutaneous transplantation in rats, as described in the embodiments of the disclosure. [Figure 14] Figure 14A is a Masson trichrome stained section of an 8-week explant described in the examples of the disclosure. Figure 14B is another Masson trichrome stained section of an 8-week explant described in the examples of the disclosure. Figure 14C is another Masson trichrome stained section of an 8-week explant described in the examples of the disclosure. [Figure 15] Figure 15 provides a microscopic image of aggregates formed in a moist adipose tissue matrix product after 1.5 years of storage. [Figure 16] Figure 16 is a graph showing the infusion strength of the lipid matrix product with and without HA as a fluid carrier. [Figure 17] Figure 17 shows H&E stained sections of 4-week HA fat exgrafts described in the examples. [Modes for carrying out the invention]

[0010] 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.

[0011] 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.

[0012] 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.

[0013] As used herein, “tissue product” refers to any human or animal tissue containing extracellular matrix proteins. “Tissue product” includes cell-free or partially decellularized tissue matrix, as well as decellularized tissue matrix rearranged with exogenous cells.

[0014] As used herein, the term "acellular tissue matrix" refers to an extracellular matrix derived from human or animal tissue that retains a substantial amount of natural collagen and glycoproteins necessary to serve as a scaffold to support tissue regeneration. "Acellular 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 microstructure properties of the tissue matrix during the purification process. Although referred to as "acellular tissue matrix," it should be understood that such tissue matrix may be combined with exogenous cells, including, for example, stem cells and cells from the patient into whom the "acellular tissue matrix" is implanted. It should be understood that "decellularized adipose tissue matrix" refers to adipose tissue from which all cells have been removed to produce an adipose extracellular matrix. The "decellularized adipose tissue matrix" may include intact matrix or matrix further processed as discussed herein, including mechanical processing, sponge formation, and / or further processing to produce a particulate matrix.

[0015] It should be understood that an "acellular" or "decellularized" tissue matrix refers to a tissue matrix in which cells cannot be seen even using an optical microscope.

[0016] Various human and animal tissues can be used to manufacture products for treating patients. For example, various tissue products have been manufactured for the regeneration, repair, augmentation, reinforcement, and / or treatment of human tissues damaged or lost due to various diseases and / or structural injuries (e.g., due to trauma, surgery, atrophy, and / or long-term wear and tear or degeneration). Such products can include, for example, acellular tissue matrix, tissue allografts or xenografts, and / or reconstituted tissue (i.e., tissue that has been at least partially decellularized and seeded with cells to provide a growth-capable material).

[0017] Various tissue products are manufactured to treat soft and hard tissues. For example, ALLODERM® and STRATTICE® (LIFECELL CORPORATION, Branchburg, New Jersey) are two dermal cell-free tissue matrices manufactured from human and porcine dermis, respectively. While such materials are very useful for treating certain types of diseases, materials with different biological and mechanical properties may be desirable for specific 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 applications. However, such materials may not be ideal for the regeneration, repair, replacement and / or augmentation of adipose tissue when the production of adipose tissue containing viable adipocytes is the desired outcome. Therefore, this disclosure provides tissue products useful for the treatment of tissue defects / flaws involving adipose tissue. This disclosure also provides methods for manufacturing such tissue products.

[0018] 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.

[0019] As described above, the tissue products of this disclosure are formed from adipose tissue. The adipose tissue is derived from human or animal sources. For example, human adipose tissue may be obtained from a cadaver. Furthermore, human adipose tissue may also be obtained from a living donor (e.g., autologous tissue). Adipose tissue may also be obtained from animals such as pigs or monkeys or from other sources. When using animal sources, the tissue can be further processed to remove antigenic components such as the 1,3-α-galactose moiety, which is present in pigs and other mammals but not in humans or primates. See "A Porcine-Derived Acellular Dermal Scaffold that Supports Soft Tissue Regeneration: Removal of Terminal Galactose-α-(1,3)-Galactose and Retention of Matrix Structure" by Xu, Hui et al., Tissue Engineering, Vol. 15, 1-13 (2009). This document is incorporated in its entirety by citation. Furthermore, the adipose tissue may be obtained from animals that have been genetically modified to remove antigenic moieties.

[0020] Figures 1 and 2 illustrate exemplary processes for manufacturing the tissue products of this disclosure. Figure 1 provides a flowchart illustrating the basic steps, which can be used to generate a suitable adipose tissue sponge and subsequently further process it to produce injectable or implantable particles. As illustrated, the process may include several steps, but it should be understood that additional or alternative steps may be added or replaced depending on the specific tissue used, the desired application, or other factors.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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 assist in the liberation or removal of lipids. For example, mechanical processing may be carried out under conditions in which 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 can cause liquefaction of lipids released from the destroyed adipocytes by mechanical destruction, which can then lead to efficient phase separation for bulk lipid removal. In one example, when processing porcine adipose tissue, the temperature reached during the process exceeds 100°F but does not exceed 122°F (50°C). The temperature range reached can be adjusted depending on the origin of the adipose tissue. For example, the temperature can be lowered further to around 80°F, 90°F, 100°F, 110°F, or even 120°F when processing less saturated tissues, such as primate tissue. Alternatively, the process can be chosen so that fat reaches a minimum temperature of, for example, 80°F, 90°F, 100°F, 110°F, or 120°F.

[0025] 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., 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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 material, 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.

[0030] The material can be formed into a porous or spongy material after the removal of lipids 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. 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. In some cases, the resuspended extracellular matrix can be further mechanically processed one or more times by grinding, cutting, mixing or other processes, and the processed material can 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.

[0031] Once the additional washing and grinding steps are complete, the resuspended material is placed in a container or mold, as shown in step 180, 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 includes: cooling the material to -10°C in 20–40 minutes; holding the sample at -10°C for 120–180 minutes, then further cooling the sample to -40°C to freeze completely; applying a vacuum; raising the temperature to -5°C and holding for 30–60 hours; and raising the temperature to 25°C and holding for 6–12 hours. The freeze-dried sample can then be removed from the freeze-dryer and packaged in a foil pouch under nitrogen.

[0032] After the formation of a solid or sponge, the material can optionally be stabilized, as shown in step 190. 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 result in 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.

[0033] In some embodiments, the material is crosslinked for stabilization. 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 a variety of ways. In one embodiment, the material can be crosslinked by contacting it with a crosslinking agent such as glutaraldehyde, genephine, carbodiimide (e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)) and diisocyanate. Furthermore, crosslinking may be carried out by heating the material in a vacuum. 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. Furthermore, other crosslinking processes, or combinations of processes, including ultraviolet irradiation, gamma-ray irradiation, and / or electron beam irradiation, may be used to produce any of the disclosed products. Furthermore, although not essential, vacuum may shorten the crosslinking time. Furthermore, lower or higher temperatures can be used as long as the matrix proteins do not melt and / or sufficient time is provided for crosslinking.

[0034] 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).

[0035] 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.

[0036] An exemplary crosslinking process may involve contacting the freeze-dried material prepared as described above with glutaraldehyde or EDC. For example, a 0.1% glutaraldehyde solution may be used, immersing the tissue in the solution for approximately 18 hours, followed by extensive rinsing with water to remove the solution. Alternatively, or in combination with this, a dehydrothermal (DHT) process may be used. For example, one exemplary dehydrothermal process involves treating the material at 100°C, approximately 20 inches of mercury, for 18 hours, followed by immersion in water. The final crosslinked tissue product can be stored in a film pouch.

[0037] After forming a solid or sponge, the tissue product can be further processed to produce an injectable form. An exemplary process for forming an injectable form is shown in Figure 2 as process 200. Note that “injectable” may include materials that are injected with a syringe, cannula, or needle, but it should be understood that the disclosed materials can be manufactured to have sizes and mechanical properties suitable for other modes of administration, including manual insertion (e.g., by hand, or by other bulk instruments such as spatulas, tubes, or other devices equipped for handling fluid materials).

[0038] The process that brings about injectability begins with obtaining a bulk sponge, as shown in step 210. Obtaining the bulk sponge is carried out using the process or a suitable variation thereof, as described with reference to Figure 1. In one aspect of this disclosure, the process begins with a stable (porcine or human) adipose tissue matrix sponge.

[0039] After the bulk sponge material is obtained, the material undergoes a size reduction or particle formation process, as shown in step 220. Size reduction or particle formation may include mechanical cutting, grinding, or mixing to produce particles of a desired size and size distribution. In some embodiments of the present disclosure, if the initial material is a dry sponge, grinding may be preferred to reduce the dry sponge to smaller particles. Size reduction can be carried out at room temperature.

[0040] In particular, it has been found that the sponge size should be reduced in order to maintain the porous sponge structure within the particles. Therefore, the particles must be large enough to maintain the sponge structure in order to support lipid production. Loss or absence of the porous structure may result in a composition that does not support lipid growth. For example, particles may be formed from the sponge so that they have a size of at least 0.5 microns, 1 micron, 2 microns, 3 microns, 4 microns, or larger. The particle size can be selected based on the microstructure of the sponge.

[0041] Continuing to refer to Figure 2, in the next step 230, size selection may be desirable. For example, once the stabilized sponge has been processed to be ground or to produce particles, it is sieved or otherwise separated to obtain particles of a desired size as a fluid / injectable adipose tissue matrix material. In some examples, one or more injectable adipose tissue matrix sponges of different particle sizes may be desirable to accommodate various needle sizes. In this case, the material is sieved to achieve a preferred particle size range. In one embodiment, the particle size ranges from 50 microns to 2,800 microns. For example, ground sponge can be sieved to recover particles of preferred dimensions, such as fine particles (e.g., 50-100 microns), medium particles (e.g., 0.4 mm-0.6 mm), large particles (e.g., 0.8 mm-1 mm), and even larger particles (e.g., 2.8 mm-3.4 mm). In some aspects of this disclosure, particle sizes within this range may not elicit a variety of biological responses. In other words, for example, particle sizes in the range of 50 microns to 2,800 microns may not produce any difference in biological response. For various applications requiring specific needle sizes, a specific particle size can be selected without needing to consider whether the biological response differs.

[0042] Once the particle size is selected, the next step 240 involves hydrating and / or adding the particles to another carrier to a desired extent to obtain fluidity and a desired degree of solids content. For example, sieved particles are hydrated with saline or other material to obtain a solids content concentration of 5–12%. In addition, other carriers, including hyaluronic acid-based materials (e.g., JUVEDERM®, ALLERGAN, or similar materials), may be used or added. In some other embodiments, the particles may be hydrated using water, saline, phosphate-buffered saline, or other suitable physiological solutions. In some embodiments of this disclosure, step 240 may be performed before step 230, and the particles may be hydrated or added to a carrier before size selection and / or before sieving the particles.

[0043] Figures 4 and 5 show exemplary particulate matter. Figure 4 is a magnified view of particulate tissue matrix having dimensions of 2–3 mm after being produced by grinding adipose tissue matrix sponge. Figure 5 shows a group of cell-free tissue matrix particles produced by grinding or crushing adipose tissue matrix sponge to yield particles of 100–300 microns, and a paste / pudding-like injectable material after hydration.

[0044] According to certain embodiments of this disclosure, materials having a desired tissue matrix solid content can be used. For example, materials with a solid content of 5% to 12% can be used, with 7.5% to 10% being preferable. In another embodiment, a suitable carrier may be used with the 5% to 10% solid content material to facilitate injection and prevent particle dissipation from the injection site. A suitable carrier is a fluid carrier, such as a fluid hyaluronic acid carrier. In some embodiments, the hyaluronic acid carrier is a non-crosslinked hyaluronic acid carrier. In some other embodiments, the hyaluronic acid carrier is a crosslinked hyaluronic acid carrier.

[0045] As used herein, “hyaluronic acid-based materials” refer to materials containing hyaluronic acid (HA). HA refers to hyaluronic acid and any salt thereof, including, but not limited to, sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate, calcium hyaluronate, and combinations thereof. Hyaluronic acid-based materials may contain both HA and its pharmaceutically acceptable salts. Exemplary HA-based materials are commercially available as JUVEDERM® and JUVEDERM VOLUMA®. It should be understood that hyaluronic acid-based materials may contain additional agents, such as lidocaine.

[0046] All numerical values ​​representing "molecular weight" for HA in this specification should be understood as Dalton's weight-average molecular weight (Mw).

[0047] The molecular weight of HA is equal to its intrinsic viscosity (m³ / kg) = 9.78 × 10⁻⁶. -5 ×Mw 0.690 It is calculated from intrinsic viscosity measurements using the Mark Houwink relation. Intrinsic viscosity is measured according to the procedure specified in the European Pharmacopoeia (HA monograph N°1472,01 / 2009).

[0048] The high molecular weight HA used herein refers to at least about 1.0 million daltons (Mw≧10). 6 This refers to HA material having a molecular weight of Da (1 MDa) to approximately 4.0 MDa. The high molecular weight HA incorporated into this tissue product composition may have a molecular weight in the range of approximately 1.5 MDa to approximately 3.0 MDa, or the high molecular weight HA may have a weight-average molecular weight of approximately 2.0 MDa. In another example, the high molecular weight HA may have a molecular weight of approximately 3.0 MDa.

[0049] As used herein, low molecular weight HA refers to HA materials having a molecular weight of less than about 1.0 MDa. Low molecular weight HA can have molecular weights ranging from about 200,000 Da (0.2 MDa) to less than 1.0 MDa, for example, from about 300,000 Da (0.3 MDa) to about 750,000 Da (0.75 MDa), but with a maximum of 0.99 MDa. Preferably, there is no overlap between the molecular weight distributions of low molecular weight HA materials and high molecular weight HA materials. Preferably, a mixture of low molecular weight HA and high molecular weight HA has a bimodal molecular weight distribution. The mixture may also have a multimodal distribution.

[0050] In one aspect of the present invention, the adipose tissue product composition comprises HA having a high molecular weight component and a low molecular weight component, wherein the high molecular weight component may have a weight-average molecular weight at least twice that of the low molecular weight component. For example, the molecular weight ratio of high molecular weight HA to low molecular weight HA in the composition may be at least 2:1. For example, the tissue product composition may include HA having a low molecular weight component having a weight-average molecular weight of about 500,000 Da and a high molecular weight component having a weight-average molecular weight of about 1.0 MDa or at least about 1.0 MDa. In another example, the tissue product composition according to the present invention may include HA having a low molecular weight component having a weight-average molecular weight of about 800,000 Da and a high molecular weight component having a weight-average molecular weight of about 1.6 MDa or at least about 1.6 MDa. It should be understood that many different types of HA can be incorporated into the adipose tissue product composition, and the examples described above are not intended to be limiting.

[0051] In some exemplary embodiments, HA is crosslinked using one or more suitable crosslinking agents. The crosslinking agent may be any agent known to be suitable for crosslinking polysaccharides and their derivatives via their hydroxyl groups. Suitable crosslinking agents include, but are not limited to, 1,4-butanediol diglycidyl ether (or 1,4-bis(2,3-epoxypropoxy)butane or 1,4-bisglycidyloxybutane; all commonly known as BDDE), 1,2-bis(2,3-epoxypropoxy)ethylene, 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (commonly known as EDC). Other suitable hyaluronic acid crosslinking agents include, but are not limited to, multifunctional PEG-based crosslinking agents such as pentaerythritol tetraglycidyl ether (PETGE), divinyl sulfone (DVS), 1,2-bis(2,3-epoxypropoxy)ethylene (EGDGE), 1,2,7,8-diepoxyoctane (DEO), phenylene bis-(ethyl)-carbodiimide and 1,6-hexamethylene bis(ethylcarbodiimide), adipic acid dihydrazide (ADH), bis(sulfosuccinimidyl)sverate (BS), hexamethylenediamine (HMDA), 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, or combinations thereof.

[0052] In one exemplary embodiment of an adipose tissue product composition formed according to the present invention, the adipose tissue product composition comprises a fluid carrier containing a hyaluronic acid-based material and a plurality of adipose tissue matrix particles mixed with the carrier. In some exemplary embodiments, the fluid carrier comprises HA that is not mixed with additional agents, and in other exemplary embodiments, the fluid carrier comprises HA that is mixed with additional agents. Additional agents include, but are not limited to, anesthetics, such as aminoamide local anesthetics and their salts, or aminoester local anesthetics and their salts. Examples include procaine, chloroprocaine, cocaine, cyclomethicaine, symethocaine, propoxycaine, procaine, proparacaine, tetracaine, or salts thereof, or any combination thereof. In some embodiments, the anesthetics include articaine, bupivacaine, cincocaine, etidocaine, levobupivacaine, lidocaine, mepivacaine, pipelocaine, prilocaine, ropivacaine, trimecaine, or salts thereof, or any combination thereof.

[0053] The fluid carrier may initially be in the form of a fluid liquid solution that can be mixed with adipose matrix particles to form a slurry. The formed slurry can then be loaded into a syringe or other infusion device for administration to a patient. In some exemplary embodiments, the fluid carrier may be a sufficient amount of non-crosslinked HA to improve the injectability of the adipose tissue product composition. In some exemplary embodiments, the fluid carrier may be a sufficient amount of crosslinked HA to improve the injectability of the adipose tissue product composition. Although the fluid carrier is described herein as comprising HA, other glycosaminoglycans (GAGs), such as HSGAGs, CSGAGs and / or keratin sulfate-type GAGs, can be used as fluid carriers.

[0054] Surgical transplantation is a suitable option for implanting adipose tissue matrix material to repair specific areas of the body, although injection may be preferable in some applications. While particleization of adipose tissue matrix has been found to improve application and injection compared to the application of adipose tissue matrix in its natural form, pure adipose tissue matrix, even when particleized, has been found not to be easily applied or injected into patients. In particular, the application of particleized tissue matrix material has been found to be difficult to control, as it tends to diffuse or aggregate after storage. Furthermore, the injection force required to inject particulate cell-free tissue matrix is ​​relatively high, and it has been found to be relatively difficult to inject all of the particulate tissue matrix loaded into an injection device such as a syringe.

[0055] To address some of the above-described problems in injecting adipose tissue matrix material, exemplary embodiments described herein provide a tissue product composition comprising adipose tissue matrix particles mixed in a fluid carrier containing a hyaluronic acid-based material. The formed tissue product composition can be applied more easily than pure adipose tissue matrix particles while maintaining properties that promote tissue growth in the transplant and / or injection area, as further described herein.

[0056] In one exemplary embodiment, adipose tissue matrix particles and a fluid carrier may be mixed in a large batch under substantially sterile conditions to form a tissue product composition according to the present invention. Mixing may include, for example, stirring the adipose tissue matrix particles and the fluid carrier together to form a slurry. The parameters and methods of mixing can be modified according to the properties of the fluid carrier and the cell-free tissue matrix particles, and their approximate amounts in the tissue product composition, and can be readily derived from routine experiments by those skilled in the art.

[0057] Various types of hyaluronic acid-based materials can be used to formulate tissue product compositions according to the present invention. In some cases, all types of HA are initially included in a solution with a concentration of 20 mg HA / mL. Exemplary HA types include HA type 1, HA type 2, HA type 3, and HA type 4. HA type 1 is a non-crosslinked hyaluronic acid with a G' value of 320 Pa, while HA type 2 and HA type 3 are, in contrast, hyaluronic acids crosslinked with an EDC crosslinking agent, having different G' and G'' values ​​depending on the degree of crosslinking. The G' value of HA type 2 was 160 Pa, and the G' value of HA type 3 was 500-550 Pa. HA type 4 is also crosslinked. The G' value of HA type 4 is 350 Pa.

[0058] Various hyaluronic acid-based materials can be mixed with adipose tissue matrix particles to produce the various tissue product compositions listed in Table 1 below. It should be noted that the hyaluronic acid-based materials described herein are merely examples, and other hyaluronic acid-based materials can be mixed with adipose tissue matrix particles. Furthermore, the compositions shown in Table 1 are merely examples, and other tissue product compositions can be formed according to the present invention. TIFF2026062652000001.tif121170

[0059] Referring here to Table 1, exemplary embodiments of tissue product compositions formed according to the present invention are described. Table 1 illustrates compositions 1 to 4, which show various tissue product compositions, but it should be understood that other tissue product compositions can be formed according to the present invention. In each of compositions 1 to 4, adipose tissue matrix particles were derived from porcine adipose tissue and, when mixed with a fluid carrier, yielded an adipose tissue matrix slurry, also called a “fluid adipose tissue matrix”. The adipose tissue matrix particles were at a concentration of 100 mg / mL in aqueous buffer before mixing with the fluid carrier, which was provided at a concentration of 20 mg HA / mL. As can be seen from Table 1, by adjusting the adipose matrix:HA ratio, slurries with various fluid properties can be produced, as will be further described herein. The ratios described herein are by volume or mass, and it should be understood that in the exemplary embodiments shown in Table 1, the ratio is given as volume adipose matrix:volume HA. As illustrated in compositions 1 and 2, the adipose matrix:HA ratio is 9:1, and as illustrated in compositions 3 and 4, the adipose matrix:HA ratio is 4:1. It should be noted that these ratios are merely illustrative, and other exemplary embodiments of the tissue product composition may have other ratios of fat matrix:HA, including any values ​​between the disclosed ratios.

[0060] According to certain aspects of this disclosure, tissue product compositions having a desired tissue matrix particle solid content can be used. For example, materials with a solid content of 5% to 15%, such as 7.5% to 10%, may be desirable depending on the type of hyaluronic acid-based material mixed with the tissue matrix particles. In some exemplary embodiments, the tissue product composition has a solid content of 10% cell-free adipose-containing matrix particles corresponding to 100 mg / mL.

[0061] As described above, tissue products must have the ability to support cell proliferation and tissue regeneration when implanted in or on a patient. Furthermore, tissue products must function as carriers for cells, including stem cells such as adipose-derived stem cells, and possess the ability to support the growth of such cells. For this reason, the above process must not alter extracellular matrix proteins in an unacceptable manner (e.g., by damaging protein structures and / or removing important glycosaminoglycans and / or growth factors). In some embodiments, the product will have normal collagen banding as demonstrated by transmission electron microscopy.

[0062] In various embodiments, the tissue product is treated with a process that retains either or both of natural hyaluronic acid and chondroitin sulfate. Therefore, the tissue product may contain either or both of hyaluronic acid and chondroitin sulfate. Furthermore, a process that retains native growth factors can be selected. For example, the tissue product may be produced to contain one or more growth factors selected from PECAM-1, HGF, VEGF, PDGF-BB, follistatin, IL-8, and FGF-basic.

[0063] Adipose tissue matrix is ​​characterized by its abundance of type IV and type VI collagen. The ratio of these two types of collagen to type I collagen may differ from that of the dermal matrix, and this may be an important factor used to distinguish between adipose and dermal matrix in vivo.

[0064] 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. For this reason, adipose tissue products are considered to offer superior regenerative capabilities when implanted in a particular tissue site compared to materials manufactured from other tissue types. In some cases, the tissue product may be implanted in a tissue site that is primarily or substantially adipose tissue. In some cases, the tissue product may be used as a facial filler to add volume or replace lost tissue, for example, to treat lines, wrinkles, gaps or depressions. 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 face, buttocks, abdomen, waist, thighs, or any other site where additional adipose tissue with a structure and feel similar to natural fat is desired. In any of those sites, the tissue can be used to reduce or eliminate wrinkles, sagging, or undesirable shapes.

[0065] When used for breast tissue replacement or augmentation, the tissue can offer advantages over other tissue products. For example, while some tissue products enable endografting and tissue formation, they do not mimic the texture and feel of normal breast tissue and can form considerable fibrous tissue that appears abnormal on radiographic images. Since the tissue products of this disclosure are formed from fat, they can help facilitate the more normal regeneration of adipose tissue.

[0066] Furthermore, the tissue product may be used as a carrier for cells. For example, the product may be transplanted or used in any of the sites described above, but with cells seeded. In some cases, such cells may include stem cells, such as adipose-derived stem cells. In addition, other pluripotent cells and cells from any tissue source (e.g., blood, bone marrow, fetal stem cells, umbilical cord blood cells, etc.) may be used. Cells may be seeded in the tissue after or before transplantation. Furthermore, cells may be cultured on the tissue product before transplantation and then transplanted in or onto the body.

[0067] As described above, particulate tissue matrix products may further include a fluid carrier to facilitate the injection of the tissue product. In various embodiments, the particulate tissue matrix and the fluid carrier are packaged in separate containers and do not come into contact until mixed immediately before injection. For example, the particulate tissue matrix and the fluid carrier are packaged in separate barrels of a multi-barrel syringe and mixed immediately before or during injection of the contents. In other embodiments, the particulate tissue matrix and the fluid carrier are pre-mixed and packaged together. The materials may be dried separately or stored in a biocompatible buffer, which can preserve the biological properties of the tissue matrix and / or carrier, prevent bacterial growth, and prevent damage during sterilization or storage.

[0068] The following embodiments are illustrative of the present disclosure and are not limiting. [Examples]

[0069] A. Manufacturing of adipose tissue material: To prepare the adipose tissue matrix material, porcine adipose tissue was first sliced ​​into 2-inch strips and roughly chopped in a food-grade meat chopper. The chopped adipose tissue may be frozen at -80°C if not ready for further processing. The frozen material can be thawed overnight at room temperature or at 4°C. The pre-processed material was coarsely ground at 2000 rpm using a RETSCH® GM300 (GRINDOMIX), and then further ground at 4000 rpm to separate the oil from the solid matrix. The adipose matrix solids were collected by centrifugation and washed with buffer. The matrix material was decellularized overnight at room temperature in EDTA-Triton solution, with the solution changed every 4 hours. The matrix proteins were washed again. During washing, the matrix pellet was centrifuged to pelletize the tissue matrix, and the used solution was decanted. The suspension was mechanically ground again to further decompose the matrix fibers. After washing, the matrix pellet was resuspended in 20% PBS at a solids concentration of approximately 2-3% w / w. The slurry was placed in a metal tray, freeze-dried to form a sponge, and then stabilized by DHT treatment. The stabilized sponge was then pulverized and sieved to obtain particles of the desired size as a fluid / injectable porcine cell-free tissue slurry (PATS) material. The particulate matter was converted into a 5-15% paste and subjected to final sterilization by electron beam.

[0070] B. The process generates intact collagen structures: The tissue samples produced by the above process were analyzed using scanning electron microscopy (SEM) and atomic force microscopy (AFM). The results showed that the tissue samples were porous scaffolds containing collagen material with a typical collagen banding pattern, including structure and porosity, as shown in Figure 6. The microscope images show intact collagen with a normal banding pattern.

[0071] The tissue samples were further subjected to differential scanning calorimetry (DSC). The DSC results, as illustrated in Figure 7A, show that the melting onset temperature of the tissue samples was 61.5°C. In this example, the melting onset temperature of the tissue samples was similar to that of natural tissues (e.g., the fat of the raw material).

[0072] The tissues were also subjected to Masson's trichrome staining and collagenase digestion assays. Collagenase digestion was performed on the tissues generated as described above, with and without electron beam sterilization. The results of staining and collagenase digestion are shown in Figures 7B and 7C, respectively. Specifically, Figure 7C shows the collagenase digestion curves for adipose tissue generated according to the examples of disclosure, with and without electron beam sterilization. In the case of no electron beam sterilization and DHT stabilization only, the percentage of residual solids as a function of digestion time decreases to approximately 18% after 8 hours. On the other hand, when electron beam sterilization and DHT stabilization are used together, the percentage of residual solids approaches approximately 25% after the same digestion period, which is slightly higher than with DHT alone.

[0073] Referring to Figures 7B and 7C, the material had trichrome staining indicating normal collagen on trichrome sections, and its collagenase sensitivity remained unchanged even after final sterilization by electron beam. Generally, when collagen is trichrome-stained, normal collagen should appear blue rather than red. Taken together, microscopic examination, DSC, staining, and collagenase digestion indicate preserved collagen.

[0074] The process described in C. is efficient for removing cells, cell debris, and oil from within the scaffold: As described, the prepared samples underwent hematoxylin and eosin staining ("H&E") as shown in Figure 8A, DNA and lipid content analysis as shown in Figure 8B, and immunostaining for MHC-1 and MHC-2 components as shown in Figure 8C (showing tissue matrix versus natural fat control). H&E histology revealed that the tissue product exhibited a porous structure with no signs of cells. Consistent with this observation, residual DNA and free oils in the tissue product were very low. Immunostaining showed that the tissue product was unaffected by MHC-1 and MHC-2 staining, indicating that the described process is efficient for decellularization.

[0075] D. The tissue product retained the major matrix components of natural fats: The tissues were subjected to immunohistochemical analysis for major extracellular matrix proteins, including types I, III, and IV. As shown in Figure 9, the adipose tissue product (bottom panel) retained the original properties of the native adipose matrix (top panel).

[0076] E. Tissue products support the growth of multiple tissue cells: To test whether the tissue product sponge has the potential to support the growth of adipose tissue, vascular structures, and other connective tissues such as dermal tissue, three different cell types were selected. Specifically, all primary cells isolated from normal human individuals were tested, including adipogenic mesenchymal stem cells, endothelial cells, and dermal fibroblasts. Cells isolated from these tissues were seeded onto the tissue product and cultured for 1, 7, and 16 days. Cell proliferation was quantified using a cell proliferation assay kit. Using the CyQUANT® Cell Proliferation Assay Kit, cell proliferation was quantified by DNA content using a fluorescent dye. The tissue product was analyzed for cell proliferation. As shown in Figure 10, the cell seeding scaffold was stained with viability stain, and cell viability and growth were observed under a fluorescence microscope. The tissue product supports the proliferation of all three types of cells in vitro.

[0077] F. The tissue product retained volume and supported in vivo fat production: The biological performance of the product was tested in a subcutaneous nude rat model. As shown in Figure 11, this study was designed to test various formulations of the product.

[0078] The products prepared as described in Example A were classified into five different size ranges: ARM1: 2.8–3.4 mm; ARM2: 0.8–1.0 mm; ARM3: 0.4–0.6 mm; ARM4: 0.05–0.1 mm; ARM5: 0.1–1 mm. The sixth ARM, ARM6, actually contained slurry and fibers, which were materials obtained immediately after the decellularization process without freeze-drying or subsequent steps. ARM6 was used as a control for comparison with tissue products having a DHT-stabilized 3D microporous structure, like ARM1–5. ARM7 contained a sponge prepared by a similar process, but it was derived from a cell-free tissue matrix of the dermis. The dermal tissue was Strattice®.

[0079] ARM1-5 were first hydrated in physiological saline to a solid content of 10% by weight to prepare an injectable paste, and ARM6 was similarly adjusted to a solid content of 10%. ARM7 was a 5mm thick, 10mm punch from dermal sponge, which was hydrated in PBS. Approximately 0.5cc aliquots of each injectable fatty material (ARM1-6) and the 10mm punch of ARM7 were implanted into the subcutaneous region of nude rats for each of the three ARMs.

[0080] At four weeks, explants were harvested for rough observation and histological analysis. Figure 11 shows rough observations of injectable adipose tissue matrix explants after subcutaneous transplantation into nude rats. The dotted line indicates the injected material. All explants were soft to palpation. Injectable fat across all particle size ranges persisted for at least four weeks. Injectable fat implants of specific particle sizes were also observed to persist well for at least 12 weeks (data not shown).

[0081] Figure 12 shows additional Masson trichrome staining of the explants at low magnification. Arms 1–4, prepared from stabilized PATS sponges, showed a potent adipogenic response with tissue infiltration. In contrast, the adipose tissue matrix slurry of ARM6, lacking 3D structure (i.e., lyophilization and stabilization omitted), did not induce adipogenic response (c).

[0082] Furthermore, ARM7 further demonstrates an intact, spongy porcine dermal tissue scaffold when transplanted into the subcutaneous space of the same nude rat model, as shown in Figure 12. Although volume was retained, Masson's trichrome staining revealed no adipose tissue regeneration in the dermal scaffold.

[0083] As shown in Figure 13, the volume retention rates of representative injectable fat matrix products (ARM1: 2.8-3.4 mm, ARM2: 0.8-1.0 mm, ARM5: 0.1-1.0 mm) were evaluated for 8 weeks, along with the ARM6 slurry. ARM1, ARM2, and ARM5, which are porous microstructured particles, all retained more than 84% of the explant volume for up to 8 weeks compared to the transplanted volume.

[0084] For ARM3: 0.4–0.6 mm and ARM4: 0.05–0.1 mm, similar results are expected, as these ARMs showed a similar biological response to ARM1, 2, and 5 over 4 weeks. Microscopically, the 0.4–0.6 mm particles have a porous microstructure similar to ARM1, 2, and 5. The 0.05–0.1 mm particles are too small to have intact micropores, but most particles are branched and may form similar pores when in contact with each other due to stabilized material containing DHT (data not shown), which is fundamentally different from the slurry material of ARM6. In contrast, grafts without microporous structure, adipose tissue slurry morphology (e.g., ARM6), were difficult to find on palpation or very flat. This indicates significant graft volume loss. The weight of the explant decreased over time, with only about 38% remaining at the end of 8 weeks. This indicates significant graft volume loss.

[0085] Figures 14A–14C show enlarged Masson trichrome-stained sections of the ARM2 explant, demonstrating good adipogenesis. Adipocytes are represented by white areas and were abundant and large. Angiogenesis was also clearly visible. Similar histological findings were observed in the ARM5 sample (not shown in the figures).

[0086] In summary, the results, as shown in Figures 11 and 13, indicate that adipose tissue matrix particles of all sizes tested (e.g., 50 μm to 3.4 mm) maintained their volume and showed good cell invasion and revascularization responses up to 8 weeks. Data for only 4 weeks are shown in Figure 12. In contrast, in the control ARM6, a slurry made from the same material prepared using the same process but without lyophilization and DHT stabilization, did not show an adipose tissue infiltration response in the graft, as shown in ARM6 in Figure 12. The lack of porosity in ARM6 suggests that microporous structures may be important or noteworthy during adipogenesis. Furthermore, ARM6 suffered significant volume loss at the end of 8 weeks (Figure 13), which further indicates that DHT stabilization of tissue products is important for volume retention in vivo. In addition, adipose tissue infiltration was specific to the adipose matrix and was not present in intact spongy scaffolds prepared with dermal cell-free matrix using a similar process, as shown in ARM7 in Figure 12.

[0087] G. Using hyaluronic acid (HA) as a carrier improved the injectability of adipose tissue products. Figure 15 provides images of fresh adipose tissue matrix compared to tissue matrix stored for 1.5 years, both prepared as described in Example A. The images are provided at various magnifications. As shown, adipose tissue matrix material in a wet configuration containing 10% solids formed aggregates after 1.5 years of storage. Biological studies have shown that the aggregation does not affect the biological properties of the material, such as volume retention or adipose tissue endografting in the subcutaneous region of nude rats. However, for non-invasive delivery in a clinical setting, the 10% adipose tissue product may require a carrier to facilitate injectability.

[0088] To enhance the injectability of adipose tissue material, a 20 mg / mL non-crosslinked hyaluronic acid (HA) carrier was mixed with newly prepared adipose tissue matrix (10%, 0.1–1.0 mm) or adipose tissue matrix (10%, 0.1–1 mm) from 1.5 years prior. The final concentration of the HA additive in the mixed material was 2 mg / mL. The compressive load (N) required for injection of the adipose tissue material was evaluated using an Instron Model 5865 material tester (Instron Corporation, Norwood, Massachusetts) for both cases with and without the HA additive. The adipose tissue material without HA was injected via either a 16G or 18G needle, while the adipose tissue material with the HA additive was injected via an 18G needle. The average compressive force over time (n=3) for products with and without the HA additive was calculated and plotted.

[0089] As shown in Table 2, when HA was not used as a carrier, the injection of fresh fat material using a 16G needle was smooth, but fat aged 1.5 years encountered significant resistance. When HA was added as a carrier, both fresh and 1.5-year-old fat material became injectable. TIFF2026062652000002.tif54170

[0090] As shown in Figure 16, without HA as a carrier, the injection of newly prepared fatty material at both speeds of 0.15 mm / sec and 0.25 mm / sec encountered significant resistance with the 18G needle, while the injection of the product containing 2 mg / mL of HA additive (final concentration) was very smooth, even at a speed of 0.25 mm / sec.

[0091] To evaluate the biological properties of a mixture of adipose tissue products and HA additives, 20 mg / mL of cross-linked hyaluronic acid (HA type 4) and adipose tissue matrix (10%) were mixed in a 1:10 ratio and injected into the subcutaneous region of nude rats. When explants of the HA-adipose tissue mixture were harvested 4 weeks after injection, strong adipose tissue endoplasia was observed in the explants, as shown in Figure 17.

[0092] The principles of this disclosure are described herein with reference to exemplary embodiments for specific uses, but it should be understood that this disclosure is not limited thereto. Those skilled in the art and those with access to the teachings provided herein will recognize that all additional modifications, applications, embodiments, and substitutions of equivalents are within the scope of the embodiments described herein. Therefore, the present invention should not be considered limited by the foregoing description.

Claims

1. A method for manufacturing tissue products, The step of selecting adipose tissue, The process involves mechanically processing adipose tissue to reduce its size, A step of processing mechanically treated tissue to remove substantially all cellular material from the tissue, The steps include suspending the tissue in a solution to form a suspension, The process involves processing a suspension to generate a stabilized three-dimensional structure having a microporous structure, A method characterized by comprising the step of mechanically processing a stabilized three-dimensional structure to generate particles.

2. In the method according to claim 1, A method for processing a suspension to produce a stabilized three-dimensional structure, characterized in that the suspension is dried.

3. In the method of claim 2, A method characterized in that drying includes freeze-drying.

4. In the method according to any one of claims 1 to 3, A method characterized by the fact that processing a suspension to produce a stabilized three-dimensional structure includes cross-linking of tissue.

5. In the method according to claim 4, A method characterized in that crosslinking includes treatment by at least one of a chemical substance, a photoactivation crosslinking process, or heating.

6. In the method according to claim 5, A method characterized in that the chemical substance comprises at least one of glutaraldehyde, genepine, carbodiimide, and diisocyanate.

7. In the method according to claim 4, A method for crosslinking, characterized by including heating of the tissue.

8. In the method of claim 7, A method characterized by heating tissue in a vacuum.

9. In the method according to claim 4 or 7, A method characterized by heating the tissue to 70°C to 120°C.

10. In the method according to claim 1, A method characterized in that a stabilized three-dimensional structure maintains its microporous structure when in contact with an aqueous environment.

11. In the method according to claim 10, A method characterized by the fact that a substance maintains a stable three-dimensional structure when implanted in the body.

12. In the method according to any one of claims 1 to 11, A method characterized by generating particles by mechanically processing a stabilized three-dimensional structure, which includes generating a group of particles with a maximum dimension of 50 microns to 4 mm.

13. In the method according to any one of claims 1 to 11, A method characterized by generating particles by mechanically processing a stabilized three-dimensional structure, which includes generating a group of particles with a maximum dimension of 1.5 mm to 4 mm.

14. In the method according to any one of claims 1 to 11, A method characterized by generating particles by mechanically processing a stabilized three-dimensional structure, which includes generating a group of particles with a maximum dimension of 2 mm to 3.5 mm.

15. In the method according to any one of claims 1 to 11, A method characterized by generating particles by mechanically processing a stabilized three-dimensional structure, which includes generating a group of particles with a maximum dimension of 2 mm to 3 mm.

16. In the method according to any one of claims 1 to 15, A method characterized in that the mechanical processing of a stabilized three-dimensional structure to generate particles includes generating a group of particles having a preserved microporous structure from the stabilized three-dimensional structure.

17. In the method according to any one of claims 1 to 16, A method characterized by further comprising the step of hydrating particles.

18. In the method according to claim 17, A method for hydrating particles, characterized in that it includes mixing particles with a predetermined amount of aqueous solution such that the particles and the aqueous solution form a composition in which the particles constitute 5% to 20% by weight.

19. In the method according to claim 18, A method for hydrating particles, characterized in that it includes mixing particles with a predetermined amount of aqueous solution such that the particles and the aqueous solution form a composition in which the particles constitute 5% to 12% by weight.

20. A tissue product manufactured by the method described in any one of claims 1 to 19.

21. A tissue product composition, A tissue product composition comprising a particulate tissue matrix, wherein the tissue product composition comprises adipose-free tissue matrix formed into particulates after being formed into a porous sponge, and the particulate tissue matrix comprises particles having a maximum dimension of about 0.05 mm to 3 mm.

22. In the tissue product composition according to claim 21, A tissue product composition characterized in that the particles contain a microporous structure derived from a porous sponge.

23. In the tissue product composition according to claim 21 or 22, A tissue product composition characterized by having dry particles.

24. In the tissue product composition according to claim 21 or 22, A tissue product composition characterized by further comprising a fluid carrier.

25. In the tissue product composition according to claim 24, A tissue product composition characterized in that the fluid carrier contains an aqueous fluid.

26. In the tissue product composition according to claim 25, A tissue product composition characterized by containing physiological saline as a fluid carrier.

27. In the tissue product composition according to claim 25 or 26, A tissue product composition characterized by containing 80 to 95% by weight of water.

28. In the tissue product composition according to claim 25 or 26, A tissue product composition characterized by containing 5% to 15% by weight of adipose tissue matrix protein.

29. In the tissue product composition according to any one of claims 24 to 26, A tissue product composition characterized in that the fluid carrier is a fluid hyaluronic acid carrier.

30. In the tissue product composition according to claim 29, A tissue product composition characterized by hyaluronic acid not being cross-linked.

31. In the tissue product composition according to claim 29, A tissue product composition characterized by cross-linked hyaluronic acid.

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