Adipose tissue products and methods of production

Abstract

To provide compositions for treating tissue.SOLUTION: A tissue product composition comprises: an adipose acellular tissue matrix that has been formed into a porous sponge and then formed into a particulate tissue composition, where the particulate composition comprises adipose tissue matrix particles and 5 wt.% to 10 wt.% of residual lipids, and where the particles comprise a microporous structure derived from the porous sponge from which the particles are produced.SELECTED DRAWING: None

Description

[Technical field]

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

[0002] This disclosure claims priority under 35 U.S.C. §119 to U.S. Provisional Application No. 62 / 573,892, filed October 18, 2017, the entire contents of which are incorporated herein by reference. [Background technology]

[0003] Currently, there is a demand for improved injectable materials for tissue treatment. For example, injectable materials such as hyaluronic acid-based materials may be used to treat various facial features (e.g., lines, wrinkles, insufficient size, or areas that are not of the desired shape or form). Such materials, no matter how effective, provide only temporary improvement and are eventually absorbed by the body and are unable to induce regeneration of tissues such as adipose tissue. Additionally, although research has been conducted to develop fat-based materials for tissue treatment and regeneration, current materials are not suitable for small volume procedures or injections or have not proven to be highly effective for fat regeneration. Summary of the Invention

[0004] Thus, the present disclosure provides compositions for injection, small volume transplantation, or larger void filling or volume addition with adipose-based tissue products. The present disclosure also provides methods of making such compositions.

[0005] The present disclosure provides a method of producing an injectable product from an adipose tissue matrix, the method including the steps of selecting adipose tissue, mechanically treating the adipose tissue to reduce the size of the tissue, treating the mechanically treated tissue to remove substantially all cellular material from the tissue, suspending the tissue in a solution to form a suspension, treating the suspension to produce a stabilized three-dimensional structure having a microporous structure, and mechanically treating the stabilized three-dimensional structure to produce particles.

[0006] The present disclosure also provides tissue product compositions, which can include a particulate tissue matrix, where the tissue product composition includes an adipose acellular tissue matrix formed into a particulate form after being formed into a porous sponge, where the particulate tissue matrix includes particles having a longest dimension of between about 0.05 mm and 3 mm.

[0007] The present disclosure also provides methods of treatment using the disclosed products.

[0008] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as defined by the appended claims. [Brief description of the drawings]

[0009] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the present disclosure and, together with the description, serve to explain the principles of the disclosure. [Figure 1] FIG. 1 is a flow chart outlining a process for producing an adipose tissue matrix sponge according to an embodiment of the present disclosure. [Diagram 2] FIG. 2 is a flow chart outlining a process for making an injectable adipose tissue matrix product according to an embodiment of the present disclosure. [Diagram 3] FIG. 3 shows a bulk piece of adipose tissue matrix sponge manufactured according to various embodiments. [Figure 4] FIG. 4 is a close-up of particulate tissue matrix having dimensions of 2-3 mm after being produced by grinding an adipose tissue matrix sponge. [Diagram 5] FIG. 5 shows a group of acellular tissue matrix particles produced by grinding or crushing an adipose tissue matrix sponge to produce particles of 100-300 microns and the resulting paste / pudding-like injectable material after hydration. [Figure 6] FIG. 6 shows scanning electron microscope (SEM) and atomic force microscope (AFM) images of adipose tissue matrix particles produced according to the process of the disclosed embodiment. [Figure 7] Figure 7A shows a differential scanning calorimetry curve of an adipose tissue matrix material produced according to disclosed embodiments, Figure 7B shows a Masson's Trichrome stained section of an adipose tissue matrix material produced according to disclosed embodiments, and Figure 7C shows a collagenase digestion curve of an adipose tissue produced according to disclosed embodiments with and without e-beam sterilization. [Figure 8] Figure 8A is a hematoxylin and eosin ("H&E") stained section of adipose tissue matrix material produced according to disclosed embodiments. Figure 8B is a table of DNA and lipid content measurements of adipose tissue produced according to disclosed embodiments. Figure 8C is an immunostained section of adipose tissue matrix material produced according to disclosed embodiments with no effect on MHC-I&II staining compared to a natural fat control. [Figure 9] FIG. 9 provides histological images of adipose tissue matrix produced according to the disclosed embodiments compared to a natural fat control and subjected to immunohistological analysis of major extracellular matrix proteins (e.g., type I, type III and type IV) collagen. [Figure 10] Figure 10 provides an optical microscopy image of an adipose tissue matrix generated according to disclosed embodiments supporting the growth of three different cell types in vitro (e.g., adipogenic mesenchymal stem cells, endothelial cells and dermal fibroblasts). [Figure 11] FIG. 11 provides a gross photograph of an injectable adipose tissue matrix explant after subcutaneous implantation in a nude rat prepared according to the disclosed embodiments. [Figure 12] FIG. 12 shows Masson's trichrome staining of the explants shown in FIG. [Figure 13] FIG. 13 is a bar graph showing the volume of explants remaining after 4 or 8 weeks of subcutaneous implantation in rats, as described in the disclosed examples. [Figure 14] Figure 14A is a Masson's Trichrome stained section of an 8-week explant as described in the disclosed examples, Figure 14B is another Masson's Trichrome stained section of an 8-week explant as described in the disclosed examples, and Figure 14C is another Masson's Trichrome stained section of an 8-week explant as described in the disclosed examples. [Figure 15] FIG. 15 provides microscopic images of aggregates formed in the wet adipose tissue matrix product after 1.5 years of storage. [Figure 16] FIG. 16 is a graph of the injection force of an adipose matrix product with and without HA as a flowable carrier. [Figure 17] FIG. 17 is an H&E stained section of a 4 week HA adipose explant as described in the Examples. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Reference will now be made in detail to certain exemplary embodiments in accordance with the present disclosure, examples of which are illustrated in the accompanying drawings, and wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts.

[0011] In this application, the use of the singular includes the plural unless specifically stated otherwise. In this application, the use of "or" means "and / or" unless specifically stated otherwise. Furthermore, the use of the term "including" as well as other forms such as "including" and "including" is not limiting. Any ranges described herein should be understood to include the endpoints and all values ​​between the endpoints.

[0012] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, articles, books, and papers, are expressly incorporated herein by reference in their entirety for any purpose.

[0013] As used herein, "tissue product" refers to any human or animal tissue that contains extracellular matrix proteins. "Tissue product" includes acellular or partially decellularized tissue matrix, as well as decellularized tissue matrix that has been repopulated with exogenous cells.

[0014] The term "acellular tissue matrix" as used herein refers to an extracellular matrix derived from human or animal tissue that retains a significant amount of native 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 are substantially free of other matrix proteins and do not retain the natural microstructural characteristics of tissue matrices due to the purification process. Although referred to as "acellular tissue matrix," it is understood that such tissue matrices are combined with exogenous cells, including, for example, stem cells and cells from the patient into whom the "acellular tissue matrix" is implanted. "Decellularized adipose tissue matrix" is understood to refer to adipose tissue from which all cells have been removed to generate an adipose extracellular matrix. "Decellularized adipose tissue matrix" may include intact matrix or matrix that has been further processed as discussed herein, including mechanical processing, forming a sponge and / or further processing to generate a particulate matrix.

[0015] It will be understood that "acellular" or "decellularized" tissue matrix refers to a tissue matrix in which no cells are visible using a light 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 regeneration, repair, augmentation, reinforcement and / or treatment of human tissues damaged or lost due to various diseases and / or structural damage (e.g., due to trauma, surgery, atrophy, and / or long-term wear and degeneration). Such products can include, for example, acellular tissue matrices, tissue allografts or xenografts, and / or reconstructed tissues (i.e., tissues that have been at least partially decellularized and seeded with cells to provide a material that can grow).

[0017] Various tissue products have been manufactured to treat soft and hard tissues. For example, ALLODERM® and STRATTICE™ (LIFECELL CORPORATION, Branchburg, NJ) are two dermal acellular 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 certain applications. For example, ALLODERM® and STRATTICE™ have been used to aid 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 regenerating, repairing, replacing and / or augmenting fat-containing tissues, where the production of adipose tissue containing viable adipocytes is the desired outcome. Thus, the present disclosure provides tissue products useful for the treatment of tissue defects / defects involving fat-containing tissues. The present disclosure also provides methods for producing such tissue products.

[0018] The tissue product may include adipose tissue that has been processed to remove at least a portion of the cellular components. In some cases, all or substantially all cellular material is removed, thereby leaving the adipose extracellular matrix proteins. The product may further be processed to remove some or all of the extracellular and / or intracellular lipids. In some cases, however, complete removal of the 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 treated for extended periods of time may have denatured or otherwise damaged collagen or may be depleted of proteins necessary for fat regeneration. Thus, in some cases, the product contains a 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. As further described below, the extracellular matrix proteins may be further processed to produce a three-dimensional porous or spongy material, and the porous or spongy material may be further processed to produce an injectable product.

[0019] As discussed above, the tissue products of the present 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 cadavers. Additionally, human adipose tissue may be obtained from a living donor (e.g., autologous tissue). Adipose tissue may also be obtained from animals, such as pigs, monkeys, or other sources. When animal sources are used, the tissue may be further processed to remove antigenic components, such as 1,3-α-galactose moieties, present in pigs and other mammals but not in humans or 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), which is incorporated by reference in its entirety. Additionally, the adipose tissue may be obtained from animals that have been genetically modified to remove antigenic moieties.

[0020] 1 and 2 illustrate an exemplary process for manufacturing the tissue products of the present disclosure. FIG. 1 provides a flow chart illustrating the basic steps to produce a suitable adipose tissue sponge that can then be further processed to produce an injectable or implantable particle. As illustrated, the process can include several steps, but it is understood that additional or alternative steps may be added or substituted depending on the particular tissue used, the desired application or other factors.

[0021] As shown, the process 100 may generally begin at step 110 with receiving tissue. The tissue may include various adipose tissue types, including, for example, human or animal adipose tissue. Suitable tissue sources include allograft tissue, autograft tissue, or xenograft tissue. When xenografts are used, the tissue may include fat from animals, including porcine, bovine, canine, feline, domestic or wild sources, and / or any other suitable mammalian or non-mammalian fat source.

[0022] The tissue may be harvested from the animal source using any desired technique, but will typically be harvested using aseptic or sterile techniques where possible. The tissue may be stored under cryogenic or frozen conditions or may be processed immediately to prevent undesirable changes due to long-term storage.

[0023] After receiving the tissue, it is first subjected to a process of mechanical size reduction at step 120 and / or mechanical defatting at step 130. Mechanical size reduction involves visible or gross cutting of the tissue using a manual blade or any other suitable comminuting process.

[0024] The mechanical defatting of step 130 is important in the preparation of tissue. Specifically, the fat is exposed to various mechanical processing conditions to aid in the removal of lipids. For example, mechanical processing can include grinding, mixing, shredding, grating, or other processing of the tissue. The mechanical processing can be performed under conditions that allow for some heating, which can aid in the liberation or removal of lipids. For example, the mechanical processing can be performed 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 thus the heat generated during mechanical disruption may be preferred to aid in lipid removal. In some examples, the heating during mechanical processing may be a pulse of elevated temperature and of short duration. This heat pulse can cause liquefaction of lipids released from fat cells destroyed by mechanical disruption, followed by 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 further reduced to about 80° F., 90° F., 100° F., 110° F., or 120° F. when processing less saturated tissue, such as primate tissue. Alternatively, the process can be selected such that the fat reaches a minimum temperature, for example, 80° F., 90° F., 100° F., 110° F., 120° F.

[0025] In some cases, mechanical defatting can be performed by mechanically treating the tissue with little or no washing solution. For example, the tissue may be mechanically treated by grinding or mixing without the use of a solvent. Alternatively, if water is required for tissue disruption, for example to increase flowability or reduce viscosity, water can be used, including pure water, saline, or other buffers, including saline or phosphate buffered saline. In some instances, the tissue is treated by adding a certain amount of a solvent that is biocompatible, 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 be appropriate.

[0026] In step 140, after mechanical treatment and lipid removal, the fat can be washed. For example, the tissue can be washed with one or more rinses with various biocompatible buffers. For example, suitable washing solutions include saline, phosphate buffered saline, or other suitable biocompatible materials or physiological solutions. In one example, water can be used as a rinse to further disrupt the cells, after which 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, and the free lipids flow to the top while the extracellular matrix proteins are deposited as a pellet. The protein pellet is then resuspended, and washing and centrifugation are repeated until a sufficient amount of lipids is removed.

[0028] After washing, the fat may be treated to remove some or all of the cells from the adipose tissue, as shown in step 150. The cell removal process may include a number of suitable processes. For example, suitable methods for removing cells from adipose tissue include treatment with a detergent, such as deoxycholic acid, polyethylene glycol, or other detergent, at a concentration and for a time sufficient to disrupt the cells and / or remove cellular components.

[0029] After cell removal, as shown in step 160, additional processing and / or washing steps can be incorporated depending on the tissue used or the desired final structure. For example, additional washing or processing can be performed to remove antigenic material, such as antigenic components, such as alpha-1,3-galactose moieties that may be present in non-primate animal tissue. Additionally, additional solutions or reagents can be used to treat the material during, before and / or after the washing steps. For example, enzymes, detergents 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 bacterial or other bioburden of the material. For example, one or more washing steps can be included using detergents such as sodium dodecyl sulfate or Triton to aid in the removal of cells and lipids. Additionally, enzymes, such as lipases, DNAses, RNAses, alpha-galactosidases or other enzymes can be used to ensure the destruction of nuclear material, antigens from xenogeneic sources, residual cellular components and / or viruses. Additionally, acidic solutions and / or peroxides may be used to further remove cellular material and to help destroy bacteria and / or viruses, or other potential infectious agents.

[0030] The material can be formed into a porous or sponge-like material after removing the lipid and cellular components. As shown in step 170, typically the extracellular matrix is ​​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 can vary based on the desired porosity of the final material. In some cases, the slurry-like material can have a solids 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 mechanically processed one or more times further 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 into a container or mold to form a porous, sponge-like product, as shown in step 180. Typically, the porous or sponge-like material is formed by drying the material to leave a three-dimensional matrix of porous structures. In some embodiments, the material is freeze-dried. Freeze-drying allows for the production of a three-dimensional structure that generally conforms to the shape of the mold, as shown in FIG. 3. The specific freeze-drying protocol can be modified based on the solvent used, sample size, and / or to optimize 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, further cooling the sample to -40°C to completely freeze; applying a vacuum; raising the temperature to -5°C and holding for 30-60 hours; 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 the solid or sponge, the material may optionally be stabilized, as shown in step 190. In some cases, stabilization may include additional processes such as cross-linking, treatment with 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 may result in loss of the desired shape and size. Additionally, the porous structure, which is important for supporting cell attachment, tissue growth, blood vessel formation, and tissue regeneration, may be lost. For this reason, the material may be further processed to stabilize the size, shape, and structure of the material.

[0033] In some embodiments, the material is crosslinked for stabilization. In some embodiments, the material is crosslinked after lyophilization. However, the material can also be crosslinked before or during the lyophilization process. Crosslinking can be performed in a variety of ways. In one embodiment, crosslinking can be performed by contacting the material with a crosslinking agent such as glutaraldehyde, genepin, carbodiimides (e.g., 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)) and diisocyanates. Additionally, crosslinking can be performed by heating the material in a vacuum. For example, in some embodiments, the material can be heated under reduced pressure or vacuum to 70°C-120°C, or 80°C-110°C, or up to about 100°C, or any value within a specified range. Additionally, other crosslinking processes, or combinations of processes, including ultraviolet radiation, gamma radiation, and / or electron beam radiation, can be used to produce any of the disclosed products. Additionally, a vacuum is not required, but may shorten the crosslinking time. Additionally, lower or higher temperatures can be used as long as melting of the matrix protein does not occur and / or sufficient time is provided for cross-linking.

[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 is controlled to produce the desired strength. Additionally, the amount of crosslinking can affect the ability of the product to maintain a desired shape and structure (e.g., porosity) upon implantation. Thus, 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 tissues or materials).

[0035] Excessive crosslinking can alter the extracellular matrix material. For example, excessive crosslinking can damage collagen or other extracellular matrix proteins. The damaged proteins may not support tissue regeneration when the tissue product is placed in an adipose tissue site or other anatomical location. Additionally, excessive crosslinking can cause the material to become brittle or weak. For this reason, the amount of crosslinking is controlled to provide a desired level of stability while maintaining the desired biological, mechanical and / or structural characteristics.

[0036] An exemplary crosslinking process may include contacting the freeze-dried material produced as described above with glutaraldehyde or EDC. For example, a 0.1% glutaraldehyde solution may be used, and the tissue is soaked in the solution for about 18 hours, followed by extensive rinsing with water and removal of the solution. Alternatively, or in combination, a dehydrothermal (DHT) process may be used. For example, one exemplary dehydrothermal process includes treating the material at 100° C., about 20 inches of mercury, for 18 hours, followed by soaking in water. The final crosslinked tissue product may be stored in a film pouch.

[0037] After formation of the 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 FIG. 2 as process 200. It should be understood that while "injectable" can include materials that are injected with a syringe, cannula, or needle, 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 other bulk equipment such as spatulas, tubes, or other devices equipped to handle flowable materials).

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

[0039] After the bulk sponge material is obtained, the material is subjected to 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 the desired size and size distribution. In some aspects of the present disclosure, when the initial material is a dry sponge, grinding may be preferred to reduce the dry sponge into smaller particles. Size reduction may be performed at room temperature.

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

[0041] 2, in the next step 230, size selection may be desirable. For example, once the stabilized sponge has been milled or processed to produce particles, it may be sieved or otherwise selected to obtain particles of a desired size for the flowable / injectable adipose tissue matrix material. In some instances, 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, the milled sponge may 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 the present disclosure, particle sizes in this range may not elicit different biological responses. In other words, particle sizes ranging from, for example, 50 microns to 2,800 microns may not result in a different biological response. For various applications requiring a specific size needle, particles of a particular size may be selected without needing to consider whether the biological response will differ.

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

[0043] Exemplary particulate materials are shown in Figures 4 and 5. Figure 4 is a close-up of particulate tissue matrix having dimensions of 2-3 mm after production by grinding an adipose tissue matrix sponge. Figure 5 shows a group of acellular tissue matrix particles produced by grinding or crushing an adipose tissue matrix sponge to yield particles of 100-300 microns, and a paste / pudding-like injectable material after hydration.

[0044] According to certain aspects of the present disclosure, a material having a desired tissue matrix solids content can be used. For example, a material having a solids content of 5% to 12% can be used, with a material having a solids content of 7.5% to 10% being preferred. In another embodiment, a suitable carrier can be used with the 5% to 10% solids material to facilitate injection and prevent particle escape from the injection site. A suitable carrier is a flowable carrier, for example, a flowable 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, a "hyaluronic acid-based material" is a material that includes hyaluronic acid (HA). HA refers to hyaluronic acid and any salts thereof, including, but not limited to, sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate, calcium hyaluronate, and combinations thereof. Hyaluronic acid-based materials can include both HA and its pharma-ceutically acceptable salts. Exemplary HA-based materials are commercially available as JUVEDERM® and JUVEDERM VOLUMA®. It is understood that hyaluronic acid-based materials can include additional agents, such as, for example, lidocaine.

[0046] All numbers herein referring to the "molecular weight" of HA should be understood to refer to the weight average molecular weight (Mw) in Daltons.

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

[0048] As used herein, high molecular weight HA is at least about 1.0 million daltons (Mw≧10 6 "High molecular weight HA" refers to HA materials having a molecular weight of from about 1.5 MDa to about 3.0 MDa. The high molecular weight HA incorporated into the tissue product composition can have a molecular weight in the range of about 1.5 MDa to about 3.0 MDa, or the high molecular weight HA can have a weight average molecular weight of about 2.0 MDa. In another example, the high molecular weight HA can have a molecular weight of about 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 a molecular weight of about 200,000 Da (0.2 MDa) to less than 1.0 MDa, for example, about 300,000 Da (0.3 MDa) to about 750,000 Da (0.75 MDa), but up to 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, the 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 embodiment of the present invention, the adipose tissue product composition comprises HA having a high molecular weight component and a low molecular weight component, the high molecular weight component having a weight average molecular weight at least twice that of the low molecular weight component. For example, the molecular weight ratio of the high molecular weight HA to the low molecular weight HA in the composition may be at least 2:1. For example, the tissue product composition may comprise 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 or at least about 1.0 MDa. In another example, the tissue product composition of the present invention may comprise 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 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 above examples 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 through 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 of which are 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 crosslinkers include, but are not limited to, multifunctional PEG-based crosslinkers 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) suberate (BS), hexamethylenediamine (HMDA), 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, or combinations thereof.

[0052] In one exemplary embodiment of the adipose tissue product composition formed according to the present invention, the adipose tissue product composition comprises a flowable carrier comprising a hyaluronic acid-based material and a plurality of adipose tissue matrix particles mixed with the carrier. In some exemplary embodiments, the flowable carrier comprises HA that is not mixed with an additional agent, and in other exemplary embodiments, the flowable carrier comprises HA mixed with an additional agent. The additional agent includes, but is not limited to, an anesthetic agent, such as an aminoamide local anesthetic and its salt, or an aminoester local anesthetic and its salt. For example, procaine, chloroprocaine, cocaine, cyclomethycaine, simetocaine, propoxycaine, procaine, proparacaine, tetracaine, or its salt, or any combination thereof. In some embodiments, the anesthetic agent includes articaine, bupivacaine, cinchocaine, etidocaine, levobupivacaine, lidocaine, mepivacaine, piperocaine, prilocaine, ropivacaine, trimecaine, or its salt, or any combination thereof.

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

[0054] Surgical implantation is a suitable option for implanting adipose tissue matrix material to repair certain areas of the body, although injection may be preferred in some applications. Particulation of adipose tissue matrix has been found to improve application and injection compared to application of adipose tissue matrix in its natural form, but pure adipose tissue matrix, even in particulated form, has been found not to be easily applied or injected into patients. In particular, application of particulate tissue matrix material has been found to be difficult to control, as particulate tissue matrix material has a tendency to diffuse or aggregate after storage. Furthermore, the injection force required to inject particulate acellular 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 problems of injecting adipose tissue matrix material, exemplary embodiments described herein provide tissue product compositions comprising adipose tissue matrix particles mixed within a flowable carrier comprising a hyaluronic acid-based material. The resulting tissue product composition can be applied more easily than pure adipose tissue matrix particles while maintaining properties that promote tissue growth at the implantation and / or injection area, as further described herein.

[0056] In an exemplary embodiment, the adipose tissue matrix particles and the flowable carrier may be mixed in a large batch under substantially sterile conditions to form a tissue product composition of the present invention. Mixing may include, for example, stirring the adipose tissue matrix particles and the flowable carrier together to form a slurry. Mixing parameters and techniques may vary according to the characteristics of the flowable carrier and acellular tissue matrix particles and the approximate amounts of each in the tissue product composition, and can be readily deduced by one of ordinary skill in the art through routine experimentation.

[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 solution at 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, in contrast, are hyaluronic acids crosslinked with an EDC crosslinker with different G' and G" values ​​depending on the degree of crosslinking. HA type 2 had a G' value of 160 Pa, and HA type 3 had a G' value of 500-550 Pa. HA type 4 is also crosslinked. It has a G' value of 350 Pa.

[0058] Various hyaluronic acid-based materials can be mixed with the adipose tissue matrix particles to produce various tissue product compositions as set forth below in Table 1. It should be understood that the hyaluronic acid-based materials described herein are merely exemplary, and that other hyaluronic acid-based materials can be mixed with the adipose tissue matrix particles. Additionally, the compositions shown in Table 1 are merely exemplary, and other formulations of tissue product compositions can be formed in accordance with the present invention. TIFF2025011092000001.tif121170

[0059] Referring now to Table 1, exemplary embodiments of tissue product compositions formed in accordance with the present invention are described. Table 1 illustrates compositions 1-4, which are representative of various tissue product compositions, however, it is understood that other tissue product compositions can be formed in accordance with the present invention. In each of compositions 1-4, the adipose tissue matrix particles were derived from porcine adipose tissue, which when combined with the flowable carrier resulted in an adipose tissue matrix slurry, also referred to as "flowable adipose tissue matrix." Prior to being combined with the flowable carrier, which was provided at a concentration of 20 mg HA / mL, the adipose tissue matrix particles were at a concentration of 100 mg / mL in aqueous buffer. As can be seen from Table 1, the ratio of fat matrix:HA can be adjusted to produce slurries with various flow characteristics, as further described herein. It is understood that the ratios described herein are either by volume or by mass, and in the exemplary embodiment shown in Table 1, the ratios are given as volume fat matrix:volume HA. As illustrated in compositions 1 and 2, the ratio of fat matrix:HA is 9:1, and as illustrated in compositions 3 and 4, the ratio of fat matrix:HA is 4:1. It should be understood that these ratios are merely exemplary and that other exemplary embodiments of the tissue product compositions may have other ratios of fat matrix:HA, including any value between the disclosed ratios.

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

[0061] As mentioned above, the tissue product should have the ability to support cell ingrowth and tissue regeneration when implanted in or on a patient. Additionally, the tissue product should be capable of acting as a carrier for cells, including stem cells such as adipose-derived stem cells, and supporting the growth of such cells. To this end, the process must not alter the extracellular matrix proteins in an unacceptable manner (e.g., by damaging the protein structure and / or removing important glycosaminoglycans and / or growth factors). In some embodiments, the product will have normal collagen banding as evidenced by transmission electron microscopy.

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

[0063] Adipose tissue matrix is ​​rich in types IV and VI collagen, and the ratio of these two collagens to type I collagen may differ from that of the dermal matrix, which may be an important factor used to distinguish adipose from 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 the present disclosure are generated from an adipose tissue matrix. For this reason, it is believed that adipose tissue products provide superior regenerative capabilities when implanted into certain tissue sites compared to materials made from other tissue types. In some cases, the tissue products may be implanted into tissue sites that are primarily or substantially adipose tissue. In some cases, the tissue products may be used as facial fillers, for example, to treat lines, wrinkles, voids, or depressions, to add volume, or to replace lost tissue. In some cases, the tissue site may include the breast (e.g., for breast augmentation, replacement of excised tissue, or placement around an implant). Additionally, any site that includes other adipose tissue may be selected. For example, the tissue products may be used for reconstructive or cosmetic applications in the face, buttocks, abdomen, hips, thighs, or any other site where additional adipose tissue with a structure and feel similar to natural fat is desired. In any of these sites, the tissue may be used to reduce or eliminate wrinkles, sagging, or undesirable shapes.

[0065] When used for breast tissue replacement or augmentation, the tissues can offer advantages over other tissue products. For example, while some tissue products allow for ingrowth and tissue formation, they do not mimic the texture and feel of normal breasts and may form significant fibrous tissue that appears abnormal on radiological images. Because the tissue products of the present disclosure are formed from fat, they can support more normal regeneration of adipose tissue.

[0066] Additionally, the tissue product may be used as a carrier for cells. For example, the product may be implanted or used at any of the sites described above, but seeded with cells. In some cases, such cells may include stem cells, such as adipose-derived stem cells. Additionally, 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 into the tissue after or before implantation. Additionally, cells may be cultured on the tissue product prior to implantation, and then implanted in or on the body.

[0067] As discussed above, the particulate tissue matrix product may further include a flowable carrier to facilitate injection of the tissue product. In various embodiments, the particulate tissue matrix and the flowable carrier are packaged in separate containers and do not come into contact until mixed immediately prior to injection. For example, the particulate tissue matrix and the flowable carrier are packaged in separate barrels of a multi-barrel syringe and mixed immediately prior to or as the contents are injected. In other embodiments, the particulate tissue matrix and the flowable carrier are premixed and packaged together. The materials are dried separately or stored in a biocompatible buffer that 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 examples are intended to illustrate the disclosure but not to limit it in any way. EXAMPLES

[0069] A. Preparation of adipose tissue material: To produce the adipose tissue matrix material, porcine adipose tissue was first sliced ​​into 2-inch strips and coarsely chopped with 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 at room temperature or 4°C overnight. The pre-processed material was coarsely ground with a RETSCH® GM300 (GRINDOMIX) at 2000 rpm and then further ground at 4000 rpm to effect phase separation of the oil from the solid matrix. The adipose matrix solids were harvested by centrifugation and washed with buffer. The matrix material was decellularized with EDTA-Triton solution overnight at room temperature, changing the solution every 4 hours. The matrix proteins were washed again. During the washes, the matrix pellet was centrifuged to pellet the tissue matrix and the spent solution was decanted. The suspension was again mechanically milled to further break down 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 and freeze-dried to form a sponge, followed by DHT treatment to stabilize the material. The stabilized sponge was then crushed and sieved to obtain particles of the desired size as a flowable / injectable porcine acellular tissue slurry (PATS) material. The particulate material was reduced to a 5-15% paste and underwent terminal sterilization by electron beam.

[0070] B. The process produces an intact collagen structure: The tissue product produced by the above process was subjected to analysis by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The results showed that the tissue product was a porous scaffold containing collagen material with a typical collagen banding pattern including structure, porosity, etc., as shown in Figure 6. The microscope shows intact collagen with a normal banding pattern.

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

[0072] The tissues were also subjected to Masson's trichrome staining and collagenase digestion assay. Collagenase digestion was performed on tissues produced as described above with and without e-beam sterilization. The staining and collagenase digestion results are shown in Figures 7B and 7C, respectively. Specifically, Figure 7C shows collagenase digestion curves of adipose tissue produced according to the disclosed embodiments with and without e-beam sterilization. In the absence of e-beam sterilization and with DHT stabilization alone, the percentage of solids remaining as a function of digestion time drops to about 18% after 8 hours. On the other hand, the combined use of e-beam sterilization and DHT stabilization results in a percentage of solids remaining approaching about 25% after the same digestion period, slightly higher than DHT alone.

[0073] With continued reference to Figures 7B and 7C, the material had trichrome staining indicating normal collagen on trichrome sections and no change in collagenase sensitivity even after terminal sterilization by electron beam. In general, when collagen is trichrome stained, normal collagen should appear blue rather than red. Taken together, the microscopy, DSC, staining and collagenase digestion indicate preserved collagen.

[0074] C. The described process is efficient in removing cells, cell debris and oil within the scaffold: Samples prepared as described were subjected to 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 I and II components, as shown in Figure 8C (showing tissue matrix vs. native fat control). H&E histology demonstrated that the tissue products exhibited a porous structure with no signs of cells. Consistent with this observation, the tissue products had very little residual DNA and free oil. Immunostaining demonstrated that the tissue products were free of effects of MHC-I and II staining, indicating that the described process is efficient for decellularization.

[0075] D. The tissue product retained the major matrix components of native fat: The tissues were subjected to immunohistological analysis of major extracellular matrix proteins including types I, III and IV. Referring to Figure 9, the adipose tissue products (bottom row) retained the original properties of the native adipose matrix (top row).

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

[0077] F. Tissue products retain volume and support adipogenesis in vivo: The biological performance of the product was tested in a subcutaneous nude rat model. As shown in Figure 11, the study was designed to test various formulations of the product.

[0078] The products manufactured 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, a material obtained immediately after the decellularization process, without freeze-drying or any subsequent steps. ARM6 was used as a control to compare with tissue products with DHT-stabilized 3D microporous structures, as in ARM1-5. ARM7 contained a sponge prepared with a similar process, but from an acellular tissue matrix of the dermis. The dermal tissue was Strattice®.

[0079] ARM1-5 were first hydrated in saline at 10% solids by weight to create an injectable paste, and ARM6 was similarly adjusted to 10% solids. ARM7 was a 10 mm punch of 5 mm thickness from a dermal sponge and hydrated with PBS. Approximately 0.5 cc aliquots of each injectable adipose material (ARM1-6) and the 10 mm punch of ARM7 were implanted into the subcutaneous region of nude rats for each ARM in triplicate.

[0080] At 4 weeks, explants were harvested for gross observation and subjected to histological analysis. FIG. 11 shows gross observation of injectable adipose tissue matrix explants after subcutaneous implantation in nude rats. The dotted line indicates the injected material. All explants were soft to palpation. Injectable fat of all particle size ranges lasted for at least 4 weeks. It was also observed that injectable fat implants of certain particle sizes lasted well for at least 12 weeks (data not shown).

[0081] Figure 12 shows additional Masson's trichrome staining of explants at low magnification. A strong adipogenic response accompanied by tissue ingrowth was observed in ARM1-4 prepared from stabilized PATS sponges. In contrast, adipose tissue matrix slurry in ARM6 lacking 3D structure (i.e., lyophilization and stabilization were omitted) did not induce an adipogenic response (c).

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

[0083] The volume retention of representative injectable adipose matrix products (ARM1: 2.8-3.4 mm, ARM2: 0.8-1.0 mm, ARM5: 0.1-1.0 mm) was also evaluated for 8 weeks along with a slurry of ARM6, as shown in Figure 13. All of the particles with porous microstructures, ARM1, 2, and 5, retained 84% or more of the explant volume compared to the implanted volume for up to 8 weeks.

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

[0085] Figures 14A-C show enlarged Masson's trichrome stained sections of ARM2 explants, showing good adipogenesis. Adipocytes were the white areas, abundant and large, and vascularization was evident. Similar histological findings were seen in ARM5 samples (not shown).

[0086] Taken together, the results showed that adipose tissue matrix particles of all sizes tested (e.g., 50 μm to 3.4 mm) maintained volume and showed good cell infiltration and revascularization responses up to 8 weeks, as shown in Figure 11 and Figure 13. Data is shown in Figure 12 for 4 weeks only. On the other hand, in the control ARM6, a slurry consisting of the same material prepared by the same process but not subjected to freeze-drying and DHT stabilization, no adipose tissue ingrowth response was observed in the grafts, as shown in ARM6 in Figure 12. As ARM6 lacks a microporous structure, the results suggest that microporous pore structure may be important or noteworthy during adipogenesis. In addition, ARM6 suffered significant volume loss at the end of 8 weeks (Figure 13), further indicating that DHT stabilization of the tissue product is important for volume retention in vivo. Furthermore, adipose tissue ingrowth was specific to the adipose matrix and was absent in intact sponge-like scaffolds prepared with dermal acellular matrix using a similar process, as shown in ARM7 in Figure 12.

[0087] G. Using hyaluronic acid (HA) as a carrier enhanced the injectability of adipose tissue products: FIG. 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, the adipose tissue matrix material in wet configuration with 10% solids formed aggregates after 1.5 years of storage. Biological testing indicates that aggregation does not affect the biological performance of the material, such as volume retention and adipose tissue ingrowth 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 facilitate the injectability of the adipose tissue material, 20 mg / mL of non-crosslinked hyaluronic acid (HA) carrier was mixed with freshly prepared adipose tissue matrix (10%, 0.1–1.0 mm) or 1.5-year-old adipose tissue matrix (10%, 0.1–1 mm). The final concentration of HA additive in the mixed material was 2 mg / mL. The compressive load (N) required to inject adipose material with and without HA additive was evaluated with an Instron Model 5865 Material Tester (Instron Corporation, Norwood, MA). Adipose material without HA was injected through either a 16G or 18G needle, and adipose material with HA additive was injected through an 18G needle. The average compressive force (n=3) over time for products with and without HA additive was calculated and plotted.

[0089] As shown in Table 2, without HA as a carrier, injection of fresh adipose material using a 16G needle was smooth, but 1.5-year-old fat encountered significant resistance. With the addition of HA as a carrier, both fresh and 1.5-year-old adipose materials became injectable. TIFF2025011092000002.tif54170

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

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

[0092] While the principles of the disclosure have been described herein with reference to exemplary embodiments for particular applications, it should be understood that the disclosure is not limited thereto. Those skilled in the art and with access to the teachings provided herein will recognize that additional modifications, applications, embodiments, and equivalent substitutions are all within the scope of the embodiments described herein. Thus, the invention should not be considered as limited by the foregoing description.

Claims

1. 1. A method for producing a tissue product, comprising: selecting adipose tissue; mechanically processing the adipose tissue to reduce the size of the tissue; treating the mechanically treated tissue to remove substantially all cellular material from the tissue; suspending the tissue in a solution to form a suspension; processing the suspension to produce a stabilized three-dimensional structure having a microporous structure; and mechanically treating the stabilized three-dimensional structure to generate particles.

2. 10. The method of claim 1 , The method, wherein treating the suspension to produce a stabilized three-dimensional structure comprises drying the suspension.

3. 3. The method of claim 2, The method, wherein drying comprises freeze-drying.

4. The method according to any one of claims 1 to 3, The method, wherein treating the suspension to produce a stabilized three-dimensional structure includes crosslinking the tissue.

5. 5. The method of claim 4, The method, wherein crosslinking comprises treatment with at least one of a chemical, a light-activated crosslinking process, or heat.

6. 6. The method of claim 5, The method, wherein the chemicals include at least one of glutaraldehyde, genepin, carbodiimide, and diisocyanate.

7. 5. The method of claim 4, The method, wherein crosslinking comprises heating the tissue.

8. 8. The method of claim 7, A method characterized in that the tissue is heated in a vacuum.

9. The method according to claim 4 or 7, The method is characterized in that the tissue is heated to between 70°C and 120°C.

10. 10. The method of claim 1 , The method is characterized in that the stabilized three-dimensional structure maintains its microporous structure when in contact with an aqueous environment.

11. 11. The method of claim 10, The method is characterized in that the material maintains a stable three-dimensional structure when implanted in the body.

12. The method according to any one of claims 1 to 11, The method, wherein mechanically treating the stabilized three-dimensional structure to produce particles includes producing particles comprising a longest dimension between 50 microns and 4 mm.

13. 12. The method according to any one of claims 1 to 11, The method, wherein mechanically treating the stabilized three-dimensional structure to produce particles includes producing particles comprising a longest dimension of between 1.5 mm and 4 mm.

14. 12. The method according to any one of claims 1 to 11, The method, wherein mechanically treating the stabilized three-dimensional structure to produce particles includes producing particles comprising a longest dimension of between 2 mm and 3.5 mm.

15. The method according to any one of claims 1 to 11, The method, wherein mechanically treating the stabilized three-dimensional structure to produce particles includes producing particles comprising a longest dimension of between 2 mm and 3 mm.

16. 16. The method according to any one of claims 1 to 15, The method of claim 1, wherein mechanically treating the stabilized three-dimensional structure to produce particles includes producing particles having a retained microporous structure from the stabilized three-dimensional structure.

17. 17. The method according to any one of claims 1 to 16, The method further comprising the step of hydrating the particles.

18. 18. The method of claim 17, The method, wherein hydrating the particles includes mixing the particles with a volume of an aqueous solution such that the particles and the aqueous solution form a composition in which the particles are 5% to 20% by weight.

19. 20. The method of claim 18, The method, wherein hydrating the particles includes mixing the particles with a volume of an aqueous solution such that the particles and the aqueous solution form a composition having between 5% and 12% particles by weight.

20. 20. A tissue product produced by the method of any one of claims 1 to 19.

21. 1. A tissue product composition comprising:

1. A tissue product composition comprising a particulate tissue matrix, the tissue product composition comprising adipose acellular tissue matrix formed into a particulate form after being formed into a porous sponge, the particulate tissue matrix comprising particles having a longest dimension of about 0.05 mm to 3 mm.

22. 22. The tissue product composition of claim 21, A tissue product composition, wherein the particles comprise a microporous structure derived from a porous sponge.

23. 23. The tissue product composition of claim 21 or 22, A tissue product composition, wherein the particles are dry.

24. 23. The tissue product composition of claim 21 or 22, A tissue product composition further comprising a flowable carrier.

25. 25. The tissue product composition of claim 24, A tissue product composition, wherein the flowable carrier comprises an aqueous fluid.

26. 26. The tissue product composition of claim 25, A tissue product composition, wherein the flowable carrier comprises saline.

27. 27. The tissue product composition of claim 25 or 26, A tissue product composition, characterized in that the composition comprises 80-95% water by weight.

28. 27. The tissue product composition of claim 25 or 26, A tissue product composition, wherein the composition comprises from 5% to 15% by weight of adipose tissue matrix protein.

29. 27. The tissue product composition of any one of claims 24 to 26, A tissue product composition, wherein the fluid carrier is a fluid hyaluronic acid carrier.

30. 30. The tissue product composition of claim 29, A tissue product composition, wherein the hyaluronic acid is not crosslinked.

31. 30. The tissue product composition of claim 29, A tissue product composition, characterized in that the hyaluronic acid is crosslinked.

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