Adipose tissue products and methods of manufacture

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] FIELD OF THE DISCLOSURE 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, which is incorporated herein by reference in its entirety. [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 are sometimes used to treat various facial features (e.g., lines, wrinkles, insufficient size, or areas that do not have the desired shape or form). However effective, such materials only provide temporary improvement, are eventually absorbed by the body, and are unable to induce the regeneration of tissues such as adipose tissue. Furthermore, while research has been conducted to develop adipose-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 for 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 adipose acellular tissue matrix formed into a particulate form after being formed into a porous sponge, the particulate tissue matrix including particles having a longest dimension of about 0.05 mm to 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 explanation 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. [Figure 2] FIG. 2 is a flow chart outlining a process for manufacturing an injectable adipose tissue matrix product according to an embodiment of the present disclosure. [Figure 3] FIG. 3 shows a bulk piece of adipose tissue matrix sponge manufactured in accordance with various embodiments. [Figure 4] FIG. 4 is a magnified view of particulate tissue matrix having dimensions of 2-3 mm after being produced by grinding the adipose tissue matrix sponge. [Figure 5] Figure 5 shows a group of acellular tissue matrix particles produced by crushing or grinding 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 example. [Figure 7] Figure 7A shows a differential scanning calorimetry curve of an adipose tissue matrix material produced according to a disclosed embodiment, Figure 7B shows a Masson's Trichrome stained section of an adipose tissue matrix material produced according to a disclosed embodiment, and Figure 7C shows a collagenase digestion curve of adipose tissue produced according to a disclosed embodiment with and without electron 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] Figure 9 provides histological images of adipose tissue matrix produced according to the disclosed examples 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 microscope image of an adipose tissue matrix produced according to an embodiment of the disclosure that supports 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 an embodiment of the disclosure. [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 described in the disclosed examples, Figure 14B is another Masson's Trichrome stained section of an 8-week explant described in the disclosed examples, and Figure 14C is another Masson's Trichrome stained section of an 8-week explant described in the disclosed examples. [Figure 15] FIG. 15 provides a microscopic image 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 adipose matrix products 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 described in the Examples. DETAILED DESCRIPTION OF THE INVENTION

[0010] Reference will now be made in detail to certain exemplary embodiments consistent with the present disclosure, examples of which are illustrated in the accompanying drawings. 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 "comprising" and other forms such as "includes" and "including" is not limiting. Any range described herein should be understood to include the 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. 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] As used herein, the term "acellular tissue matrix" 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" differs 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. While referred to as "acellular tissue matrix," it is understood that such tissue matrices are combined with exogenous cells, including, for example, stem cells or 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 produce an adipose extracellular matrix. "Decellularized adipose tissue matrix" can include intact matrix or matrix that has been further processed as discussed herein, including mechanical processing, sponge formation, and / or further processing to produce 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 the regeneration, repair, augmentation, reinforcement, and / or treatment of human tissue 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., at least partially decellularized tissues seeded with cells to provide a material upon which growth can occur).

[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 acellular tissue matrices manufactured from human and porcine dermis, respectively. While such materials are highly 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 tissue, where the desired outcome is the production of adipose tissue containing viable adipocytes. Therefore, the present disclosure provides tissue products useful for treating tissue defects / defects involving fat-containing tissue. The present disclosure also provides methods for manufacturing such tissue products.

[0018] Tissue products can include adipose tissue that has been processed to remove at least a portion of its cellular components. In some cases, all or substantially all cellular material is removed, thereby leaving behind adipose extracellular matrix proteins. The product can further be processed to remove some or all of the extracellular and / or intracellular lipids. However, in some cases, complete removal of the extracellular and / or intracellular lipids can damage the structure and function of the adipose matrix. For example, adipose tissue that has been chemically or enzymatically processed for extended periods of time can denature or otherwise damage collagen or be depleted of proteins necessary for adipose regeneration. Therefore, in some cases, the product contains a level of residual lipid. The residual lipid content can be, for example, about 5%, 6%, 7%, 8%, 9%, or 10% by weight of the product. As further described below, the extracellular matrix proteins can be further processed to produce three-dimensional porous or spongy materials, and the porous or spongy materials can be further processed to produce injectable products.

[0019] As described above, the tissue products of the present disclosure are formed from adipose tissue. The adipose tissue can be derived from human or animal sources. For example, human adipose tissue can be obtained from cadavers. Additionally, human adipose tissue can be obtained from living donors (e.g., autologous tissue). Adipose tissue can also be obtained from animals, such as pigs or monkeys, or other sources. When using animal sources, the tissue can be further processed to remove antigenic components, such as 1,3-α-galactose moieties, which are 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, pp. 1-13 (2009), which is incorporated by reference in its entirety. Additionally, the adipose tissue can 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. Figure 1 provides a flow chart illustrating the basic steps to produce a suitable adipose tissue sponge, which can then be further processed to produce an injectable or implantable particle. As illustrated, the process may include several steps, but it should be understood that additional or alternative steps may be added or substituted depending on the particular tissue used, the desired application, or other factors.

[0021] As shown, process 100 generally may 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] Tissues may be harvested from animal sources using any desired technique, but will typically be harvested using aseptic or sterile techniques where possible. Tissues 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 mechanical size reduction process at step 120 and / or a mechanical defatting process at step 130. Mechanical size reduction involves visible or large cuts of the tissue using a manual blade or any other suitable comminution process.

[0024] Mechanical defatting, step 130, is important in tissue preparation. Specifically, fat is subjected to various mechanical processing conditions to aid in lipid removal. For example, mechanical processing can include grinding, blending, shredding, grating, or other processing of the tissue. Mechanical processing can be performed under conditions that allow for some heating, which can aid in lipid liberation or removal. For example, mechanical processing can be performed under conditions that heat the adipose tissue to a maximum of 122°F (50°C). Application of external heat may be insufficient to release lipids, and thus, heat generated during mechanical disruption may be preferred to aid lipid removal. In some instances, heating during mechanical processing may be a short-duration pulse of elevated temperature. This heat pulse can cause liquefaction of lipids released from adipocytes disrupted by mechanical disruption, subsequently resulting in efficient phase separation for bulk lipid removal. In one example, when processing porcine adipose tissue, temperatures reached during the process exceed 100°F, but do not exceed 122°F (50°C). The temperature range reached can be adjusted depending on the source 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 so that the fat reaches a minimum temperature of, for example, 80°F, 90°F, 100°F, 110°F, or 120°F.

[0025] In some cases, mechanical delipidation can be performed by mechanically processing the tissue with little or no washing solution. For example, the tissue may be mechanically processed by grinding or mixing without the use of a solvent. Alternatively, if water is required for tissue disruption, e.g., to increase fluidity 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 processed 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, 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 wash 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, followed by the introduction of phosphate-buffered saline or other suitable saline 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 adipose tissue 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 multiple 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 materials, such as alpha-1,3-galactose moieties, that may be present in non-primate 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 materials, and / or reduce the bacterial or other bioburden of the material. For example, one or more washing steps can be included that use 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-galactosidase, or other enzymes, can be used to ensure the destruction of nuclear material, antigens from foreign sources, residual cellular components, and / or viruses. Additionally, acidic solutions and / or peroxides may be used to further remove cellular material and help destroy bacteria and / or viruses, or other potential infectious agents.

[0030] After removing lipid and cellular components, the material can be formed into a porous or sponge-like material. As shown in step 170, the extracellular matrix is ​​typically 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 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 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. Specific freeze-drying protocols 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; 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 solid or sponge is formed, the material can optionally be stabilized, as shown in step 190. In some cases, stabilization can include additional processes such as crosslinking, treatment with a dehydrothermal (DHT) process, or other suitable stabilization methods. For example, mechanically processed tissues, when formed into a porous matrix, typically form a more putty- or paste-like material when implanted in the body, wetted, or placed in solution. This can result in a loss of the desired shape and size. Furthermore, the porous structure, which is important for supporting cell attachment, tissue growth, vascularization, and tissue regeneration, can be lost. Therefore, 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 lyophilization. However, the material can also be crosslinked before or during the lyophilization process. Crosslinking can be accomplished in a variety of ways. In one embodiment, crosslinking can be achieved 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 achieved by heating the material under vacuum. For example, in some embodiments, the material can be heated under reduced pressure or vacuum to 70°C to 120°C, or 80°C to 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 irradiation, gamma irradiation, and / or electron beam irradiation, can also be used to produce any of the disclosed products. Furthermore, 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. Furthermore, 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. Damaged proteins may not support tissue regeneration when the tissue product is placed in an adipose tissue site or other anatomical location. Furthermore, excessive crosslinking can cause the material to become brittle or weak. Therefore, 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 can include contacting the freeze-dried material produced as described above with glutaraldehyde or EDC. For example, a 0.1% glutaraldehyde solution can be used, and the tissue is immersed in the solution for approximately 18 hours, followed by extensive rinsing with water and removal of the solution. Alternatively, or in combination, a dehydrothermal (DHT) process can be used. For example, one exemplary dehydrothermal process involves treating the material at 100°C and 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 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 a spatula, tube, or other device equipped to handle flowable materials).

[0038] The process of creating 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 a desired size and size distribution. In some aspects of the present disclosure, if 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 sponge size reduction 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 lipogenesis.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] Continuing with reference to FIG. 2 , size selection may be desirable in the next step 230. For example, once the stabilized sponge has been crushed or processed to generate 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 with different particle sizes may be desired 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 range is between 50 microns and 2,800 microns. For example, the crushed sponge may be sieved to recover particles of a preferred size, 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 within 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 specific size may be selected without needing to consider whether a different biological response will occur.

[0042] Once the particle size is selected, the next step 240 involves hydrating the particles and / or adding them to another carrier to the desired extent to achieve flowability and a desired level of solids content. For example, the sieved particles may be hydrated with saline or other materials to achieve a solids concentration of 5-12%. Additionally, other carriers, including hyaluronic acid-based materials (e.g., JUVEDERM®, ALLERGAN®, or similar materials), may be used or added. 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 may be performed before step 230, and the particles may 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 being produced 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 the paste / pudding-like injectable material after hydration.

[0044] According to certain aspects of the present disclosure, materials having a desired tissue matrix solids content can be used. For example, materials having a solids content of 5% to 12% can be used, with 7.5% to 10% being preferred. In another embodiment, a suitable carrier may be used with a 5% to 10% solids material to facilitate injection and prevent particle escape from the injection site. A suitable carrier is a flowable carrier, such as 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" refers to a material 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 can include both HA and its pharmaceutically acceptable salts. Exemplary HA-based materials are commercially available under the trademarks JUVEDERM® and JUVEDERM VOLUMA®. It should be understood that hyaluronic acid-based materials can include additional agents, such as lidocaine.

[0046] All figures 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 No. 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 an HA material 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 ranging from 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 an HA material 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 not more than 0.99 MDa. Preferably, there is no overlap between the molecular weight distributions of the low molecular weight HA material and the high molecular weight HA material. 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, wherein the high molecular weight component has 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 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 1.0 MDa 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 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 above examples are not intended to be limiting.

[0051] In some exemplary embodiments, the HA is cross-linked using one or more suitable cross-linking agents. The cross-linking agent may be any agent known to be suitable for cross-linking polysaccharides and their derivatives through their hydroxyl groups. Suitable cross-linking agents include, but are not limited to, 1,4-butanediol diglycidyl ether (also known as 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 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 an 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 without any additional agent mixed therewith, while in other exemplary embodiments, the flowable carrier comprises HA mixed with an additional agent. The additional agent may include, but is not limited to, an anesthetic, such as an aminoamide local anesthetic and its salt, or an aminoester local anesthetic and its salt. For example, procaine, chloroprocaine, cocaine, cyclomethicaine, simetocaine, propoxycaine, procaine, proparacaine, tetracaine, or a salt thereof, or any combination thereof. In some embodiments, the anesthetic may include articaine, bupivacaine, cinchocaine, etidocaine, levobupivacaine, lidocaine, mepivacaine, piperocaine, prilocaine, ropivacaine, trimecaine, or a salt thereof, 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 the flowable carrier.

[0054] While surgical implantation is a suitable option for implanting adipose tissue matrix material to repair specific areas of the body, injection may be preferred for some applications. While particulating adipose tissue matrix has been shown to improve application and injection compared to application of the adipose tissue matrix in its natural form, pure adipose tissue matrix, even in particulated form, has been found to be difficult to apply or inject into patients. In particular, the application of particulated tissue matrix material has been found to be difficult to control, as particulated tissue matrix materials tend to diffuse or aggregate after storage. Furthermore, the injection force required to inject particulate acellular tissue matrix is ​​relatively high, making it 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 with 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 compositions can be applied more easily than pure adipose tissue matrix particles while maintaining properties that promote tissue growth in the implantation and / or injection area, as further described herein.

[0056] In one 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 determined 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 contained in a 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 uncrosslinked hyaluronic acid with a G' value of 320 Pa. HA Type 2 and HA Type 3, in contrast, are hyaluronic acid crosslinked with an EDC crosslinker, with G' and G" values ​​varying depending on the degree of crosslinking. HA Type 2 had a G' value of 160 Pa, while HA Type 3 had a G' value of 500-550 Pa. HA Type 4 is also crosslinked; its G' value is 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 in Table 1 below. 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. Furthermore, the compositions shown in Table 1 are merely exemplary, and other tissue product compositions can be formed in accordance with the present invention. TIFF0007785886000001.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 represent various tissue product compositions, but it should be 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 and, when combined with the flowable carrier, resulted in an adipose tissue matrix slurry, also referred to as "flowable adipose tissue matrix." Prior to mixing 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 should be understood that the ratios described herein are either by volume or by mass, and that in the exemplary embodiment shown in Table 1, the ratios are given as volume fat matrix:volume HA. As exemplified in compositions 1 and 2, the ratio of fat matrix:HA is 9:1, and as exemplified 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 fatty 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 with 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 10% acellular fat matrix particle solids content, corresponding to 100 mg / mL.

[0061] As noted above, tissue products should be capable of supporting cellular ingrowth and tissue regeneration when implanted in or on a patient. Furthermore, the tissue products should be capable of acting as carriers 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 extracellular matrix proteins in an unacceptable manner (e.g., by damaging protein structure and / or removing important glycosaminoglycans and / or growth factors). In some embodiments, the products will have normal collagen banding as evidenced by transmission electron microscopy.

[0062] In various embodiments, the tissue product is processed to preserve either or both of native hyaluronic acid and chondroitin sulfate. Thus, the tissue product may contain either or both of hyaluronic acid and chondroitin sulfate. Additionally, processes can be selected that preserve native growth factors. 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 ​​enriched in types IV and VI collagen. 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 and dermal matrices 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, adipose tissue products are believed to offer 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 significantly composed of 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, excised tissue replacement, or peri-implant placement). Additionally, any site containing other adipose tissue can be selected. For example, tissue products can 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 can be used to reduce or eliminate wrinkles, sagging, or undesirable contours.

[0065] When used for breast tissue replacement or augmentation, the tissue 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 a normal breast and can form significant fibrous tissue that appears abnormal on radiographic images. Because the tissue products of the present disclosure are formed from fat, they can support more normal regeneration of adipose tissue.

[0066] Additionally, tissue products may be used as carriers for cells. For example, the product may be implanted or used at any of the aforementioned sites and then 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 tissue before or after implantation. Additionally, cells may be cultured on the tissue product before implantation and then implanted in or onto the body.

[0067] As described above, the particulate tissue matrix product can 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 at the time of injection. In other embodiments, the particulate tissue matrix and the flowable carrier are premixed and packaged together. The materials can 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 examples are intended to illustrate, but not limit, the present disclosure in any way. [Example]

[0069] A. Preparation of adipose tissue material: To prepare the adipose tissue matrix material, porcine adipose tissue was first sliced ​​into 2-inch strips and coarsely minced using a food-grade meat chopper. The minced 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 using a RETSCH® GM300 (GRINDOMIX) grinder at 2000 rpm, followed by further grinding at 4000 rpm to achieve phase separation of 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 with an EDTA-Triton solution, with the solution changed every 4 hours. The matrix proteins were washed again. During the wash, the matrix pellet was centrifuged to pellet the tissue matrix, and the spent solution was decanted. The suspension was again mechanically triturated 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 made into a 5-15% paste and subjected to 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 scanning electron microscopy (SEM) and atomic force microscopy (AFM) analysis. The results showed that the tissue product was a porous scaffold containing collagen material with a typical collagen banding pattern, including structure and porosity, 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 was 61.5°C. In this example, the onset melting temperature of the tissue product was similar to that of the native tissue (e.g., the source fat).

[0072] The tissues were also subjected to Masson's trichrome staining and collagenase digestion assays. Collagenase digestion was performed on tissues prepared 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 for adipose tissue prepared according to the disclosed embodiments, with and without e-beam sterilization. Without e-beam sterilization and with DHT stabilization alone, the percentage of remaining solids as a function of digestion time drops to approximately 18% after 8 hours. In contrast, with e-beam sterilization and DHT stabilization combined, the percentage of remaining solids approaches approximately 25%, slightly higher than DHT alone, after the same digestion period.

[0073] With continued reference to Figures 7B and 7C, the material had trichrome staining, indicating intact collagen on trichrome sections, and its collagenase sensitivity was unchanged even after terminal sterilization by electron beam. Generally, when collagen is trichrome stained, intact 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 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 I and II components, as shown in Figure 8C (showing tissue matrix vs. native fat control). H&E histology revealed that the tissue product had a porous structure with no signs of cells. Consistent with this observation, the tissue product had very little residual DNA and free oil. Immunostaining revealed that the tissue product was free of MHC I and II staining, indicating that the described process is efficient for decellularization.

[0075] D. The tissue product retained the primary matrix component 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 product (bottom row) retained the original properties of the native adipose matrix (top row).

[0076] E. Tissue products support the growth of multiple tissue cells: To test the potential of the tissue product sponge to support the growth of adipose tissue, vasculature, and other connective tissues, such as dermal tissue, three different cell types were selected. Specifically, primary cells isolated from normal human individuals were tested: 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. Cell proliferation was quantified by DNA content using a fluorescent dye using the CyQUANT® Cell Proliferation Assay Kit. The tissue product was analyzed for cell proliferation. As shown in Figure 10, cell-seeded scaffolds were stained with a live / dead stain and observed for cell viability and growth under a fluorescent microscope. The tissue product supported the growth of all three cell types in vitro.

[0077] F. Tissue products retain volume and support in vivo adipogenesis: 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 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; and ARM5: 0.1-1 mm. The sixth ARM, ARM6, actually contained slurry and fibers, a material obtained immediately after the decellularization process, without lyophilization or any subsequent steps. ARM6 was used as a control for comparison with tissue products with DHT-stabilized 3D microporous structures, like ARM1-5. ARM7 contained a sponge prepared by a similar process, but derived from a dermal acellular tissue matrix. The dermal tissue was Strattice®.

[0079] ARMs 1-5 were first hydrated in saline to a solids content of 10% by weight to create an injectable paste, and ARM 6 was similarly adjusted to a solids content of 10%. ARM 7 was a 10 mm punch, 5 mm thick, from a dermal sponge and hydrated with PBS. Approximately 0.5 cc aliquots of each injectable fat material (ARMs 1-6) and the 10 mm punch of ARM 7 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. Figure 11 shows the 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 persisted for at least 4 weeks. It was also observed that injectable fat implants of certain particle sizes persisted 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 ARMs 1-4, which were prepared from stabilized PATS sponges. In contrast, the adipose tissue matrix slurry in ARM 6, which lacked a 3D structure (i.e., lyophilization and stabilization were omitted), did not induce an adipogenic response (c).

[0082] Furthermore, 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, Masson's trichrome staining revealed no adipose tissue regeneration in the dermal scaffold.

[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. ARM1, 2, and 5, which are particles with porous microstructures, all 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 due to the stabilized material containing DHT (data not shown), they may form similar pores when in contact with each other, which is fundamentally different from the slurry material of ARM6. In contrast, grafts without a microporous structure, such as the adipose tissue slurry morphology (e.g., ARM6), were difficult to detect or very flat when palpated, indicating significant graft volume loss. Explant weight decreased over time, with only approximately 38% remaining at the end of 8 weeks, indicating significant graft volume loss.

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

[0086] Taken together, the results, as shown in Figures 11 and 13, demonstrated that adipose tissue matrix particles of all sizes tested (e.g., 50 μm to 3.4 mm) maintained their volume and demonstrated favorable cell infiltration and revascularization responses for up to 8 weeks. Data are shown for only 4 weeks in Figure 12. In contrast, in the control ARM6, a slurry composed of the same material prepared using the same process but without freeze-drying and DHT stabilization, no adipose tissue ingrowth response was observed in the implants, as shown for ARM6 in Figure 12. Given that ARM6 lacks a microporous structure, these results suggest that microporous structures may be important or noteworthy during adipogenesis. Furthermore, ARM6 underwent 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 for ARM7 in Figure 12.

[0087] G. Using hyaluronic acid (HA) as a carrier enhanced 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, the adipose tissue matrix material in a wet configuration containing 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 noninvasive delivery in a clinical setting, the 10% adipose tissue product may require a carrier to facilitate injectability.

[0088] To facilitate the injectability of adipose tissue materials, 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 materials with and without HA additive was evaluated using an Instron Model 5865 Materials Tester (Instron Corporation, Norwood, MA). Adipose materials without HA were injected through either a 16-gauge or 18-gauge needle, while adipose materials with HA additive were injected through an 18-gauge 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 the 1.5-year-old fat encountered significant resistance. When HA was added as a carrier, both fresh and 1.5-year-old adipose material became injectable. TIFF0007785886000002.tif54170

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

[0091] To evaluate the biological performance of the mixture of adipose product and HA additive, 20 mg / mL cross-linked hyaluronic acid (HA type 4) was mixed with adipose tissue matrix (10%) at a ratio of 1:10, 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 present disclosure have been described herein with reference to illustrative 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 present invention should not be deemed limited by the foregoing description.

Claims

1. A tissue product composition comprising:

1. A tissue product composition comprising an adipose acellular tissue matrix formed into a porous sponge and then formed into a particulate tissue composition, the particulate composition comprising particles of adipose tissue matrix and 5% to 10% by weight of residual lipids, the particles comprising a microporous structure derived from the porous sponge from which they were produced.

2. In the tissue product composition according to claim 1, A tissue product composition wherein the particles are dry.

3. In the tissue product composition according to claim 1 or 2, A tissue product composition, wherein the composition comprises 5% to 15% by weight of a solid adipose acellular tissue matrix.

4. The tissue product composition according to any one of claims 1 to 3, A tissue product composition wherein said particles have a longest dimension of between 0.05 mm and 4 mm.

5. The tissue product composition according to claim 4, A tissue product composition wherein said particles of adipose tissue matrix have a longest dimension of between 50 microns and 4 mm.

6. The tissue product composition according to claim 4, A tissue product composition wherein said particles of adipose tissue matrix have a longest dimension of between 1 mm and 1.5 mm.

7. The tissue product composition of claim 4, A tissue product composition wherein said particles of adipose tissue matrix have a longest dimension of between 2 mm and 3.5 mm.

8. The tissue product composition of claim 1, A tissue product composition wherein said particles of adipose tissue matrix have a longest dimension of between 2 mm and 3.5 mm.

9. A tissue product composition according to claim 1, further comprising a fluid carrier.

10. The tissue product composition of claim 9, A tissue product composition wherein the flowable carrier comprises an aqueous fluid.

11. The tissue product composition of claim 10, A tissue product composition wherein the fluid carrier comprises saline.

12. The tissue product composition of claim 10 or 11, A tissue product composition, wherein the composition comprises 80% to 95% water by weight.

13. The tissue product composition of claim 9 or 10, A tissue product composition, wherein the fluid carrier is a fluid hyaluronic acid carrier.

14. The tissue product composition of claim 13, A tissue product composition characterized in that the hyaluronic acid is not cross-linked.

15. The tissue product composition of claim 13, A tissue product composition characterized in that the hyaluronic acid is crosslinked.

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