WO2015103054A1

WO2015103054A1 - Adipose tissue biocomposites

Info

Publication number: WO2015103054A1
Application number: PCT/US2014/072260
Authority: WO
Inventors: John Raymond CHAPMAN
Original assignee: MicroAire Surgical Instruments LLC
Current assignee: MicroAire Surgical Instruments LLC
Priority date: 2013-12-31
Filing date: 2014-12-23
Publication date: 2015-07-09
Anticipated expiration: 2016-06-30

Abstract

Aspects of the present disclosure include adipose tissue biocomposites. Adipose tissue biocomposites according to certain embodiments include adipose tissue in a fibrin network such that the fibrin network is configured to break down after a period of time to release the adipose tissue component from the biocomposite. Adipose tissue biocomposites in certain embodiments include two layers, a first layer having an adipose tissue component and a second layer having fibrin and thrombin. Methods for preparing and using adipose tissue biocomposites of the disclosure are also described.

Description

ADIPOSE TISSUE BIOCOMPOSITES AND METHODS FOR MAKING AND

USING THE SAME

CROSS-REFERENCE TO RELATED APPLICATIONS Pursuant to 35 U.S.C. § 1 19 (e), this application claims priority to the filing date of United States Provisional Patent Application No. 61/922,1 15, filed December 31 , 2013; United States Provisional Patent Application No. 61/925,240 filed on January 9, 2014 and United States Provisional Patent Application No. 61/925,246 filed on January 9, 2014; the disclosures of which applications are herein incorporated by reference.

INTRODUCTION

When the serous or underlying vascularized layers of the human body are disrupted, either by a traumatic injury or a deliberate surgical procedure, the body mounts a complex inflammatory wound healing response to repair the defect. Initial trauma leads to increased histamine-mediated vascular permeability and bathing of the injured tissue's local environment in inflammatory exudates, resulting in repair characterized by mesothelial and fibroblast proliferation and, depending on the extent of injury, cell-mediated contraction.

Adhesion formation is a complication of serosal repair following surgery, ischemia, or infection, and leads to conditions such as intestinal obstruction, severe abdominal pain, and infertility. Intraperitoneal adhesions, for example, occur in 67 to 93% of general abdominal surgeries and at an even higher rate following open gynecological pelvic surgeries. Adhesions have been shown to form in many body site sites including the peritoneal, pleural, and pelvic cavities. In addition to surgical trauma, ischemia from surgical repair (grafting, suturing), mechanical effects of handling body tissues, foreign materials (e.g., starch), inflammation-induced peritonitis, blood, and serosal drying have been shown to lead to adhesion formation. Postoperative adhesions often require removal by additional surgical procedures. For example, when

adhesiolysis is performed to remove an intestinal obstruction, adhesions reform and create a subsequent obstruction in approximately 1 1 to 21 percent of postoperative surgeries. SUMMARY

Aspects of the present disclosure include adipose tissue biocomposites. Adipose tissue biocomposites according to certain embodiments include an adipose tissue component in a fibrin network such that the fibrin network is configured to break down after a period of time to release the adipose tissue component from the biocomposite. Methods for preparing and using adipose tissue biocomposites of the disclosure are also described.

In embodiments of the present disclosure, the adipose tissue biocomposites component includes adipose tissue, such as adipose tissue fragments from lipoaspirate, viable stem cells and tissue plasminogen activator. The fibrin network includes thrombin and fibrin. In some embodiments, the fibrin network also includes plasminogen. In some embodiments, the adipose tissue biocomposite is layered. In some instances, the adipose tissue biocomposite includes a first layer having an adipose tissue component in a fibrin network and a second layer having thrombin and fibrin. In other instances, the adipose tissue biocomposite includes a first layer having the adipose tissue component and a second layer having the fibrin network of thrombin and fibrin.

Aspects of the disclosure also include methods for preparing the subject adipose tissue biocomposites. Methods according to certain embodiments include contacting an adipose tissue component with thrombin and fibrinogen in a manner sufficient to form the adipose tissue biocomposite such that the fibrin network formed by the thrombin and fibrinogen is configured to break down after a period of time to release the adipose tissue component from the biocomposite. In some embodiments, methods include applying the adipose tissue component with thrombin to a body site of a subject (e.g., a wound site) in a manner sufficient to produce the adipose tissue biocomposite at the body site. In other embodiments, methods include mixing the adipose tissue component with thrombin and fibrinogen in a three-dimensional mold. In certain embodiments, methods include preparing adipose tissue biocomposites having two or more layers. In some instances, the adipose tissue component is contacted with thrombin and fibrinogen in a container followed by subsequent formation of two layers in the container, where the first layer includes the adipose tissue component in a fibrin network and a second layer includes thrombin and fibrin. In other instances, the adipose tissue component is contacted with thrombin and fibrinogen in a container followed by subsequent formation of two layers in the container, where the first layer includes the adipose tissue component and the second layer includes the fibrin network of thrombin and fibrin. Forming the two layers may include contacting the adipose tissue component with thrombin and fibrinogen in a container and allowing the adipose tissue component to separate into a layer from the thrombin and fibrinogen during conversion of fibrinogen to fibrin by thrombin. In other embodiments, forming the two layers may include contacting the adipose tissue component with thrombin and fibrinogen in a container and allowing the adipose tissue component to separate into a layer from the thrombin and fibrinogen before conversion of fibrinogen to fibrin by thrombin.

Aspects of the disclosure also include methods for using the adipose tissue biocomposites. Methods according to certain embodiments include applying one or more of the subject adipose tissue biocomposites to a body site of a subject where fibrin network is configured to break down at the body site after a period of time to release the adipose tissue component from the biocomposite. In some embodiments the body site is a wound site (e.g., trauma or surgery), such as a wound site that is susceptible to forming an adhesion during wound repair. In some instances, the adipose tissue biocomposite is prepared and applied directly to the wound site, such as by applying the adipose tissue component with thrombin to a body site of a subject in a manner sufficient to produce the adipose tissue biocomposite at the body site. In other instances, the adipose tissue biocomposite is first prepared in a container (e.g., in a three-dimensional mold) and applied to the body site.

In some embodiments, methods include inhibiting adhesions from occurring at a wound site (e.g., surgical sites). In certain instances, inhibiting adhesion of apposing internal human body tissue layers after a surgical procedure is achieved by means of applying an adipose tissue graft, where the method includes the steps of: a) performing lipoplasty to derive a plurality of adipose tissue fragments from a donor; b) harvesting the plurality of adipose tissue fragments from the donor; c) contacting the plurality of adipose tissue fragments with an amount of thrombin; and d) applying the mixture of the adipose tissue fragments and the thrombin between opposing internal human body tissue layers. In some embodiments, the subject adipose tissue biocomposites inhibit adhesion formation in two different ways. In some instances, the adipose tissue biocomposite is configured as a biocompatible physical barrier separating the opposing tissue layers at risk of forming an adhesion. In other instances, the adipose tissue biocomposite is configured to be bioactive and capable of causing fibrinolysis with the passage of time. Specifically, adipose tissue biocomposites of interest activate the fibrinolysis cascade. In some embodiments, dissolving of the fibrin network in the subject adipose tissue biocomposites corresponded with the viability of the cells in the adipose tissue fragments. The living cells within the biocomposite actively secrete proteins that promote fibrinolysis by leading to the conversion of plasminogen to plasmin. The fibrin structure stabilizes these molecules and acts as substrate. In embodiments, the subject adipose tissue biocomposites exhibit biocompatibility, barrier and bioactivity in fibrinolysis. Adipose tissue biocomposites are also configured, in some embodiments, to control both the fibrin deposition/degradation equilibrium, such as in the treatment of adhesion formation. In certain embodiments, adipose tissue biocomposites of interest are configured as a bioactive barrier to inhibit adhesion formation, where one or more components of the adipose tissue biocomposite is derived from the patient's own body (i.e., one or more components are autologous).

In some embodiments, the disclosed adipose graft biocomposite temporarily prevents apposition of serosal tissue surfaces by separating the adhesiogenic tissue while the normal tissue repair process occurs. Subsequent degradation and clearance of the body's own adipose tissue, in certain instances, prevents a foreign body response involving fibrosis of the implant or local toxicity. The disclosed adipose graft

biocomposite is safe, effective, nonimmunogenic, noninflammatory, and is configured to separate adhesiogenic tissue during remesothelialization, biodegrades, and remains functional in the presence of blood products. Further, the disclosed adipose graft biocomposite does not interfere with the healing process, nor does it promote infection or abscess formation. In some embodiments, adipose tissue biocomposites of interest are configured to inhibit formation of adhesions. In other embodiments, the adipose tissue biocomposites exhibit ease of surgical use with respect to handling, application, retention at the wound site and applicability to both open and minimally invasive surgical procedures and facilitate combination with local drug application when necessary. In embodiments, the present invention is bioactive due to it having living cells and these cells secrete proteins that achieve fibrinolysis which, in some instances, inhibit adhesion formation beyond being a simple passive barrier.

In some embodiments, the subject adipose tissue biocomposites are prepared by combining adipose tissue, plasma and thrombin and can be readily mixed and molded into stable, three-dimensional structures. In some instances, blood plasma serves as a source for both plasminogen for fibrinolysis and fibrinogen for forming the fibrin network. In some embodiments, the adipose tissue biocomposite has a structure that is transient. In some instances, adipose tissue biocomposites of interest include an adipose tissue component in a fibrin network where the fibrin network is configured to break down after a period of time to release the adipose tissue component from the biocomposite. In one example, the fibrin network holding the biocomposite together is stable for hours at room temperature and 37°C and begins to break down after about 6 to 10 hours. In these embodiments, the fibrin network breaks down and thereby releases the adipose tissue fragments from the fibrin network. Some other tissues, for example blood, do not possess the ability to cause dissolution of the fibrin network even after storage for days. In some embodiments, the adipose tissue biocomposite passively inhibits adhesion formation by acting as a physical barrier separating the two opposing layers that are at risk of forming an adhesion. In other embodiments, the adipose tissue biocomposite actively inhibits adhesion formation by secreting proteins including tissue plasminogen activator that activates plasminogen into plasmin and promotes fibrinolysis. In some embodiments, the subject adipose tissue biocomposites include endothelial cells that are activated by the contact with thrombin and fibrin and produce greater production of tissue plasminogen activator than inhibitors.

In some embodiments, methods include performing lipoplasty to derive a plurality of adipose tissue fragments containing at least one viable stem cell from a donor, harvesting the plurality of adipose tissue fragments from the donor, placing the plurality of adipose tissue fragments in contact with an amount of thrombin, fibrinogen to achieve an appropriate gelling reaction and plasminogen and applying the mixture of the adipose tissue fragments, fibrin and plasminogen to a body site (e.g., wound site) of a subject, such as to inhibit adhesion formation by the plasminogen being converted to plasmin which results in localized fibrinolysis which attacks the root cause of adhesion formation (nascent fibrin strands).

In some embodiments, each of the above steps occurs within the same surgical procedure at the point of care, and the adipose tissue biocomposite may be applied to the subject in the form of a liquid biocomposite, a molded gel biocomposite and gel biocomposite fragments. In some instances, a liquid adipose tissue biocomposite is prepared by mixing adipose tissue fragments, thrombin and fibrinogen in liquid form such that the combination solidifies into a hydrogel. In other instances, a mixture of adipose tissue fragments, thrombin and fibrinogen is introduced into a three-dimensional mold cavity so that the adipose tissue biocomposite forms into a three-dimensional shaped gel biocomposite. In these instances, the molded adipose tissue biocomposite can be positioned between two layers of tissue, such as at a wound site at risk of forming an adhesion, so as to create a physical barrier separating the tissue layers.

In certain embodiments, the physical characteristics of the subject adipose tissue biocomposites is achieved by controlling the relative percentage of the graft volume derived from adipose tissue fragments and controlling the type and amount of the fibrinogen and the thrombin. In some instances, adipose tissue biocomposites of interest include an adipose tissue component that is 5% to 95% of the total volume of the adipose tissue biocomposite. In embodiments, the fibrin network is configured to break down over a period of time to release the adipose tissue component from the

biocomposite. In certain instances, a greater concentration of the adipose tissue component in the biocomposite results in more rapid fibrin network dissolution. As discussed in greater detail below, the relative amount of components in the subject adipose tissue biocomposite may vary, such as to maintain the passive physical barrier (e.g., decreased adipose tissue = longer barrier action) and the activation of the fibrinolysis pathway (e.g., more adipose tissue = more vigorous fibrinolytic activation).

In some embodiments, one or more supplements are added to the adipose tissue biocomposite, such as to enhance the therapeutic potential of the biocomposite. These supplements may be added to the mixture prior to the initiation of the gelling reaction. By supplementing the graft with such materials, customized and more therapeutic grafts can be prepared. In accordance with another aspect of the present invention, a method for preparing an improved adipose tissue biocomposite graft with a wound-healing promoter to inhibit adhesion formation is disclosed. In one example, the present invention provides an adipose tissue biocomposite graft that inhibits the occurrence of postsurgical adhesions. In a second example, the present invention provides an adipose tissue biocomposite graft characterized by sufficient tensile strength to be easily handled and placed by an operator. In a third example, the present invention provides a biocomposite graft, which is easily processed, molded, and customized to precise dimensions. In another example, the present invention provides an adipose tissue biocomposite that is supplemented with additives to obtain customized and more therapeutic grafts, including the addition of antibiotics to the mixture. In yet another example, the present invention provides a method for generating fully autologous adipose tissue composite grafts from a donor to treat surgical sites, such as to prevent adhesions. Still in another example, the present invention, provides three-dimensional molds to prepare multiple castings of the adipose tissue biocomposite grafts for an individual donor.

BRIEF DESCRIPTION OF THE FIGURES

The invention may be best understood from the following detailed description when read in conjunction with the accompanying drawings. Included in the drawings are the following figures:

FIG. 1 is a first operational flow chart for preparing an adipose tissue

biocomposite according to certain embodiments.

FIG. 2 is a second operational flow chart for preparing an adipose tissue biocomposite according to certain embodiments.

FIG. 3 is an operational flow chart for preparing an adipose tissue biocomposite with a wound-healing promoter according to certain embodiments.

FIG. 4 is an operational flow chart for preparing an adipose tissue biocomposite utilizing a syringe according to certain embodiments.

FIG. 5 illustrates an example of an adipose tissue biocomposite graft prepared using a rectangular three dimensional mold cavity according to certain embodiments.

FIG. 6 illustrates an example of an adipose tissue biocomposite graft prepared using a circular three dimensional mold cavity according to certain embodiments.

FIG. 7A-7E depict photographs of adipose tissue biocomposites prepared according to certain embodiments.

DETAILED DESCRIPTION

Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. As summarized above, the present disclosure provides adipose tissue biocomposites. In further describing embodiments of the disclosure, adipose tissue biocomposites that include an adipose tissue component in a fibrin network are first described in greater detail. Next, methods for preparing the subject adipose tissue biocomposites from an adipose tissue component, fibrinogen and thrombin are described. Methods for using the subject biocomposites as well as kits are also provided.

ADIPOSE TISSUE BIOCOMPOSITES

As summarized above, aspects of the present disclosure include adipose tissue biocomposites. Adipose tissue biocomposites according to certain embodiments include an adipose tissue component in a fibrin network such that the fibrin network is configured to break down after a period of time to release the adipose tissue component from the biocomposite. By "period of time" is meant that the subject adipose tissue biocomposites are configured such that the fibrin network breaks apart (e.g., by fibronlysis) over a period of time, such as over the course of hours, days and including weeks. In some embodiments, the subject adipose tissue biocomposites are configured to break down over the course of hours, such as 1 hour or longer, such as 2 hours or longer, such as 3 hours or longer, such as 5 hours or longer, such as 6 hours or longer and including over the course of 12 hours or longer. In other embodiments, the fibrin network in the adipose tissue biocomposites is configured to break down over the course of days, such as 1 day or longer, such as 2 days or longer, such as 3 days or longer, such as 5 days or longer and including 7 days or longer. As described in greater detail below, in certain embodiments the adipose tissue biocomposites are prepared such that the fibrin network is configured to break down over a predetermined period of time such as after 6 hours or longer, such as after 12 hours or longer, such as after 24 hours or longer, such as after 48 hours or longer, such as after 72 hours or longer, such as after 96 hours or longer, such as after 120 hours or longer, such as after 144 hours or longer and including after 168 hours or longer. By "break down" is meant degrade or disrupt such that the biocomposite no longer exists in its original structure, and instead has been disrupted into components thereof which are not unified in structure.

In embodiments, adipose tissue biocomposites may be configured to be applied to a body site of a subject, such as a wound site. In describing the subject

biocomposites, the term "subject" is meant the person or organism to which the adipose tissue biocomposite is applied and maintained in contact. As such, subjects may include but are not limited to mammals, e.g., humans and other primates, such as chimpanzees and other apes and monkey species; and the like, as well as non-human subjects such as, but not limited to, birds, mice, rats, dogs, cats, livestock and horses. In certain embodiments, the subject is a human. The adipose tissue biocomposites may be configured to be applied to any convenient internal or external location on the subject, such as to organ tissue including but not limited to integumentary tissue (e.g. sections of the skin), oral tissue (e.g., buccal, tongue, palatal, gums), respiratory tissue (e.g., pharynx, larynx, trachea, bronchi, lungs, diaphragm) gastrointestinal tissue (e.g., esophagus, stomach, liver, gallbladder, pancreas, intestines, colon, rectum and anus.), cardiovascular tissue (e.g., heart, blood vessels), endocrine tissue (e.g., hypothalamus, pituitary gland, pineal body or pineal gland, thyroid, parathyroids, adrenal glands) and genitourinary tissue (kidneys, ureters, bladder, urethra, ovaries, fallopian tubes, uterus, vagina, mammary glands, testes, vas deferens, seminal vesicles, prostate, penis), muscular tissue, nervous tissue (e.g., brain, spinal cord, nerves) as well as soft skeletal tissue (cartilage, ligaments, tendons). Furthermore, the adipose tissue biocomposites may be applied to any type of organismic tissue, including both healthy and diseased tissue (e.g., cancerous, malignant, necrotic, etc.), where desired.

In embodiments, adipose tissue biocomposites include an adipose tissue component. In some embodiments, the adipose tissue component includes adipose tissue, such as in the form of adipose tissue fragments. The term "adipose tissue" is used herein in its conventional sense to refer to the lipophilic connective tissue in the body that contains adipocytes as well as the stromal vascular fraction having

preadipocytes, fibroblasts, vascular endothelial cells, mesenchymal stem cells, immune cells (e.g., adipose tissue macrophages) as well as endothelial precursor cells that can secrete tissue repair proteins (e.g., tissue plasminogen activator, tissue plasminogen inhibitor). Adipose tissue, in some instances, also includes the hormones produced by the adipose tissue, such as leptin, estrogen, resistin, and the cytokine TNFa. In some embodiments, adipose tissue is fat obtained from the body of the subject, such as abdominal fat, epicardial fat, subcutaneous fat and ectopic fat, among other types of fats.

In some embodiments, the adipose tissue is in the form of a plurality of adipose tissue fragments. The fragments may be homogeneous in shape and size or more may be different. In some embodiments, the adipose tissue fragments have the same shapes and sizes. In other embodiments, the adipose tissue fragments have different shapes and sizes. The size of the fragments vary depending on the source of the adipose tissue as well as any processing following obtaining the adipose tissue fragments and may have a median diameter which ranges, such as from 1 μηι to 5000 μηι, such as from 10 μηι to 4500 μηι, such as from 50 μηι to 4000 μηι, such as from 75 μηι to 3500 μηι, such as from 100 μηι to 3000 μηι, such as from 250 μηι to 2500 μηι and including a median diameter from 500 μηι to 1500 μηι. The amount of adipose tissue in the adipose tissue biocomposites may vary ranging from 1 g to 10,000 g, such as from 5 g to 9000 g, such as from 10 g to 8000 g, such as from 15 g to 7000 g, such as from 20 g to 6000 g, such as from 25 g to 5000 g, such as from 30 g to 4000 g, such as from 35 g to 3000 g, such as from 40 g to 2000 g, such as from 50 g to 1000 g and including from 100 g to 500 g. In embodiments, and 70% or more of the cells contained in these same the adipose tissue fragments are viable at the time of application to the body as the subject adipose tissue biocomposite, such as 75% or more, such as 80% or more, such as 85% or more, such as 90% or more, such as 95% or more and including 99% or more of the adipose tissue fragments are viable at the time of application to the body as the subject adipose tissue biocomposite.

In some instances, the adipose tissue component also includes viable stem cells. The term "stem cells" is used herein in its conventional sense to refer to undifferentiated biological cells that can differentiate into specialized cells and can divide to produce more stem cells. In some embodiments, the adipose tissue component includes viable stem cells that are present in the adipose tissue, such as mesenchymal stem cells and stromal stem cells. In other embodiments, the adipose tissue component includes viable stem cells that have been added to harvested adipose tissue and may include purified hematopoietic and non-hematopoietic stem cells. By "viable" is meant that the stem cells in the adipose tissue component are living and capable of maintaining or recovering the potentialities of stem cell activity (e.g., dividing, differentiating, etc.)

Depending on the type (e.g., body region of harvesting) and amount of adipose tissue, the number of viable stem cells in the subject adipose tissue biocomposites may vary, such as 1 viable stem cell or greater, such as10 viable stem cells or greater, such as 1 x10² viable stem cells or greater, such as 5x10² viable stem cells or greater, such as 1 x10³ viable stem cells or greater, such as 5x10³ viable stem cells or greater, such as 1 x10⁴ viable stem cells or greater, such as 5x10⁴ viable stem cells or greater, such as 1 x10⁵ viable stem cells or greater, such as 5x10⁵ viable stem cells or greater, such as 1 x10⁶ viable stem cells or greater, such as 5x10⁶ viable stem cells or greater, such as 1 x10⁷ viable stem cells or greater, such as 1 x10⁸ viable stem cells or greater, such as 1 x10⁹ viable stem cells or greater and including 1 x10¹⁰ viable stem cells or greater.

In some embodiments, the adipose tissue component includes tissue

plasminogen activator. Tissue plasminogen activator refers to the serine protease protein that catalyzes the conversion of plasminogen to plasmin. In some embodiments, the adipose tissue component includes tissue plasminogen activator that is present in the adipose tissue. In other embodiments, the adipose tissue component includes tissue plasminogen activator added to the adipose tissue, such as purified tissue plasminogen activator as well as recombinant tissue plasminogen activator. The amount of tissue plasminogen activator in the subject adipose tissue biocomposites may vary, ranging from 0.01 μg to 100 μg, such as from 0.05 μg to 90 μg, such as from 0.1 μg to 80 μg, such as from 0.5 μg to 70 μg, such as from 1 μg to 60 μg and including from 5 μg to 50 μg. In these embodiments, the expressed activity of the tissue plasminogen activator may range from 1 x10² lU/mg to 1 x10⁸ lU/mg, such as from 5x10² lU/mg to 5x10⁷ lU/mg, such as from 1 x10³ lU/mg to 1 x10⁷ lU/mg, such as from 5x10³ lU/mg to 5x10⁶ lU/mg and including from 1 x10⁴ lU/mg to 1 x10⁶ lU/mg.

In some embodiments, the adipose tissue component includes lipoaspirate from a subject. For example, the adipose tissue component may include lipoaspirate obtained from a subject by a lipoplasty protocol such as suction assisted lipoplasty

(SAL), ultra-sound assisted lipoplasty (USAL), power assisted lipoplasty (PAL), syringe assisted lipoplasty (SAL), laser assisted lipoplasty (LAL) or water jet assisted lipoplasty (WJAL), among other lipoplasty protocols.

The amount of adipose tissue component in the subject adipose tissue biocomposites may vary, depending on the size and application and may range from 1 g to 10,000 g, such as from 5 g to 9000 g, such as from 10 g to 8000 g, such as from 15 g to 7000 g, such as from 20 g to 6000 g, such as from 25 g to 5000 g, such as from 30 g to 4000 g, such as from 35 g to 3000 g, such as from 40 g to 2000 g, such as from 50 g to 1000 g and including from 100 g to 500 g. In embodiments, the percentage of adipose tissue component in the adipose tissue biocomposite may range from 5% to 95% w/w, such as from 10% to 90% w/w, such as from 15% to 85% w/w, such as from 20% to 80% w/w, such as from 25% to 75% w/w, such as from 30% to 70% w/w, such as from 35% to 65% w/w and including from 40% to 50% w/w. As summarized above, adipose tissue biocomposites of interest include an adipose tissue component in a fibrin network. By "network" is meant a crosslinked array of polymerized fibrin sufficient to retain the adipose tissue component. The term

"crosslinked" is used its conventional sense to refer to the physical (e.g., intermolecular interactions or entanglements, such as through hydrophobic interactions) or chemical (e.g., covalent bonding) interaction between backbone components of polymer precursors. In some embodiments, the fibrin network includes polymerized strands of fibrin. In other embodiments, the fibrin network includes polymerized strands of fibrin and platelets. In yet other embodiments, the fibrin network includes polymerized strands of fibrin that are crosslinked by Factor XIII.

As described in greater detail below, the fibrin network in adipose tissue biocomposites of interest is prepared from thrombin and fibrinogen. The amount of thrombin may vary depending on the desired mechanical and tensile strength and malleability of the subject adipose tissue biocomposite and may include 0.01 μg to 100 μg, such as from 0.05 μg to 90 μg, such as from 0.1 μg to 80 μg, such as from 0.5 μg to 70 μg, such as from 1 μg to 60 μg and including from 5 μg to 50 μg. In these

embodiments, the expressed activity of the thrombin in preparing the subject

biocomposites may range from 1 x10² lU/mg to 1 x10⁸ lU/mg, such as from 5x10² lU/mg to 5x10⁷ lU/mg, such as from 1 x10³ lU/mg to 1 x10⁷ lU/mg, such as from 5x10³ lU/mg to 5x10⁶ lU/mg and including from 1 x10⁴ lU/mg to 1 x10^s lU/mg.

The amount of fibrin in the subject adipose tissue biocomposites may vary depending on the size and application and may range from 1 g to 1000 g, such as from 5 g to 900 g, such as from 10 g to 800 g, such as from 15 g to 700 g, such as from 20 g to 600 g, such as from 25 g to 500 g, such as from 30 g to 400 g, such as from 35 g to 300 g, such as from 40 g to 200 g and including from 50 g to 100 g. In embodiments, the percentage of fibrin in the adipose tissue biocomposite may range from 5% to 95% w/w, such as from 10% to 90% w/w, such as from 15% to 85% w/w, such as from 20% to 80% w/w, such as from 25% to 75% w/w, such as from 30% to 70% w/w, such as from 35% to 65% w/w and including from 40% to 50% w/w.

In certain embodiments, the fibrin network includes plasminogen. In some embodiments, the fibrin network includes plasminogen that is present in one or more of the thrombin source and fibrinogen source. In other embodiments, the fibrin network includes plasminogen added to one or more of the thrombin source and fibrinogen source, such as purified plasminogen as well as recombinant plasminogen. The amount of plasminogen in the subject adipose tissue biocomposites may vary, ranging from 0.01 μg to 100 μg, such as from 0.05 μg to 90 μg, such as from 0.1 μg to 80 μg, such as from 0.5 μg to 70 μg, such as from 1 μg to 60 μg and including from 5 μg to 50 μg. In these embodiments, the expressed activity of plasminogen may range from 1 x10² lU/mg to 1 x10⁸ IU/mg, such as from 5x10² lU/mg to 5x10⁷ lU/mg, such as from 1 x10³ lU/mg to 1 x10⁷ lU/mg, such as from 5x10³ lU/mg to 5x10⁶ lU/mg and including from 1 x10⁴ lU/mg to 1 x10^s lU/mg.

In other embodiments, the fibrin network includes plasmin. In some

embodiments, the fibrin network includes plasmin that is present in one or more of the thrombin source and fibrinogen source. In other embodiments, the fibrin network includes plasmin added to one or more of the thrombin source and fibrinogen source, such as purified plasmin as well as recombinant plasmin. The amount of plasmin in the subject adipose tissue biocomposites may vary, ranging from 0.01 μg to 100 μg, such as from 0.05 μg to 90 μg, such as from 0.1 μg to 80 μg, such as from 0.5 μg to 70 μg, such as from 1 μg to 60 μg and including from 5 μg to 50 μg. In these embodiments, the expressed activity of plasmin may range from 1 x10² lU/mg to 1 x10⁸ lU/mg, such as from 5x10² lU/mg to 5x10⁷ lU/mg, such as from 1 x10³ lU/mg to 1 x10⁷ lU/mg, such as from 5x10³ lU/mg to 5x10⁶ lU/mg and including from 1 x10⁴ lU/mg to 1 x10^s lU/mg.

Depending on the mechanical and physicochemical properties desired and the concentrations of thrombin and fibrinogen used, the fibrin network may have a crosslink density which ranges from 1 x10^~15 moles/cm³ to 1 x10 ³ moles/cm³, such as 1 x10^~14 moles/cm³ to 1 x10 ³ moles/cm³, such as 1 x10^~13 moles/cm³ to 1 x10 ³ moles/cm³, such as 1 x10^~12 moles/cm³ to 1 x10 ³ moles/cm³, such as 1 x10 moles/cm³ to 1 x10^~3 moles/cm³, such as 1 x10^~10 moles/cm³ to 1 x10 ³ moles/cm³, such as 1 x10^~9 moles/cm³ to 1 x10^~3 moles/cm³, such as 1 x10^"8 moles/cm³ to 1 x10^~3 moles/cm³, such as 1 x10^"11 moles/cm³ to 1 x10^"7 moles/cm³, and including 1 x10^~6 moles/cm³ to 1 x10 ³ moles/cm³.

Likewise, the compressive modulus of the subject fibrin network may vary. By compressive modulus is meant the capacity of the fibrin network in the subject adipose tissue biocomposite to withstand axially directed pushing forces and is the value of uniaxial compressive stress reach when the material fails completely (e.g., crushed). In some embodiments, the compressive modulus of the fibrin network ranges from 1 kPa to 35 kPa, such as from 2 kPa to 33 kPa, such as from 3 kPa to 30 kPa, such as from 4 kPa to 28 kPa, such as form 5 kPa to 25 kPa, such as from 6 kPa to 22 kPa, such as from 7 kPa to 20 kPa and including a compressive modulus ranging from 10 kPa to 20 kPa.

The pore sizes of the fibrin network may also vary depending on the structure of the fibrin in the biocomposite (e.g., crosslink density, fibrinogen content, thrombin concentration). In some embodiments, the pore sizes range from 0.1 microns to 1000 microns, such as 0.5 microns to 900 microns, such as 1 micron to 800 microns, such as 5 microns to 750 microns, such as 10 microns to 600 microns, such as 25 microns to 500 microns, such as 50 microns to 400 microns and including from 100 microns to 300 microns.

In embodiments, the source of thrombin may be any convenient source including, but not limited to, autologous thrombin serum, autologous thrombin serum supplemented with ethanol, allogeneic thrombin serum, allogeneic thrombin serum supplemented with ethanol, bovine thrombin, recombinant thrombin, and human thrombin derived from pooled plasma. In some embodiments, thrombin is from whole blood. In other embodiments, thrombin is from plasma. The source of fibrinogen, plasminogen and plasmin may also vary, as desired, including by not limited to autologous whole blood anti-coagulated with a calcium-chelating agent, platelet rich plasma with its associated growth factors, autologous plasma, autologous platelet rich plasma, plasma and collagen mixture as represented by Vitagel by Orthovita; purified allogeneic fibrinogen as represented by Tisseel/Tissucol and Beriplast products or by Quixil® consisting of a cross-linked allogeneic fibrinogen-fibronectin multimers and other naturally occurring adhesive glycoproteins to promote adhesion to collagen. In some embodiments, one or more of fibrinogen, plasminogen and plasmin are from whole blood. In other

embodiments, one or more of fibrinogen, plasminogen and plasmin are from plasma. As discussed in greater detail below, in certain instances, the subject adipose tissue biocomposites are prepared in a body site (e.g., would site) and the fibrin network is prepared from thrombin, fibrinogen, plasminogen and plasmin present at the body site.

In embodiments, the fibrin network in the subject adipose tissue biocomposites is configured to break down over a period of time to release the adipose tissue component from the biocomposite. As discussed above, the fibrin network is configured to break down by fibrinolysis into fibrin degradation products break down over the course of hours, such as 1 hour or longer, such as 2 hours or longer, such as 3 hours or longer, such as 5 hours or longer, such as 6 hours or longer and including over the course of 12 hours or longer. In other embodiments, the adipose tissue biocomposites are configured to break down over the course of days, such as 1 day or longer, such as 2 days or longer, such as 3 days or longer, such as 5 days or longer and including 7 days or longer. In certain embodiments the fibrin network is configured to break down by fibrinolysis over a predetermined period of time such as after 6 hours or longer, such as after 12 hours or longer, such as after 24 hours or longer, such as after 48 hours or longer, such as after 72 hours or longer, such as after 96 hours or longer, such as after 120 hours or longer, such as after 144 hours or longer and including after 168 hours or longer. In certain embodiments, the subject fibrin network is structurally designed to degrade under physiological conditions (e.g., in vivo) over a predetermined duration, such as after 6 hours or longer, such as after 12 hours or longer, such as after 24 hours or longer, such as after 48 hours or longer, such as after 72 hours or longer, such as after 96 hours or longer, such as after 120 hours or longer, such as after 144 hours or longer and including after 168 hours or longer. The fibrin network may be configured to break down at a predetermined rate, such as at a substantially zero-order fibrinolysis rate, such as at a substantially first order fibrinolysis rate and including at a substantially second-order fibrinolysis rate. Breakdown of the fibrin network to release the adipose tissue component from the subject adipose tissue biocomposites may be measured by any convenient protocol, such as by thrombin clotting time (TCT), thromboelastometry (TEM), as well as by euglobulin lysis time (ELT) assay.

In other embodiments, adipose tissue biocomposites may be prepared into desired planar shape, such a circle, oval, half-circle, crescent-shaped, star-shaped, square, triangle, rhomboid, pentagon, hexagon, heptagon, octagon, rectangle or other suitable polygon. In other embodiments, adipose tissue biocomposites are three- dimensional, such as in the shape of a cube, cone, half sphere, star, triangular prism, rectangular prism, hexagonal prism or other suitable polyhedron as well as in the shape of thin tubes, such as where the adipose tissue biocomposite is molded using tubing.

In some embodiments, adipose tissue biocomposites are castable compositions where the term "castable" is used in its conventional sense to refer to a composition that can be molded into a desired shape (e.g., by placing the composition into a shaped mold or body cavity) and may be subsequently hardened to form the final adipose tissue biocomposite. As such, the subject adipose tissue biocomposites may be formed into any convenient shape and size. For example, adipose tissue biocomposites may be planar and in the shape of a triangle, square, rectangle, rhomboid, pentagon, hexagon, heptagon, octagon, half circle, crescent-shaped, star shaped, or some other convenient shape. In other embodiments, adipose tissue biocomposites are three-dimensional, such as in the shape of a cube, cone, half sphere, star, triangular prism, rectangular prism, hexagonal prism or other polyhedron as well as in the shape of tubes, such as where the adipose tissue biocomposite is molded using tubing. In yet other

embodiments, adipose tissue biocomposites are prepared (as described in greater detail below) in a body site and the adipose tissue biocomposite takes on the shape (2-D or 3- D) of the body site (e.g., abdominal cavity) In certain instances, the subject adipose tissue biocomposites may be produced by cutting sheets of the adipose tissue biocomposite into the desired shape. For example, an adipose tissue biocomposite may be cast as a square, circular, rectangular (or some other shaped) sheet and cutting out the desired shape (such as by scissors or any other convenient cutting tool). In certain instances, where the desired shape of the adipose tissue biocomposite is a polygon, the sheet from which it is cut may be a rectangle, square or some other polygon, as convenient.

The size of the adipose tissue biocomposite may vary. The width of the adipose tissue biocomposite may range from 10 mm to 5000mm, such as from 25 mm to 4000 mm, such as from 50 mm to 3000 mm, such as from 75 mm to 2000 mm and including from 100 mm to 1000 mm. The length of the adipose tissue biocomposite may also vary, ranging from 10 mm to 5000mm, such as from 25 mm to 4000 mm, such as from 50 mm to 3000 mm, such as from 75 mm to 2000 mm and including from 100 mm to 1000 mm.

Where the adipose tissue biocomposite is planar, the surface area may range from 0.1 to 100 cm², such as 0.5 to 75 cm², such as 1 .0 to 50 cm², such as 1 .5 to 45 cm², such as 2.0 to 40 cm², such as 2.5 to 35 cm², and including 2 to 30 cm². Where the adipose tissue biocomposite is three-dimensional, the size may range from 0.1 to 100 cm³, such as 0.5 to 75 cm³, such as 1 .0 to 50 cm³, such as 1 .5 to 45 cm³, such as 2.0 to 40 cm³, such as 2.5 to 35 cm³, and including 2 to 30 cm³. The thickness of the adipose tissue biocomposite may be 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more, such as 2 mm or more, such as 3 mm or more, such as 5 mm or more, such as 10 mm or more, such as 25 mm or more, such as 50 mm or more and including 100 mm or more. For example, the overall thickness of the adipose tissue biocomposite may range from 1 mm to 100 mm, such as from 2 mm to 90 mm, such as from 3 mm to 75 mm and including a thickness of from 5 mm to 50 mm. In some embodiments, adipose tissue biocomposites are configured into one or more layers, such as two or more layers, such as three or more layers, such as 4 or more layers and including 5 or more layers. In certain embodiments, the adipose tissue biocomposite includes two layers. In some instances, the adipose tissue biocomposite includes a first layer having an adipose tissue component and a fibrin network and a second fibrin network layer having fibrin and thrombin. In other instances, the adipose tissue biocomposite includes a first layer that includes only the adipose tissue component and a second layer that only includes the fibrin network. In still other instances, the adipose tissue biocomposite includes a first layer of adipose tissue, viable stem cells and tissue plasminogen activator and a second layer having a fibrin network.

The thickness of each layer may vary depending on the amount of each component and the overall size of the adipose tissue biocomposite, and may be 0.1 mm or greater, such as 0.2 mm or greater, such as 0.5 mm or greater, such as 1 mm or greater, such as 2 mm or greater, such as 5 mm or greater, such as 10 mm or greater, such as 25 mm or greater and including 50 mm or greater. For example, the thickness of each layer may range from 0.1 mm to 100 mm, such as from 0.5 mm to 90 mm, such as from 1 mm to 75 mm and including a thickness of from 5 mm to 50 mm. Each layer may be the same size or different sizes or some combination thereof. For example, where adipose tissue biocomposites include two layers, a first layer having an adipose tissue component and a second layer having the fibrin network may have the same thickness. In other embodiments, a first layer having an adipose tissue component may be thicker that a second layer having the fibrin network by 5% or more, such as 10% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, including by 2-fold or more, such as 3-fold or more and including by 5-fold or more. In yet other embodiments, a second layer having the fibrin network may be thicker than a first layer having an adipose tissue component by 5% or more, such as 10% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, including by 2-fold or more, such as 3-fold or more and including by 5- fold or more.

In some embodiments, aspects of the present disclosure further include one or more bioactive agents adsorbed or absorbed within the subject adipose tissue biocomposites, such as being configured to deliver the one or more bioactive agent to a site of administration, such as by applying the subject adipose tissue biocomposite to a body site (e.g., wound site) and delivering the bioactive agent to the body site. The adipose tissue biocomposite may include one or more types of bioactive agents, such as two or more types, such as three or more types, such as four or more types, such as five or more types and including ten or more types of bioactive agents.

Example bioactive agents according to embodiments of the disclosure may include but are not limited to interferon, interleukin, erythropoietin, granulocyte-colony stimulating factor (GCSF), stem cell factor (SCI:), leptin (OB protein), interferon (alpha, beta, gamma), antibiotics such as vancomycin, gentamicin ciprofloxacin, amoxycillin, lactobacillus, cefotaxime, levofloxacin, cefipime, mebendazole, ampicillin, lactobacillus, cloxacillin, norfloxacin, tinidazole, cefpodoxime, proxctil, azithromycin, gatifloxacin, roxithromycin, cephalosporin, anti-thrombogenics, aspirin, ticlopidine, sulfinpyrazone, heparin, warfarin, growth factors, differentiation factors, hepatocyte stimulating factor, plasmacytoma growth factor, glial derived neurotrophic factor (GDNF), neurotrophic factor 3 (NT3), fibroblast growth factor (FGF), transforming growth factor (TGF), platelet transforming growth factor, milk growth factor, endothelial growth factors, endothelial cell-derived growth factors (ECDGF), alpha-endothelial growth factors, beta-endothelial growth factor, neurotrophic growth factor, nerve growth factor (NGF), vascular endothelial growth factor (VEGF), 4-1 BB receptor (4- IBBR), TRAIL (TNF-related apoptosis inducing ligand), artemin (GFRalpha3-RET ligand), BCA-I (B cell-attracting chemokinel), B lymphocyte chemoattractant (BLC), B cell maturation protein (BCMA), brain-derived neurotrophic factor (BDNF), bone growth factor such as osteoprotegerin (OPG), bone-derived growth factor, thrombopoietin, megakaryocyte derived growth factor (MDGF), keratinocyte growth factor (KGF), platelet-derived growth factor (PDGF), ciliary neurotrophic factor (CNTF), neurotrophin 4 (NT4), granulocyte colony-stimulating factor (GCSF), macrophage colony-stimulating factor (mCSF), bone morphogenetic protein 2 (BMP2), BRAK, C-IO, Cardiotrophin 1 (CTI), CCR8, anti- inflammatory:

paracetamol, salsalate, diflunisal, mefenamic acid, diclofenac, piroxicam, ketoprofen, dipyrone, acetylsalicylic acid, anti-cancer drugs such as aliteretinoin, altertamine, anastrozole, azathioprine, bicalutarnide, busulfan, capecitabine, carboplatin, cisplatin, cyclophosphamide, cytarabine, doxorubicin, epirubicin, etoposide, exemestane, vincristine, vinorelbine, hormones, thyroid stimulating hormone (TSH), sex hormone binding globulin (SHBG), prolactin, luteotropic hormone (LTH), lactogenic hormone, parathyroid hormone (PTH), melanin concentrating hormone (MCH), luteinizing hormone (LHb), growth hormone (HGH), follicle stimulating hormone (FSHb), haloperidol, indomethacin, doxorubicin, epirubicin, amphotericin B, Taxol, cyclophosphamide, cisplatin, methotrexate, pyrene, amphotericin B, anti- dyskinesia agents, Alzheimer vaccine, antiparkinson agents, ions, edetic acid, nutrients, glucocorticoids, heparin, anticoagulation agents, antivirus agents, anti-HIV agents, polyamine, histamine and derivatives thereof, cystineamine and derivatives thereof, diphenhydramine and derivatives, orphenadrine and derivatives, muscarinic antagonist, phenoxybenzamine and derivatives thereof, protein A, streptavidin, amino acid, beta-galactosidase, methylene blue, protein kinases, beta-amyloid, lipopolysaccharides, eukaryotic initiation factor-4G, tumor necrosis factor (TNF), tumor necrosis factor-binding protein (TNF -bp), interleukin- 1 (to 18) receptor antagonist (IL-lra), granulocyte macrophage colony stimulating factor (GM-CSF), novel erythropoiesis stimulating protein (NESP), thrombopoietin, tissue plasminogen activator (TPA), urokinase, streptokinase, kallikrein, insulin, steroid, acetaminophen, analgesics, antitumor preparations, anti-cancer preparations, anti-proliferative preparations or pro-apoptotic preparations, among other types of bioactive agents.

The amount of each bioactive agent in the adipose tissue biocomposite may vary depending on the type of bioactive agent and the size of the biocomposite and may be 0.01 μg or more, such as 0.05 μg or more, such as 0.1 μg or more, such as 0.5 μg or more, such as 1 μg or more, such as 5 μg or more, such as 10 μg or more, such as 25 μg or more, such as 50 μg or more, such as 100 μg or more, such as 250 μg or more, such as 1000 μg or more, such as 10 g or more, such as 25 g or more and including 100 g of bioactive agent or more. Where the bioactive agent is incorporated into the adipose tissue biocomposite as a liquid, the concentration of each bioactive agent may be 0.0001 μg/mL or greater, such as 0.001 μg/mL or greater, such as 0.01 μg/mL or greater, such as 0.1 μg/mL or greater, such as 0.5 μg/mL or greater, such as 1 μg/mL or greater, such as 2 μg/mL or greater, such as 5 μg/mL or greater, such as 10 μg/mL or greater, such as 25 μg/mL or greater, such as 50 μg/mL or greater, such as 100 μg/mL or greater such as 500 μg/mL or greater, such as 1 g/mL or greater such as 5 g/mL or greater and including 10 g/mL or greater.

Where more than one bioactive agent is delivered, the amount (i.e., mass) of each of bioactive agent may vary, ranging from 0.001 mg to 1000 mg, such as 0.01 mg to 500 mg, such as 0.1 mg to 250 mg, such as 0.5 mg to 100 mg, such as 1 mg to 50 mg, including 1 mg to 10 mg. As such, in compositions of the invention, the mass ratio of the first bioactive agent to other (i.e., second or more) bioactive agent may vary, and in some instances may range between 1 :1 and 1 :2.5; 1 :2.5 and 1 :5; 1 :5 and 1 :10; 1 :10 and 1 :25; 1 :25 and 1 :50; 1 :50 and 1 :100; 1 :100 and 1 :150; 1 :150 and 1 :200; 1 :200 and 1 :250; 1 :250 and 1 :500; 1 :500 and 1 :1000, or a range thereof. For example, the mass ratio of the first bioactive agent to other (i.e., second or more) bioactive agents may range between 1 :1 and 1 :10; 1 :5 and 1 :25; 1 :10 and 1 :50; 1 :25 and 1 :100; 1 :50 and 1 :500; or 1 :100 and 1 :1000.

Where the subject adipose tissue biocomposites are configured into layers, each layer may have one or more different types of bioactive agents, such as two or more types, such as three or more types, such as four or more types, such as five or more types and including ten or more types of bioactive agents. In certain embodiments, the adipose tissue biocomposite includes a first layer having an adipose tissue component that includes one or more lipophilic bioactive agents and a second fibrin network layer that includes one or more hydrophilic bioactive agents. The amount of bioactive agent in each layer may vary and may be 0.01 μg or more, such as 0.05 μg or more, such as 0.1 μg or more, such as 0.5 μg or more, such as 1 μg or more, such as 5 μg or more, such as 10 μg or more, such as 25 μg or more, such as 50 μg or more, such as 100 μg or more, such as 250 μg or more, such as 1000 μg or more, such as 10 g or more, such as 25 g or more and including 100 g of bioactive agent or more. Where the bioactive agent is incorporated into each layer as a liquid, the concentration of each bioactive agent may be 0.0001 μg/mL or greater, such as 0.001 μg/mL or greater, such as 0.01 μg/mL or greater, such as 0.1 μg/mL or greater, such as 0.5 μg/mL or greater, such as 1 μg/mL or greater, such as 2 μg/mL or greater, such as 5 μg/mL or greater, such as 10 μg/mL or greater, such as 25 μg/mL or greater, such as 50 μg/mL or greater, such as 100 μg/mL or greater such as 500 μg/mL or greater, such as 1 g/mL or greater such as 5 g/mL or greater and including 10 g/mL or greater.

Depending on the composition of the adipose tissue biocomposite or specific layer (e.g., adipose tissue component composition, fibrin network crosslink density, etc.), the release of the one or more bioactive agents may vary. For example, the adipose tissue biocomposites (e.g., one or more of the layers) may be configured to provide a sustained release or pulsatile release of the one or more bioactive agents. By

"sustained release" is meant that the adipose tissue biocomposite (or one or more of the layers when present) is structured (e.g., adipose tissue content, crosslink density) to provide for constant and continuous delivery of one or more bioactive agents over the entire time the adipose tissue biocomposite is maintained in contact with the site of administration (e.g., abdominal cavity), such as over the course of 1 day or longer, such as 2 days or longer, such as 3 days or longer, such as 5 days or longer and including 7 days or longer. In other instances, crosslinked copolymer hydrogels of the present invention are configured to provide a pulsatile release of the one or more bioactive agents. By "pulsatile release" is meant that the adipose tissue biocomposite is configured to release one or more bioactive agents into the site of administration incrementally (e.g., at discrete times), such as every 1 hour, such as every 2 hours, such as every 5 hours, such as every 12 hours, such as every 24 hours, such as every 36 hours, such as every 48 hours, such as every 72 hours, such as every 96 hours, such as every 120 hours, such as every 144 hours and including every 168 hours.

In other instances, the subject adipose tissue biocomposites (or one or more of the layers when present) are configured to deliver one or more bioactive agents after certain percentages of the fibrin network has been broken down by fibrinolysis. For example, an amount of the one or more bioactive agents may be delivered after every 10% of the crosslinks of the fibrin network has been broken down by fibrinolysis, such as after every 15% of the fibrin network has been broken down by fibrinolysis, such as after every 20% of the crosslinks of the fibrin network has been broken down by fibrinolysis, such as after every 25% of the fibrin network has been broken down by fibrinolysis, such as after every 30% of the fibrin network has been broken down by fibrinolysis and including after every 33% of the fibrin network has been broken down by fibrinolysis at the site of administration.

In yet other instances, adipose tissue biocomposites (or one or more of the layers when present) may be configured to release a large amount of the one or more bioactive agents immediately upon contact with the site of administration, such as for example 50% or more, such as 60% or more, such as 70% or more and including 90% or more of the one or more bioactive agents are released immediately upon contact with the site of administration. In yet other instances adipose tissue biocomposites may be configured to release the one or more bioactive agents at a predetermined rate, such as at a substantially zero-order release rate, such as at a substantially first-order release rate or at a substantially second-order release rate.

Adipose tissue biocomposites of interest may also be configured to deliver bioactive agent at a substantially linear rate over a predetermined dosage interval (e.g., 4 weeks or longer). By "substantially linearly" is meant that the cumulative amount of bioactive agent released from the adipose tissue biocomposites increases at a substantially constant rate (i.e., defined by first-order kinetics). As such, the change in rate of cumulatively delivered bioactive agent increases or decreases by 10% or less at any given time, such as 8% or less, such as 7% or less, such as 6% or less, such as 5% or less, such as 3% or less, such as 2.5% or less, such as 2% or less, and including 1 % or less.

In other embodiments, depending on the size of the adipose tissue biocomposite applied, adipose tissue biocomposites may be configured to deliver an average cumulative amount of bioactive agent of 5 μg/cm² or greater over an extended period of time. The term "cumulative amount" is meant the total quantity of bioactive agent delivered by the adipose tissue biocomposites. In these embodiments, adipose tissue biocomposites of interest may be configured to deliver an average cumulative amount of bioactive agent may be 25 μg/cm² or greater, such as 50 μg/cm² or greater, such as 75 μg/cm² or greater over a 4 week delivery interval, such as 100 μg/cm² or greater, such as 125 μg/cm² or greater, such as 150 μg/cm² or greater and including 200 μg/cm² over a predetermined delivery interval.

In yet other embodiments, adipose tissue biocomposites are configured to deliver a target dosage of bioactive agent, such as for example as characterized by total bioactive agent exposure or by average daily bioactive agent exposure. The term target dosage is meant the amount of bioactive agent which is delivered to the subject and may vary depending on the physicochemical properties, mechanical properties, and break down rates of the adipose tissue biocomposite as well as the site of application. For example, the target dosage of bioactive agent delivered by the subject hydrogels may be 0.01 mg/day or greater, such as 0.04 mg/day or greater, such as 0.5 mg/day or greater over a predetermined delivery interval, such as 1 .0 mg/day or greater, such as 2 mg/day or greater, such as 5 mg/day or greater and including 10 mg/day over a predetermined delivery interval.

Therefore, the dosage of bioactive agent delivered using the subject adipose tissue biocomposites may vary, ranging from about 0.01 mg/kg to 500 mg/kg per day, such as from 0.01 mg/kg to 400 mg/kg per day, such as 0.01 mg/kg to 200 mg/kg per day, such as 0.1 mg/kg to 100 mg/kg per day, such as 0.01 mg/kg to 10 mg/kg per day, such as 0.01 mg/kg to 2 mg/kg per day, including 0.02 mg/kg to 2 mg/kg per day. In other embodiments, the dosage may range from 0.01 to 100 mg/kg four times per day (QID), such as 0.01 to 50 mg/kg QID, such as 0.01 mg/kg to 10 mg/kg QID, such as 0.01 mg/kg to 2 mg/kg QID, such as 0.01 to 0.2 mg/kg QID, depending on the dosage protocol as desired. In other embodiments, the dosage may range from 0.01 mg/kg to 50 mg/kg three times per day (TID), such as 0.01 mg/kg to 10 mg/kg TID, such as 0.01 mg/kg to 2 mg/kg TID, and including as 0.01 mg/kg to 0.2 mg/kg TID. In yet other embodiments, the dosage may range from 0.01 mg/kg to100 mg/kg two times per day (BID), such as 0.01 mg/kg to 10 mg/kg BID, such as 0.01 mg/kg to 2 mg/kg BID, including 0.01 mg/kg to 0.2 mg/kg BID.

METHODS FOR PREPARING ADIPOSE TISSUE BIOCOMPOSITES

As summarized above, the subject disclosure provides adipose tissue

biocomposites. Aspects of the disclosure also include methods for preparing the subject adipose tissue biocomposites. In certain embodiments, methods include contacting one or more of the adipose tissue components as described above with thrombin and fibrinogen in a manner sufficient to produce the adipose tissue biocomposite such that the fibrin network is configured to break down after a period of time to release the adipose tissue from the biocomposite. In other embodiments, the adipose tissue biocomposites include two layers and the method may be characterized by: 1 ) contacting an adipose tissue component with a composition having fibrinogen and thrombin; 2) allowing the adipose tissue component to float to the top of the fibrinogen and thrombin composition; and 3) forming a fibrin network from the composition of fibrinogen and thrombin to form an adipose tissue biocomposite having an adipose tissue component layer and a fibrin network layer. In still other embodiments, methods include: 1 ) contacting an adipose tissue component with a composition having fibrinogen and thrombin; 2) allowing the adipose tissue component to float to the top of the fibrinogen and thrombin composition; 3) forming a fibrin network from the composition of fibrinogen and thrombin to form an adipose tissue biocomposite having an adipose tissue component layer and a fibrin network layer; and removing any remaining thrombin and fibrinogen composition from the adipose tissue biocomposite (e.g., such as by wiping or blotting with gauze).

In some embodiments, methods include preparing an improved adipose tissue biocomposite by contacting an adipose tissue component with thrombin and applying the resultant composition to a body site of a subject. Referring to FIG. 1 , an operational flow chart of the method preparing an adipose tissue biocomposite graft in accordance with one aspect of the present invention is illustrated. Initially, lipoplasty is performed to derive an adipose tissue component having a plurality of adipose tissue fragments from a donor, as shown in block 100. The plurality of adipose tissue fragments are harvested from the donor as indicated at block 102. In this embodiment, the plurality of adipose tissue fragments contains at least one viable stem cell. After harvesting the adipose tissue fragments, appropriate concentration of thrombin source is contacted with the adipose tissue fragments as shown in block 104. Finally, as indicated at block 106, the mixture of the adipose tissue fragments and the thrombin source is applied to a wound site of the donor so as to promote wound healing. In this embodiment, the adipose tissue component (e.g., adipose tissue fragments, viable stem cells and tissue plasminogen activator) is contacted with a fibrinogen source from the wound site of the donor. The promotion of wound healing includes at least one of the effects of: promoting hemostasis, reducing time for wound closure, reducing post-surgical wound

complications and reducing scarring.

In some embodiments, one or more of the adipose tissue component, thrombin, fibrinogen and plasminogen is obtained from the subject to which the adipose tissue biocomposite will be applied (i.e., is autologous). In certain instances, the present adipose tissue biocomposites are fully autologous. In one embodiment of the present invention, all the biological constituents in the adipose tissue biocomposite are autologous. The present invention teaches the method for preparing fully autologous biocomposite as explained below. The adipose tissue fragments thus derived by lipoplasty may have excess liquid which can be removed by draining, by removal of supernatant after gentle centrifugation, by filtration or after spontaneous phase separation due to density differences in the tissue fragments and suspending fluid as occurs with adipose tissue. The structure of the adipose tissue biocomposite graft thus prepared, is controlled by controlling the relative percentage of the graft volume derived from adipose tissue fragments and controlling concentrations of the thrombin source.

In practicing the subject methods, the amount of adipose tissue component used in preparing the subject adipose tissue biocomposite may vary depending on the desired size as well as mechanical properties and may range from 1 g to 10,000 g, such as from 5 g to 9000 g, such as from 10 g to 8000 g, such as from 15 g to 7000 g, such as from 20 g to 6000 g, such as from 25 g to 5000 g, such as from 30 g to 4000 g, such as from 35 g to 3000 g, such as from 40 g to 2000 g, such as from 50 g to 1000 g and including from 100 g to 500 g. The volume of adipose tissue used to prepare the subject adipose tissue biocomposites may be 1 ml. or more, such as 2 ml. or more, such as 5 ml. or more, such as 10 mL or more, such as 25 mL or more, such as 50 mL or more, such as 100mL or more, such as 250 mL or more, such as 500 mL or more, such as 750 mL or more and including 1000 mL or more. For example, the volume of the adipose tissue component contacted with the source of thrombin and source of fibrinogen (and plasminogen when present) may range from 1 mL to 1000 mL, such as from 5 mL to 900 mL, such as from 10 mL to 800 mL, such as from 15 mL to 700 mL, such as from 20 mL to 600 mL and including from 25 mL to 500 mL. When preparing the subject adipose tissue biocomposites, the adipose tissue component may be contacted with the source of thrombin, fibrinogen and plasminogen such that the percentage of adipose tissue component in the final adipose tissue biocomposite may range from 5% to 95% w/w, such as from 10% to 90% w/w, such as from 15% to 85% w/w, such as from 20% to 80% w/w, such as from 25% to 75% w/w, such as from 30% to 70% w/w, such as from 35% to 65% w/w and including from 40% to 50% w/w.

In embodiments, the concentration of thrombin contacted with the adipose tissue component ranges from 0.1 to 1000 units/gram of adipose tissue component, such as from 0.5 to 900 units/gram, such as from 1 to 800 units/gram, such as from 5 to 750 units/gram, such as from 10 to 600 units/gram and including a concentration of thrombin from 25 to 500 units/gram of the adipose tissue component. Depending on the desired mechanical and tensile strength and malleability of the subject adipose tissue biocomposite, methods may include contacting 0.01 μg to 100 μg of thrombin with the adipose tissue component, such as from 0.05 μg to 90 μg, such as from 0.1 μg to 80 μg, such as from 0.5 μg to 70 μg, such as from 1 μg to 60 μg and including from 5 μg to 50 μg. In these embodiments, the expressed activity of the thrombin in preparing the subject biocomposites may range from 1 x10² lU/mg to 1 x10⁸ lU/mg, such as from 5x10² lU/mg to 5x10⁷ lU/mg, such as from 1 x10³ lU/mg to 1 x10⁷ lU/mg, such as from 5x10³ lU/mg to 5x10⁶ lU/mg and including from 1 x10⁴ lU/mg to 1 x10^s lU/mg.

In embodiments, the amount of fibrinogen contacted with the adipose tissue component ranges from 0.001 g to 1000 g of fibrinogen with the adipose tissue component, such as from 0.005 g to 900 g, such as from 0.01 g to 800 g, such as from 0.05 g to 700 g, such as from 0.1 g to 600 g, such as from 0.5 g to 500 g, such as 1 g to 400 g, such as from 2 g to 300 g, such as from 3 g to 200 g, and including from 5 g to 100 g of fibrinogen. Where fibrinogen is contacted with the adipose tissue component as a liquid, the concentration of fibrinogen may be 0.0001 μg/mL or greater, such as 0.001 μg/mL or greater, such as 0.01 μg/mL or greater, such as 0.1 μg/mL or greater, such as 0.5 μg/mL or greater, such as 1 μg/mL or greater, such as 2 μg/mL or greater, such as 5 μg/mL or greater, such as 10 μg/mL or greater, such as 25 μg/mL or greater, such as 50 μg/mL or greater, such as 100 μg/mL or greater such as 500 μg/mL or greater, such as 1 mg/mL or greater such as 5 mg/mL or greater and including 10 mg/mL or greater.

Where the adipose tissue biocomposite includes plasminogen, the amount of plasminogen may be 0.001 μg or greater, such as 0.005 μg or greater, such as 0.01 μg or greater, such as 0.05 μg or greater, such as 0.1 μg or greater, such as 0.5 μg or greater, such as 1 μg or greater, such as 10 μg or greater, such as 100 μg or greater, such as 1000 μg or greater, such as 10,000 μg or greater, such as 100,000 μg or greater and including 1 ,000,000 μg or greater. Where plasminogen is contacted with the adipose tissue component as a liquid, the concentration of plasminogen may be 0.0001 μg/mL or greater, such as 0.001 μg/mL or greater, such as 0.01 μg/mL or greater, such as 0.1 μg/mL or greater, such as 0.5 μg/mL or greater, such as 1 μg/mL or greater, such as 2 μg/mL or greater, such as 5 μg/mL or greater, such as 10 μg/mL or greater, such as 25 μg/mL or greater, such as 50 μg/mL or greater, such as 100 μg/mL or greater such as 500 μg/mL or greater, such as 1 mg/mL or greater such as 5 mg/mL or greater and including 10 mg/mL or greater.

The ratio by volume of adipose tissue component to composition having fibrinogen and thrombin (and plasminogen, when present) may vary depending on the specific properties of the adipose tissue biocomposite desired and may range between 1 :1 and 1 :1 .5; 1 :1 .5 and 1 :2; 1 :2 and 1 :2.5; 1 :2.5 and 1 :3; 1 :3 and 1 :3.5; 1 :3.5 and 1 :4; 1 :4 and 1 :4.5; 1 :4.5 and 1 :5; 1 :5 and 1 :5.5; 1 :5.5 and 1 :6; 1 :6 and 1 :6.5; 1 :6.5 and 1 :7; 1 :7 and 1 :7.5; 1 :7.5 and 1 :8; 1 :8 and 1 :8.5; 1 :8.5 and 1 :9; 1 :9 and 1 :9.5; 1 :9.5 and 1 :10 or a range thereof. For example, the ratio by volume of the adipose tissue component to composition having fibrinogen and thrombin ranges from 1 :1 and 1 :10, such as 1 :1 and 1 :8, such as 1 :1 and 1 :5, such as 1 :1 and 1 :4, and including from 1 :1 and 1 :2. In certain instances, the ratio of adipose tissue component to composition having fibrinogen and thrombin ranges between 10:1 and 9.5:1 ; 9.5:1 and 9:1 ; 9:1 and 8.5:1 ; 8.5:1 and 8:1 ; 8:1 and 7.5:1 ; 7.5:1 and 7:1 ; 7:1 and 6.5:1 ; 6.5:1 and 6:1 ; 6:1 and 5.5:1 ; 5.5:1 and 5:1 ; 5:1 and 4.5:1 ; 4.5:1 and 4:1 ; 4:1 and 3.5:1 ; 3.5:1 and 3:1 ; 3:1 and 2.5:1 ; 2.5:1 and 2:1 ; 2:1 and 1 .5:1 ; 1 .5:1 and 1 :1 or a range thereof. For example, the ratio by volume of adipose tissue component to composition having fibrinogen and thrombin ranges from 10:1 and 1 :1 , such as 8:1 and 1 :1 , such as 5:1 and 1 :1 , such as 4:1 and 1 :1 , and including from 2:1 and 1 :1 .

For example, the concentration of thrombin source is 0.5 to 500 units/gram of the adipose tissue biocomposite graft. The adipose tissue fragments constitute 5% to 95% of the total volume of the adipose tissue biocomposite graft depending upon the clinical judgment of graft stability for maintaining physical separation of a (achieved by adding less adipose tissue to the graft) versus fibrinolysis activity (achieved by adding more adipose tissue to the graft).

The components of the adipose tissue biocomposite may be contacted with each other by any suitable protocol. In certain embodiments, methods include mixing one or more components in a syringe and injecting the mixture into a body site (e.g., wound site) of a subject. In other embodiments, one or more components are contacted in a three-dimensional mold. Each component may be put into the mold continuously (e.g., with a syringe pump) or in discrete intervals. Where a component of the adipose tissue biocomposite (e.g., thrombin serum, plasma, adipose tissue lipoaspirate, etc.) is placed into the mold continuously, the rate of inputting the component may vary depending on the size of the adipose tissue biocomposite desired as well as the viscosity of the component and may be 1 mL/minute or greater, such as 2 mL/minute or greater, such as 3 mL/minute or greater, such as 10 mL/minute or greater, such as 25 mL/minute or greater and including 100 mL/minute or greater. Where a component is placed into the mold at discrete intervals, the amount of the component inputted into the mold at any given time may vary and may be 0.1 mL or greater, such as 0.5 mL or greater, such as 1 mL or greater, such as 5 mL or greater, such as 25 mL or greater, such as 50 mL or greater and including 100 mL or greater.

In some embodiments, the adiopose tissue component includes autologous adipose tissue fragments contained in lipoaspirate. The adipose tissue fragments provide an abundant source of living cells for tissue engineering purposes and are safe for the donor so that even large amounts of adipose tissue can be removed from the body without significant untoward effect. In certain instances, the purpose of the adipose biocomposite graft is to be a biological volume replacement material that fills voids made at sites of injury and enhances wound healing. As described above, the adipose tissue fragments may contain adipocytes, fibroblasts, endothelial cells, mesenchymal stem cells and endothelial precursor cells that can secrete proteins important to wound healing. Turning now to FIG. 2, another operational flow chart of a method for preparing an adipose tissue biocomposite to serve a wide range of medical applications in accordance with another aspect of the present invention is illustrated. Initially, lipoplasty is performed to derive an adipose tissue component having a plurality of adipose tissue fragments from a donor, as shown in block 108. The plurality of adipose tissue fragments are harvested from the donor as indicated at block 110. In next step, as shown in block 112, the plurality of adipose tissue fragments are contacted with appropriate concentrations of a thrombin source and a fibrinogen source to achieve an appropriate gelling reaction. Finally, the mixture of the adipose tissue fragments, the thrombin source and the fibrinogen source are applied to a surgical site of the donor so as to inhibit adhesion formation as indicated at block 114.

In embodiments of the present disclosure, the adipose tissue biocomposite may be prepared as a liquid (e.g., injectable) biocomposite, fragments of an adipose tissue biocomposite or into mold three-dimensional shapes. Depending on the crosslink density and concentration of fibrin network of the biocomposite, the viscosity of the liquid biocomposite may and may be 1 x10² cP or greater, such as 5x10² cP or greater, such as, 1 x10³ cP or greater, such as 5x10³ cP or greater, such as 1 x10⁴ cP or greater, such as 5x10⁴ cP or greater, such as 1 x10⁵ cP or greater, such as 5x10⁵ cP, such as 1 x10⁶ cP or greater, such as 5x10⁶ cP or greater and including 1 x10⁷ cP or greater. For example, liquid adipose tissue biocomposites of interest may have a viscosity ranging from 1 x10² cP to 1 x10⁷ cP, such as from 5x10² cP to 5x10⁶ cP, such as from 1 x10³ cP to 1 x10⁶ cP, such as from 5x10³ cP to 5x10⁵ cP, including from 1 x10⁴ cP to 1 x10⁵ cP.

In other embodiments, the adipose tissue biocomposite may be prepared as adipose tissue biocomposite fragments. The fragments may be homogeneous in shape and size or more may be different. In some embodiments, the adipose tissue biocomposite fragments have the same shapes and sizes. In other embodiments, the adipose tissue biocomposite fragments have different shapes and sizes. The size of the fragments vary ranging from 0.1 to 10 mm³, such as 0.5 to 7.5 mm³, such as 1 .0 to 5.0 mm³, such as 1 .5 to 4.5 mm³, such as 2.0 to 4.0 mm³, such as 2.5 to 3.5 mm³, and including 2 to 3.0 mm³.

In certain embodiments, the adipose tissue biocomposite is prepared into three- dimensional shapes such as by using a three-dimensional mold. The three-dimensional mold may be any suitable container for contacting the adipose tissue component with one or more of the source of thrombin, fibrinogen and plasminogen and may be in the shape of a circle, oval, half-circle, crescent-shaped, star-shaped, square, triangle, rhomboid, pentagon, hexagon, heptagon, octagon, rectangle or other suitable polygon. For example, the three-dimensional mold may be a container having the shape of a cube, cone, half sphere, star, triangular prism, rectangular prism, hexagonal prism or other suitable polyhedron. Depending on the desired size adipose tissue biocomposite, the mold may be planar having a surface area ranging from 0.1 to 100 cm², such as 0.5 to 75 cm², such as 1 .0 to 50 cm², such as 1 .5 to 45 cm², such as 2.0 to 40 cm², such as 2.5 to 35 cm², and including 2 to 30 cm². The three-dimensional mold may have a volume that ranges from 0.1 to 100 cm³, such as 0.5 to 75 cm³, such as 1 .0 to 50 cm³, such as 1 .5 to 45 cm³, such as 2.0 to 40 cm³, such as 2.5 to 35 cm³, and including 2 to 30 cm³.

The mold may include one or more ports for inputting the adipose tissue component as well as the source of thrombin, fibrinogen and plasminogen. Any suitable port configuration may be employed, where examples of ports include channels, orifices, channels having a check valve, a Luer taper fitting, a port with a breakable seal (e.g., single use ports) among other types of ports. In some embodiments, the port is configured to connect to a syringe. In other embodiments, the port is configured to facilitate access for a needle into the cavity of the container to aspirate, mix and remove components from the container. In certain embodiments, the port is configured with a Luer taper fitting, such as a Luer-Lok or a Luer-slip.

The mixture of the adipose tissue component having adipose tissue fragments, the thrombin source and the fibrinogen source is applied to the surgical site as shown in block 114, in the form of: a liquid biocomposite, a molded gel biocomposite a gel biocomposite fragments or a combination thereof. In some embodiments, the liquid biocomposite is prepared by mixing the adipose tissue fragments, the thrombin source, and the fibrinogen source in a liquid and applied (e.g., injected by a syringe or other liquid dispensing device) to fill the desired site in the donor's body. The biocomposite graft can also be applied in the form of a molded gel biocomposite, which is prepared by (a) injecting the mixture of the adipose tissue component having adipose tissue fragments, the thrombin source and the fibrinogen source into a three dimensional mold cavity to achieve the gelling reaction ex vivo, (b) removing the adipose tissue

biocomposite from the three dimensional mold cavity and (c) applying the adipose tissue biocomposite graft to the surgical of the donor such as to create a physical barrier between tissue layers at risk of forming an adhesion. In some embodiments of the present invention, the gelling reaction may be achieved by placing a fluid containing thrombin in contact with a fluid containing fibrinogen and plasminogen with an adipose tissue component suspending fluid. As shown in block 112, the adipose tissue fragments are contacted with selected concentrations of thrombin and fibrinogen in to achieve an appropriate gelling reaction. The concentration of the thrombin and the fibrinogen source is kept optimum to achieve the intended gelling reaction. In some embodiments, the amount of thrombin is selected to provide sufficient time for transferring the reaction mixture into the mold before the gelling reaction occurs but the amount should not be so low as to take too long for the gelling reaction to cure within a reasonable time. In embodiments, the time for reaction to form the fibrin network (i.e., gelling reaction) ranges from 0.5 minutes to 30 minutes, such as from 1 minute to 25 minutes, such as from 2 minutes to 20 minutes, such as from 3 minutes to 15 minutes and including from 5 minutes to 10 minutes. In some embodiments, it is desirable to have a time period of at least 15 seconds to load the mold or deliver to the body of the donor. In some embodiments, time required for the gelling reaction (i.e., formation of the fibrin network from thrombin and fibrinogen with the adipose tissue component) to occur is less than 10 minutes and sometimes in less than 3 minutes. For example, a suitable adipose graft composite can be prepared by combining 20 grams of adipose tissue, 16 ml of plasma and 4 ml of thrombin serum containing 40 international units/mL.

For the embodiment of molding the adipose biocomposite or applying the biocomposite as a liquid to the body, the most preferred amount of thrombin is that which causes a clot time of 15 to 30 seconds of human plasma at room temperature when added in equal volumes. The amount of thrombin selected for contact with the fibrinogen should provide the operator sufficient time to transfer the mixture to the wound site where the gelling reaction is desired to occur (e.g., in the range of 1 -20 units of thrombin/mL). This concentration of thrombin provides adequate time for the operator to dispense the mixture to the mold or body site prior to the gelling reaction occurring. If too much thrombin is added, the gelling reaction will take place before the mixture is added to the mold or delivered to the body. Because the gelling reaction is an irreversible process and the nascent gels can readily be disrupted if disturbed during the curing process, excessive speed of gelling may be avoided by ensuring there is not too much thrombin (relative to amount of fibrinogen present) in the mixture. The concentration of thrombin may in certain embodiments be sufficiently high (relative to fibrinogen) that a relatively small volume compared to the graft volume is required to achieve the intended gelling reaction. The concentration of fibrinogen determines in positive fashion the overall tensile strength of the biocomposite. A higher concentration of fibrinogen may be employed to prepare a stronger more persistent gel biocomposite. For example, the concentration of fibrinogen may be less than 15 mg/mL. In this method, the

concentration of thrombin source is 0.5 to 500 units/gram of the adipose tissue biocomposite graft and the concentration of fibrinogen source is 0.1 to 80 mg/gram of the adipose tissue biocomposite graft.

In practicing the subject methods, the thrombin source is selected from:

autologous thrombin serum, autologous thrombin serum supplemented with ethanol, allogeneic thrombin serum, allogeneic thrombin serum supplemented with ethanol, bovine thrombin, recombinant thrombin, and human thrombin derived from pooled plasma. In certain embodiments, the source of thrombin is autologous thrombin obtained from the subject to which the adipose tissue biocomposite will be applied. The fibrinogen and plasminogen source is selected from: autologous whole blood anti- coagulated with a calcium-chelating agent, platelet rich plasma with its associated growth factors, autologous plasma, autologous platelet rich plasma, plasma and collagen mixture as represented by Vitagel by Orthovita; purified allogeneic fibrinogen as represented by Tisseel/Tissucol and Beriplast products or by Quixil® consisting of a cross-linked allogeneic fibrinogen-fibronectin multimers and other naturally occurring adhesive glycoproteins to promote adhesion to collagen. In certain embodiments, the source of fibrinogen and plasminogen for the practice of the current invention is autologous whole blood or plasma.

In one means for practicing the invention, the tissue fragments are first contacted with plasma. The ratio of volume of plasma used to rinse the graft in these embodiments is sufficient that the remaining extra-cellular fluid in the tissue fragments does not significantly dilute the plasma. After exposing the tissue fragments to the plasma, the excess plasma may be removed by draining the produced tissue composite, introducing an absorbent material to wick away excess plasma from the graft, or gentle

centrifugation of the graft followed by aspiration of the excess plasma. The graft suspended in plasma may then be contacted with a thrombin source. The tissue fragments can be mixed with plasma in one example by utilizing two syringes connected by a female-to-female luer lock connector. FIG. 3 illustrates another operational flow chart of a method for preparing an adipose tissue biocomposite with a wound-healing promoter to serve as an inhibitor of adhesion formation in accordance with another aspect the present invention. In this embodiment, lipoplasty is performed to derive an adipose tissue component having a plurality of adipose tissue fragments from a donor, as shown in block 116. The plurality of adipose tissue fragments are harvested from the donor as indicated at block 118. In next step, as shown in block 120, the plurality of adipose tissue fragments are contacted with appropriate concentrations of a thrombin source, a fibrinogen source and a wound- healing promoter to achieve an appropriate gelling reaction (i.e., formation of the fibrin network). Finally, as shown in block 122, the mixture of the adipose tissue fragments, the thrombin source, the fibrinogen source and the wound-healing promoter are applied to a surgical site of the donor so as to inhibit adhesion formation.

While practicing the above-disclosed method, the wound-healing promoter is employed to inhibit adhesion formation. In these embodiments, the wound-healing promoter may include, but is not limited to, cytokines, hormones, drugs including germicides, antibiotics, analgesics, local anesthetic agents, and biological response modifiers,.

Referring to FIG. 4, another operational flow chart of a method for preparing an adipose tissue biocomposite graft utilizing a syringe to serve a wide range of medical applications in accordance with another aspect of the present invention is illustrated. In this embodiment, lipoplasty is performed to derive an adipose tissue component having a plurality of adipose tissue fragments from a donor, as shown in block 124. The adipose tissue component having a plurality of adipose tissue fragments is harvested from the donor as indicated at block 126. The adipose tissue component in this embodiment includes a plurality of adipose tissue fragments and at least one viable stem cell. Next, the plurality of adipose tissue fragments is contacted with appropriate concentrations of a thrombin source and a fibrinogen source in the syringe as shown in block 128. Finally, as indicated at block 130, the mixture of the adipose tissue fragments, the thrombin source and the fibrinogen source are injected in the form of gel fragments to a body site (e.g., wound site) of the donor so as to promote wound healing.

With reference to the above-discussed methods, the reaction constituents of adipose tissue component having adipose tissue fragments, thrombin, and fibrinogen may be mixed in liquid form to fill a cavity in or on the body. This type of molding is known as in situ molding. In this instant case, the wound site in the body serves as an in situ mold where the gelling reaction occurs and the tissue biocomposite graft takes the structure of the wound site. The wound site can be a normal anatomical structure, a cavity present as part of pathology or birth defect or a cavity formed by trauma, injury or wound to the body. Alternatively, the wound site can be formed by the injection of the mixture into the body through a cannula. When in situ molding is performed, in some embodiments the tissue biocomposite graft can serve as a sealant in which oozing bleeding can be arrested. In certain embodiments, the adipose tissue biocomposite is applied in situ in topical applications. Such topical applications include spraying, painting, pouring, spreading or injecting the adipose tissue biocomposite into the body. In this case, the mixture of the adipose tissue component having adipose tissue fragments and viable stems cells, thrombin source and fibrinogen source are delivered prior to its gelling reaction so that the mixture is still in its liquid phase when

administered. After delivery, the gelling reaction progresses and the biocomposite graft becomes a solid or semi-solid in the three-dimensional structure of the body site it occupies.

In some instances, the biocomposite graft is prepared by harvesting

disassociated tissue fragments from a body and then reconstructing the tissue fragments into a molded, three-dimensional structure in which a portion of the volume of the graft is living tissue. In certain embodiments, the adipose tissue fragments include 5% to 95% of the total volume of the adipose tissue biocomposite graft. In some embodiments, the fraction of the adipose tissue fragments having the greatest collective volume of the biocomposite have a median diameter greater than 100 microns and less than 5000 microns and these same tissue fragments have a median cell number of greater than 10² cells per fragment but less than 10¹⁰ cells per fragment. In embodiments, 70% or more of the cells contained in the adipose tissue fragments are viable at the time of application to the body as the subject adipose tissue biocomposite, such as 75% or more, such as 80% or more, such as 85% or more, such as 90% or more, such as 95% or more and including 99% or more of the adipose tissue fragments are viable at the time of application to the body as the subject adipose tissue biocomposite.

In some embodiments, the subject adipose tissue biocomposite may be prepared and used within the one surgical procedure and within the operation theatre. This simplicity of sourcing and processing the living tissue within a single surgical procedure in these embodiments enables significant savings in costs to the healthcare system. By doing so, the adipose tissue biocomposite includes a large number of living cells that promote wound repair and reduces the risk of post-surgical complications.

In the present invention, one method of generating adipose tissue fragments from the donor's body may be lipoplasty. Briefly, lipoplasty utilizes a cannula, suction source and a harvest chamber to harvest the generated tissue fragments. In

embodiments, the lipoplasty is carried out in such a way that the substantial majority of the cells in adipose tissue fragments remain viable. The adipose tissue fragments are derived using lipoplasty methods selected from a group consisting of: suction assisted lipoplasty (SAL), ultra-sound assisted lipoplasty (USAL), power assisted lipoplasty (PAL), syringe assisted lipoplasty (SAL), laser assisted lipoplasty (LAL) and water jet assisted lipoplasty (WJAL).

FIG. 5 illustrates the adipose tissue biocomposite graft 134 prepared using a rectangular three dimensional mold cavity 132 in accordance with certain embodiments of the present invention. The adipose tissue biocomposite graft 134 is prepared by injecting the mixture of the adipose tissue fragments, the thrombin source and the fibrinogen source into the rectangular three dimensional mold cavity 132 to achieve the gelling reaction and to confer a three dimensional shape to the adipose tissue biocomposite graft. As shown, the adipose tissue biocomposite graft is removed from the three dimensional mold cavity 132 by an operator using a hand tool 136. The hand tool 136 is selected from a group consisting of: forceps and a pair of tweezers. After molding, the adipose tissue biocomposite graft 134 retains the rectangular shape of the mold cavity 132. The molded strip of the adipose tissue biocomposite graft 134 can be used by a surgeon to reduce post surgical complications.

FIG. 6 illustrates the adipose tissue biocomposite graft 138 prepared using a circular three dimensional mold cavity 140 in accordance with certain embodiments of the present invention. The adipose tissue biocomposite graft 138 is removed from the circular mold cavity 140 by the operator using the hand tool 136. The hand tool 136 is used around the edges of the circular mold 140 to release the graft 138 from the circular mold 140. In this case, the adipose tissue biocomposite graft 138 retains the three dimensional shape of the circular mold cavity 140. The molded biocomposite 138 thus formed possess high tensile strength and elasticity, which allows it to be easily moved and handled by the operator without breaking or tearing.

The method of preparing molded adipose tissue biocomposite employing the above-specified three-dimensional molds is referred to herein as ex vivo molding. The three dimensional mold used in this type of molding is of a size, shape and dimension necessary to control structure of the adipose tissue biocomposite graft. The substantially stable three-dimensional shape is derived by the delivery of the biocomposite elements in a liquid state to the mold cavity. After introduction to the mold, the gelling reaction occurs by the chemistry of the reactants, catalysts and substrates contained in the tissue fragment-suspending medium. The gel thus formed retains the three dimensional shape of the mold when carefully removed from the mold against surface tension forces and gravity for a substantial period of time. The molded biocomposite graft demonstrates elasticity and can be sutured or held in place by it acting as a sealant. Therefore, the adipose tissue graft material can be obtained in any desired size, shape, or dimension by selecting the appropriate mold apparatus, as described in detail above.

In some embodiments of the present invention, specific molds can be customized by surgeons to carry out defined surgical procedures that will reduce the time and increase reproducibility and reliability of the adhesion prevention. The method and materials described for producing the disclosed tissue biocomposite graft preparation are rapid, reliable and easy to use. In some embodiments, the present method minimizes the amount of hands-on time and total time for grafting procedure. The present invention also provides for a single mold device to provide multiple castings of tissue biocomposite grafts for an individual donor.

The embodiments of the adipose tissue biocomposite graft and variations described herein may be used for plastic surgery, urology, neurosurgery, orthopedics, OB/GYN, dentistry and a wide variety of other medical applications. Such medical applications may include cosmetic, therapeutic and surgical procedures. Where the subject adipose tissue biocomposites include one or more autologous components, the autologous adipose tissue biocomposite represents a safe and cost effective way to prevent surgical adhesions and may be used for civilian and military wounds, trauma, burn and reconstruction applications in remote locations. In some embodiments, the adipose biocomposite graft is characterized by good handling properties such as adequate elasticity and tensile strength to enable placement at surgical sites.

In some embodiments, the structure of the biocomposite graft is dependent on the volume of the adipose tissue fragments and concentrations of the fibrinogen source and the thrombin source, and can be practiced in orchestration with several wound- healing promoters to promote wound-healing process. Promotion of wound healing includes at least one of the effects of: promoting hemostasis, reducing time for wound closure, reducing post-surgical wound complications, reduced scarring and achieving a desirable cosmetic effect.

As discussed above, in certain embodiments, the subject adipose tissue biocomposite is supplemented with supplements such as a single cell suspension, drug or other graft-modifying agent. The adipose tissue biocomposite may include one or more types of bioactive agents, such as two or more types, such as three or more types, such as four or more types, such as five or more types and including ten or more types of bioactive agents. Examples of bioactive agents may include stem cell concentrates, platelet rich plasma, cytokines, growth factors, antibiotics, analgesics, and other bioactive agents as described in detail above. By supplementing the graft with such materials, customized and more therapeutic grafts can be prepared.

The bioactive agent may be added to the adipose tissue biocomposite during preparation of the adipose tissue biocomposite, such as to one or more of the adipose tissue component or the source of thrombin and fibrinogen or may be added to the produced adipose tissue biocomposite. The bioactive agent may be added as any convenient formulation, such as a liquid, a solid, a lyophilized powder, a suspension, a syrup, or any combination thereof. The amount of each bioactive agent incorporated into the adipose tissue biocomposite may vary depending on the type of bioactive agent and the size of the biocomposite and may be 0.01 μg or more, such as 0.05 μg or more, such as 0.1 μg or more, such as 0.5 μg or more, such as 1 μg or more, such as 5 μg or more, such as 10 μg or more, such as 25 μg or more, such as 50 μg or more, such as 100 μg or more, such as 250 μg or more, such as 1000 μg or more, such as 10 g or more, such as 25 g or more and including 100 g of bioactive agent or more. Where the bioactive agent is incorporated into the adipose tissue biocomposite as a liquid, the concentration of each bioactive agent may be 0.0001 μg/mL or greater, such as 0.001 μg/mL or greater, such as 0.01 μg/mL or greater, such as 0.1 μg/mL or greater, such as 0.5 μg/mL or greater, such as 1 μg/mL or greater, such as 2 μg/mL or greater, such as 5 μg/mL or greater, such as 10 μg/mL or greater, such as 25 μg/mL or greater, such as 50 μg/mL or greater, such as 100 μg/mL or greater such as 500 μg/mL or greater, such as 1 g/mL or greater such as 5 g/mL or greater and including 10 g/mL or greater. In certain embodiments, the supplements are added prior to the initiation of the gelling reaction.

In one example, preparation of an adipose biocomposite graft for adhesion prevention can be performed in the following manner. In preparation, three 30 ml syringes designated A, B and C and a three-way stopcock are prepared as described below. Examples of molds used for casting the adipose biocomposite are also provided below.

In this example, a syringe A is filled with 20 ml of saline washed adipose tissue fragments wherein less than 12% of the adipose tissue volume is aqueous phase. The adipose tissue was obtained from a suction canister containing lipoaspirate following lipoplasty. In this example, the cannula for harvesting the adipose tissue from the body has an opening of 3 to 5 mm. The syringe is stored in an upright position with its plunger in the top position for approximately 10 to 30 minutes such that the lighter adipose tissue fragments and excess tumescent fluid become separated due to differences in density. The excess tumescent fluid is removed from the syringe by pressing down the plunger forcing the aqueous tumescent fluid to leave the syringe while retaining desired volume of adipose tissue fragments.

A syringe B is filled with 16 ml volume of normal human whole blood or plasma having a plasma fibrinogen concentration of 2 - 6 mg/mL and plasminogen (physiologic levels). As discussed above, plasma may be obtained by centrifuging whole blood anticoagulated with a calcium chelating agent such as citrate. For example,

centrifugation of a vacutainer containing sodium citrate as the anticoagulant and 60 ml of whole blood for 2,000G for 15 minutes is sufficient to cause separation of the blood elements from the plasma. The plasma may be selectively removed from the centrifuge vessel using a needle and syringe. The desired amount of plasma (16 ml) is harvested into a 30 ml. syringe.

A syringe C is filled with 4 ml of thrombin solution containing 100 U/mL of bovine thrombin serum. In certain embodiments, a source of medical grade thrombin is distributed by King Pharmaceuticals of Bristol, Tennessee designated as Thrombin, Topical (BOVINE ORIGIN), U.S. P., with the trade name THROMBIN-JMI. A vial containing 5,000 international units of bovine thrombin is preferably first reconstituted with 5 ml of saline diluent to create a 1 ,000 U/ml solution. This solution is further diluted 10 fold by adding 1 ml of 1 ,000 U/mL thrombin to 9 ml of saline to create a 100 U/mL solution. Four ml of this thrombin solution is aspirated into a 30 ml syringe. In other embodiments, 4 ml of autologous thrombin serum may be prepared according to the method disclosed in International Patent Application No. PCT/US2013/061756 which published as WO2014/052496 on April 3, 2014, the disclosure of which is herein incorporated by reference. Next, a standard three-way stopcock may be selected for allowing the mixing the contents of the three syringes. For example, a suitable medical grade stopcock is sold by Qosina from Edgewood, New York with the part number 13813 and that includes 2 female luer locks and 1 male luer lock.

In certain embodiments, the mold is tubing (e.g., plastic or metal pipe). Where tubing is used as the mold for the adipose tissue biocomposite (e.g., spaghetti shape), in some embodiments, an extension tubing such as Qosina part number 33061 may be utilized which is 20 inches in length and has internal diameter of 0.094 inches.

In other embodiments, if it is desired to make a circular adipose biocomposite, i.e., pancake shaped, one example includes using a petri dish such as those sold by Sigma Aldrich, St. Louis including Corning CLS3295 culture dishes having a depth of 60 mm and height of 15 mm as a mold to prepare the subject adipose tissue biocomposite.

In still other embodiments, if it is desired to make a columnar adipose graft, one example includes using a 30 ml syringe as a mold.

In certain embodiments, once the three syringes are prepared, the adipose tissue fragment syringe A is attached to the stopcock to one luer lock port and the plasma syringe B is attached to a second luer lock port on the stopcock. The contents of the syringes A and B are then intermingled by passing the full contents of the syringes in and out several times between the two syringes until thoroughly mixed by alternatively depressing the plunger of the two syringes with the stopcock handle turned to allow connection between the two syringes. For example, 3 to 6 times of passing the fluids between the two syringes may be sufficient to achieve good intermingling of the adipose tissue and plasma. After mixing, the combined fluids are fully delivered into Syringe A and the empty syringe B is removed from the stopcock. The thrombin containing syringe C is then attached to the stopcock. In some embodiments, the process of mixing the thrombin fluid with the plasma and adipose fragment mixture is repeated by passaging of the fluids between Syringes A and C. For example, 3 times of passing the fluid is sufficient to achieve mixing. After mixing, the fluid contents are fully loaded into Syringe A. With these concentrations of thrombin and fibrinogen, gelling reaction (i.e., fibrin network formation) will occur in approximately one to two minutes.

If it is desired to cast a mold, the contents of the syringe A now containing thrombin, fibrinogen, adipose tissue fragments may be passed into the mold (e.g., within 10 seconds of mixing). The mold is left undisturbed for a period of one minute or more to allow the gelling reaction to occur undisturbed. After gelling has occurred, the molded adipose tissue biocomposite may then be removed (e.g., using forceps). If tubing is used as the mold, the adipose composite may be removed from the tubing in certain embodiments by flushing with saline solution dispelling the composite out of the tubing.

If it is desired to deliver the adipose biocomposite as gel fractions, the gelling reaction may be allowed to occur in the syringe containing the mixture of thrombin, fibrinogen and adipose tissue fragments. The gel fragments can be ejected from the syringe by depressing the plunger at the desired tissue site to treat a wound. The columnar graft can be sliced into sections according to the desired thickness. In certain embodiments, the adipose tissue biocomposites include two layers and the methods for preparing the subject adipose tissue biocomposites may be

characterized by: 1 ) contacting an adipose tissue component with a composition having fibrinogen and thrombin; 2) allowing the adipose tissue component to float to the top of the fibrinogen and thrombin composition; and 3) forming a fibrin network from the composition of fibrinogen and thrombin to form an adipose tissue biocomposite having an adipose tissue component layer and a fibrin network layer. In these embodiments, the fibrinogen and thrombin may be present in a fluid composition together, such as in whole blood, autologous whole blood plasma anti-coagulated with a calcium-chelating agent, platelet rich plasma with its associated growth factors, autologous plasma, autologous platelet rich plasma, plasma and collagen mixture as represented by Vitagel by Orthovita; purified allogeneic fibrinogen with added thrombin. In certain

embodiments, the fibrinogen is present in plasma (e.g., platelet rich plasma, platelet- poor plasma), such as plasma obtained prepared using a centrifugation vessel such as described in co-pending United States Patent Application Serial No. 13/199,129 filed on August 19, 201 1 , United States Patent Application Serial No. 13/199,1 1 1 filed on August 19, 201 1 , United States Patent Application Serial No. 13/199,1 19 filed on August 19, 201 1 as well as United States Provisional Patent Application No. 62/069,783 filed on October 28, 2014, the disclosures of which are herein incorporated by reference. In some embodiments, thrombin is prepared such as described in International Patent Application No. PCT/US2013/061756 published as WO2014/052496 on April 3, 2014, the disclosure of which is herein incorporated by reference.

After contacting the adipose tissue component with the composition containing fibrinogen and thrombin (e.g., plasma), the adipose tissue component is allowed to float to the top of the fibrinogen and thrombin composition so as to separate the adipose tissue component and fibrinogen and thrombin composition into two layers. The adipose tissue component may be allowed to float to the top of the fibrinogen and thrombin composition for any suitable duration, depending on the density and size of the adipose tissue components as well as the concentration of fibrinogen and thrombin and may be 1 minute or longer, such as 2 minutes or longer, such as 3 minutes or longer, such as 5 minutes or longer, such as 10 minutes or longer, such as 15 minutes or longer, such as 30 minutes or longer and including allowing the adipose tissue component to float to the top of the fibrinogen and thrombin composition for 45 minutes or longer.

In some embodiments, the fibrin network is being formed in the fibrinogen and thrombin composition while the adipose tissue components are floating to the top of the fibrinogen and thrombin composition. For example 5% or more of the fibrin network in the fibrinogen and thrombin composition may have formed after the adipose tissue components have floated to the top of the fibrinogen and thrombin composition, such as 10% or more, such as 15% or more, such as 25% or more, such as 35% or more, such as 50% or more and including 75% or more of the fibrin network. In certain

embodiments, the fibrin network does not begin to form until all of the adipose tissue components have floated to the top of the fibrinogen and thrombin composition.

In certain embodiments, 50% or less of the fibrin network in the fibrinogen and thrombin composition has formed while the adipose tissue components float to the top of the fibrinogen and thrombin composition, such as 45% or less, such as 40% or less, such as 35% or less, such as 30% or less, such as 25% or less, such as 20% or less, such as 15% or less, such as 10% or less and including 5% or less of the fibrin network in the fibrinogen and thrombin composition has formed while the adipose tissue components float to the top of the fibrinogen and thrombin composition. In certain embodiments, all of the adipose tissue components float to the top of the fibrinogen and thrombin composition before the formation of the fibrin network.

The thickness of each layer formed may vary depending on the ratio of adipose tissue component contacted with the composition having fibrinogen and thrombin and may be 0.1 mm or greater, such as 0.2 mm or greater, such as 0.5 mm or greater, such as 1 mm or greater, such as 2 mm or greater, such as 5 mm or greater, such as 10 mm or greater, such as 25 mm or greater and including 50 mm or greater. Each layer may be the same size or different sizes or some combination thereof. For example, where adipose tissue biocomposites include two layers, a first layer having an adipose tissue component and a second layer having the fibrin network may have the same thickness. In other embodiments, a first layer having an adipose tissue component may be thicker that a second layer having the fibrin network by 5% or more, such as 10% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, including by 2-fold or more, such as 3-fold or more and including by 5-fold or more. In yet other embodiments, a second layer having the fibrin network may be thicker than a first layer having an adipose tissue component by 5% or more, such as 10% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, including by 2-fold or more, such as 3-fold or more and including by 5-fold or more.

The ratio by volume of adipose tissue component to composition having fibrinogen and thrombin may vary depending on the specific properties of the adipose tissue biocomposite desired and may range between 1 :1 and 1 :1 .5; 1 :1 .5 and 1 :2; 1 :2 and 1 :2.5; 1 :2.5 and 1 :3; 1 :3 and 1 :3.5; 1 :3.5 and 1 :4; 1 :4 and 1 :4.5; 1 :4.5 and 1 :5; 1 :5 and 1 :5.5; 1 :5.5 and 1 :6; 1 :6 and 1 :6.5; 1 :6.5 and 1 :7; 1 :7 and 1 :7.5; 1 :7.5 and 1 :8; 1 :8 and 1 :8.5; 1 :8.5 and 1 :9; 1 :9 and 1 :9.5; 1 :9.5 and 1 :10 or a range thereof. For example, the ratio by volume of the adipose tissue component to composition having fibrinogen and thrombin ranges from 1 :1 and 1 :10, such as 1 :1 and 1 :8, such as 1 :1 and 1 :5, such as 1 :1 and 1 :4, and including from 1 :1 and 1 :2. In certain instances, the ratio of adipose tissue component to composition having fibrinogen and thrombin ranges between 10:1 and 9.5:1 ; 9.5:1 and 9:1 ; 9:1 and 8.5:1 ; 8.5:1 and 8:1 ; 8:1 and 7.5:1 ; 7.5:1 and 7:1 ; 7:1 and 6.5:1 ; 6.5:1 and 6:1 ; 6:1 and 5.5:1 ; 5.5:1 and 5:1 ; 5:1 and 4.5:1 ; 4.5:1 and 4:1 ; 4:1 and 3.5:1 ; 3.5:1 and 3:1 ; 3:1 and 2.5:1 ; 2.5:1 and 2:1 ; 2:1 and 1 .5:1 ; 1 .5:1 and 1 :1 or a range thereof. For example, the ratio by volume of adipose tissue component to composition having fibrinogen and thrombin ranges from 10:1 and 1 :1 , such as 8:1 and 1 :1 , such as 5:1 and 1 :1 , such as 4:1 and 1 :1 , and including from 2:1 and 1 :1 .

In these embodiments, after a sufficient duration has passed to allow formation of the fibrin network, the two-layer adipose tissue biocomposite may be removed from the mold or may be further processed. In some instances, further processing includes drying the adipose tissue biocomposite, such as under an air drier or may be wiped or cleaned with a sterile pad, gauze or other wipe or rinsed with a sterile saline solution. In certain instances, the two-layer adipose tissue biocomposite may be cut into different shapes, as desired.

In certain embodiments, a two-layer adipose tissue biocomposite is prepared by contacting an adipose tissue component with plasma with thrombin (aqueous phase) at a ratio of 2 parts to 1 part of adipose tissue component (lipophilic phase) such that the adipose tissue floats to the top of the plasma before the formation of fibrin occurs. For example, 20 ml of adipose tissue (with less than 10% aqueous phase), 40 ml of plasma (mixed these two components by passing 3 times in two 60 ml syringes connected by a stopcock and then adding 1 ml of thrombin (to achieve 1 -5 unit/ml of thrombin in the aqueous phase of the mixture), mix together and then immediately caste the mixture into a mold for it to have fibrin polymerize for 5 minutes or more minutes. In certain embodiments, the adipose tissue biocomposite is blotted to remove excess plasma with a sterile gauze or rinsed with saline.

METHODS FOR APPLYING AN ADIPOSE TISSUE BIOCOMPOSITE TO A SUBJECT

As summarized above, the subject disclosure provides adipose tissue biocomposites. Aspects of the disclosure also include methods for applying one or more of the subject adipose tissue biocomposites to a subject. Methods of using the subject adipose tissue biocomposites include administering one or more of the adipose tissue biocomposites to a body site of the subject in order to treat a subject for a target condition of interest such as adhesions formed during wound repair (in trauma wounds or surgical wounds). By "treating" or "treatment" is meant at least a suppression or amelioration of the symptoms associated with the condition affecting the subject, where suppression and amelioration are used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., symptom, associated with the condition being treated. As such, treatment also includes situations where the condition is completely inhibited, e.g., prevented from happening, or stopped, e.g., terminated, such that the subject no longer experiences the condition. As such, treatment includes both preventing and managing a condition.

In certain embodiments, the subject adipose tissue biocomposites as described herein are used in the treatment of a wound site, such as where the wound occurs by injury or by surgery. In other embodiments, the subject adipose tissue biocomposites are used to separate tissues during tissue repair (e.g., remesothelialization) such as during healing from a wound or to prevent the formation of tissue adhesions. In other embodiments, the subject adipose tissue biocomposites are used to prevent or treat fistulas. In yet other embodiments, the subject adipose tissue biocomposites are used to promote hemostasis. In still other embodiments, the subject adipose tissue

biocomposites are used to reduce time for wound closure. In still other embodiments, the subject adipose tissue biocomposites are used to reduce scarring from a healed wound. In still other embodiments, the subject adipose tissue biocomposites are used to treat a skin excision. In other embodiments, the subject adipose tissue biocomposites are used to treat skin ulcers. In yet other embodiments, the subject adipose tissue biocomposites are used to treat tissue burns (e.g., skin burns). In still other

embodiments, the subject adipose tissue biocomposites are used to deliver one or more bioactive agents to the body site, such as by sustained or pulsatile release, as described above.

In embodiments, methods include applying one or more of the subject adipose tissue biocomposites a body site of a subject, such as a wound site. The term "subject" is meant the person or organism to which the adipose tissue biocomposite is applied and maintained in contact. As such, subjects may include but are not limited to mammals, e.g., humans and other primates, such as chimpanzees and other apes and monkey species; and the like, as well as non-human subjects such as, but not limited to, birds, mice, rats, dogs, cats, livestock and horses. In certain embodiments, the subject is a human.

In practicing the subject methods, the adipose tissue biocomposites may be applied to any convenient internal or external location on the subject, such as to organ tissue including but not limited to integumentary tissue (e.g. sections of the skin), oral tissue (e.g., buccal, tongue, palatal, gums), respiratory tissue (e.g., pharynx, larynx, trachea, bronchi, lungs, diaphragm) gastrointestinal tissue (e.g., esophagus, stomach, liver, gallbladder, pancreas, intestines, colon, rectum and anus.), cardiovascular tissue (e.g., heart, blood vessels), endocrine tissue (e.g., hypothalamus, pituitary gland, pineal body or pineal gland, thyroid, parathyroids, adrenal glands) and genitourinary tissue (kidneys, ureters, bladder, urethra, ovaries, fallopian tubes, uterus, vagina, mammary glands, testes, vas deferens, seminal vesicles, prostate, penis), muscular tissue, nervous tissue (e.g., brain, spinal cord, nerves) as well as soft skeletal tissue (cartilage, ligaments, tendons). Furthermore, the adipose tissue biocomposites may be applied to any type of organismic tissue, including both healthy and diseased tissue (e.g., cancerous, malignant, necrotic, etc.), where desired.

The size of the body site (e.g., wound site) treated with the subject adipose tissue biocomposites may vary, such as having a surface area ranging from 0.1 to 100 cm², such as 0.5 to 75 cm², such as 1 .0 to 50 cm², such as 1 .5 to 45 cm², such as 2.0 to 40 cm², such as 2.5 to 35 cm², and including 2 to 30 cm². Where body site treated is three-dimensional cavity, the size of the body site may range from 0.1 to 100 cm³, such as 0.5 to 75 cm³, such as 1 .0 to 50 cm³, such as 1 .5 to 45 cm³, such as 2.0 to 40 cm³, such as 2.5 to 35 cm³, and including 2 to 30 cm³.

The subject adipose tissue biocomposites may be applied and maintained at the application site over an extended period of time, as desired. For example, the adipose tissue biocomposite may be maintained at the body site (e.g., wound site) over the course of hours, days and including weeks. As described in detail above, the adipose tissue biocomposite is prepared such that the fibrin network is configured to break down in the body site over a predetermined period of time such as after 6 hours or longer, such as after 12 hours or longer, such as after 24 hours or longer, such as after 48 hours or longer, such as after 72 hours or longer, such as after 96 hours or longer, such as after 120 hours or longer, such as after 144 hours or longer and including after 168 hours or longer.

In some embodiments, methods include applying and maintaining the subject adipose tissue biocomposites at a body site of a subject in a manner where the fibrin network breaks down (e.g., by fibrinolysis) at a predetermined rate, such as at a substantially zero-order fibrinolysis rate, such as at a substantially first order fibrinolysis rate and including at a substantially second-order fibrinolysis rate. As discussed above, the breakdown of the fibrin network to release the adipose tissue component from the subject adipose tissue biocomposites during the subject methods may be measured by any convenient protocol, such as by thrombin clotting time (TCT), thromboelastometry (TEM), as well as by euglobulin lysis time (ELT) assay.

In practicing the subject methods, the adipose tissue biocomposite may be applied a single time or a plurality of times over a given time period, e.g., during the course of wound repair (e.g., to prevent adhesion formation), where the application schedule when a plurality of biocomposites are applied over a given time period may be hourly, daily, weekly, etc. For example, the subject methods include multiple application intervals. By "multiple application intervals" is meant more than one adipose tissue biocomposite is applied and maintained in contact with the subject in a sequential manner. As such, a first adipose tissue biocomposite is removed from contact with the subject and a adipose tissue biocomposite is reapplied to the subject. In practicing methods of the invention, treatment regimens may include two or more application intervals, such as three or more application intervals, such as four or more application intervals, such as five or more application intervals, including ten or more application intervals.

The duration between application intervals in a multiple application interval treatment regimen may vary, as determined by a qualified health care professional. For example, the duration between application intervals in a multiple application treatment regimen may be predetermined and follow at regular intervals. As such, the time between application intervals may vary and may be 1 hour or longer, such as 2 hours or longer, such as 3 hours or longer, such as 6 hours or longer, such as 12 hours or longer, such as 24 hours or longer, such as 48 hours or longer, such as 72 hours or longer, including 168 hours or longer.

In certain instances, a subsequent application interval in a treatment regimen may employ the same or a different formulation of adipose tissue biocomposite as the previous application interval. For example, the fibrin network (e.g., crosslink density or amount of fibrin) or the composition of the adipose tissue component of adipose tissue biocomposite may be increased in subsequent application intervals by 10% or greater, such as 20% or greater, such as 50% or greater, such as 75% or greater, such as 90% or greater and including 100% or greater. On the other hand, the fibrin network (e.g., crosslink density or amount of fibrin) or the composition of the adipose tissue component of adipose tissue biocomposite may be decreased in subsequent application intervals, such as by 10% or greater, such as 20% or greater, such as 50% or greater, such as 75% or greater, such as 90% or greater and including 100% or greater.

In some embodiments, methods include applying a two-layered adipose tissue biocomposite (as described above). In these embodiments, methods may include contacting the body site (e.g., wound site) of the subject with the adipose tissue component side of the two-layer adipose tissue composite. In other embodiments, methods may include contacting the body site (e.g., wound site) of the subject with the fibrin network side of the two-layer adipose tissue composite.

In certain embodiments, the subject methods include assessing a subject as in need of treatment with one or more of the subject adipose tissue biocomposites described above. Individuals may be assessed using any convenient protocol. For example, methods may include determining that a wound site of the subject is susceptible to adhesion formation, such as post-surgical adhesion formation. Diagnosis or assessment of target condition can be performed using any convenient diagnostic protocol as determined by a qualified health care professional. In certain embodiments, the subject adipose tissue biocomposites can be applied concurrent with other therapeutic protocols, such as for example, for management of blood clotting (e.g., anticoagulation protocols, anti-thrombotic protocols), as well as with devices to physically separate tissues at the body site (e.g., to inhibit adhesion formation). By "concurrent application" is intended administration to a subject such that the therapeutic effect of the combination is caused in the subject undergoing therapy.

In certain embodiments, methods include delivering one or more bioactive agents to the body site from with the subject adipose tissue biocomposites. In these

embodiments, method may include delivering one or more types of bioactive agents, such as two or more types, such as three or more types, such as four or more types, such as five or more types and including ten or more types of bioactive agents. Where the subject adipose tissue biocomposites are configured as layers, methods may include delivering one more bioactive agents from each layer. The amount of bioactive agent delivered may be 0.001 mg or more, such as 0.01 mg or more, such as 0.1 mg or more, such as 0.5 mg or more, such as 1 mg or more and including 1 mg or more. For example, the amount of bioactive agent delivered from each layer in a multi-layer (e.g., two-layer) adipose tissue biocomposite may range from 0.001 mg to 1000 mg, such as 0.01 mg to 500 mg, such as 0.1 mg to 250 mg, such as 0.5 mg to 100 mg, such as 1 mg to 50 mg, including 1 mg to 10 mg

As discussed above, example bioactive agents that may be delivered with the subject adipose tissue biocomposites, include but are not limited to, interferon, interleukin, erythropoietin, granulocyte-colony stimulating factor (GCSF), stem cell factor (SCI:), leptin (OB protein), interferon (alpha, beta, gamma), antibiotics such as vancomycin, gentamicin ciprofloxacin, amoxycillin, lactobacillus, cefotaxime,

levofloxacin, cefipime, mebendazole, ampicillin, lactobacillus, cloxacillin, norfloxacin, tinidazole, cefpodoxime, proxctil, azithromycin, gatifloxacin, roxithromycin,

cephalosporin, anti-thrombogenics, aspirin, ticlopidine, sulfinpyrazone, heparin, warfarin, growth factors, differentiation factors, hepatocyte stimulating factor, plasmacytoma growth factor, glial derived neurotrophic factor (GDNF), neurotrophic factor 3 (NT3), fibroblast growth factor (FGF), transforming growth factor (TGF), platelet transforming growth factor, milk growth factor, endothelial growth factors, endothelial cell-derived growth factors (ECDGF), alpha-endothelial growth factors, beta-endothelial growth factor, neurotrophic growth factor, nerve growth factor (NGF), vascular endothelial growth factor (VEGF), 4-1 BB receptor (4- IBBR), TRAIL (TNF-related apoptosis inducing ligand), artemin (GFRalpha3-RET ligand), BCA-I (B cell-attracting chemokinel), B lymphocyte chemoattractant (BLC), B cell maturation protein (BCMA), brain-derived neurotrophic factor (BDNF), bone growth factor such as osteoprotegerin (OPG), bone- derived growth factor, thrombopoietin, megakaryocyte derived growth factor (MDGF), keratinocyte growth factor (KGF), platelet-derived growth factor (PDGF), ciliary neurotrophic factor (CNTF), neurotrophin 4 (NT4), granulocyte colony-stimulating factor (GCSF), macrophage colony-stimulating factor (mCSF), bone morphogenetic protein 2 (BMP2), BRAK, C-IO, Cardiotrophin 1 (CTI), CCR8, anti- inflammatory: paracetamol, salsalate, diflunisal, mefenamic acid, diclofenac, piroxicam, ketoprofen, dipyrone, acetylsalicylic acid, anti-cancer drugs such as aliteretinoin, altertamine, anastrozole, azathioprine, bicalutarnide, busulfan, capecitabine, carboplatin, cisplatin,

cyclophosphamide, cytarabine, doxorubicin, epirubicin, etoposide, exemestane, vincristine, vinorelbine, hormones, thyroid stimulating hormone (TSH), sex hormone binding globulin (SHBG), prolactin, luteotropic hormone (LTH), lactogenic hormone, parathyroid hormone (PTH), melanin concentrating hormone (MCH), luteinizing hormone (LHb), growth hormone (HGH), follicle stimulating hormone (FSHb), haloperidol, indomethacin, doxorubicin, epirubicin, amphotericin B, Taxol, cyclophosphamide, cisplatin, methotrexate, pyrene, amphotericin B, anti- dyskinesia agents, Alzheimer vaccine, antiparkinson agents, ions, edetic acid, nutrients, glucocorticoids, heparin, anticoagulation agents, antivirus agents, anti-HIV agents, polyamine, histamine and derivatives thereof, cystineamine and derivatives thereof, diphenhydramine and derivatives, orphenadrine and derivatives, muscarinic antagonist, phenoxybenzamine and derivatives thereof, protein A, streptavidin, amino acid, beta-galactosidase, methylene blue, protein kinases, beta-amyloid, lipopolysaccharides, eukaryotic initiation factor-4G, tumor necrosis factor (TNF), tumor necrosis factor-binding protein (TNF -bp), interleukin- 1 (to 18) receptor antagonist (IL-lra), granulocyte macrophage colony stimulating factor (GM-CSF), novel erythropoiesis stimulating protein (NESP), thrombopoietin, tissue plasminogen activator (TPA), urokinase, streptokinase, kallikrein, insulin, steroid, acetaminophen, analgesics, antitumor preparations, anti-cancer preparations, anti-proliferative preparations or pro-apoptotic preparations, among other types of bioactive agents.

Depending on the composition of the adipose tissue biocomposite or specific layer (e.g., adipose tissue component composition, fibrin network crosslink density, etc.), methods may include delivering the one or more bioactive agents by sustained or pulsatile release, as described above. For example, where methods include delivering one or more bioactive agents by "sustained release" a constant and continuous delivery of one or more bioactive agents is maintained while the adipose tissue biocomposite is in contact with the site of administration (e.g., abdominal cavity), such as over the course of 1 day or longer, such as 2 days or longer, such as 3 days or longer, such as 5 days or longer and including 7 days or longer. In other instances, methods include delivering one or more bioactive agents by "pulsatile release" such as by releasing one or more bioactive agents into the site of administration incrementally (e.g., at discrete times), such as every 1 hour, such as every 2 hours, such as every 5 hours, such as every 12 hours, such as every 24 hours, such as every 36 hours, such as every 48 hours, such as every 72 hours, such as every 96 hours, such as every 120 hours, such as every 144 hours and including every 168 hours.

In other instances, methods include delivering one or more bioactive agents from the subject adipose tissue biocomposites (or one or more of the layers when present) after certain percentages of the fibrin network has been broken down by fibrinolysis. For example, methods may include delivering an amount of the one or more bioactive agents after every 10% of the crosslinks of the fibrin network has been broken down by fibrinolysis, such as after every 15% of the fibrin network has been broken down by fibrinolysis, such as after every 20% of the crosslinks of the fibrin network has been broken down by fibrinolysis, such as after every 25% of the fibrin network has been broken down by fibrinolysis, such as after every 30% of the fibrin network has been broken down by fibrinolysis and including after every 33% of the fibrin network has been broken down by fibrinolysis at the site of administration.

Depending on the size of the adipose tissue biocomposite applied, methods may include delivering an average cumulative amount of bioactive agent of 5 μg/cm² or greater over an extended period of time. In these embodiments, methods include delivering an average cumulative amount of bioactive agent may be 25 μg/cm² or greater, such as 50 μg/cm² or greater, such as 75 μg/cm² or greater over a

predetermined delivery interval, such as 100 μg/cm² or greater, such as 125 μg/cm² or greater, such as 150 μg/cm² or greater and including 200 μg/cm² over a predetermined delivery interval.

In yet other embodiments, adipose tissue biocomposites are configured to deliver a target dosage of bioactive agent, such as for example as characterized by total bioactive agent exposure or by average daily bioactive agent exposure. The term target dosage is meant the amount of bioactive agent which is delivered to the subject and may vary depending on the physicochemical properties, mechanical properties, and break down rates of the adipose tissue biocomposite as well as the site of application. For example, the target dosage of bioactive agent delivered by the subject hydrogels may be 0.01 mg/day or greater, such as 0.04 mg/day or greater, such as 0.5 mg/day or greater over a 4 week dosage interval, such as 1 .0 mg/day or greater, such as 2 mg/day or greater, such as 5 mg/day or greater and including 10 mg/day over a 4 week dosage interval.

In some embodiments, methods of the present disclosure includes using the subject adipose tissue biocomposites to deliver a dosage of about 0.01 mg/kg to 500 mg/kg of the bioactive agent per day, such as from 0.01 mg/kg to 400 mg/kg per day, such as 0.01 mg/kg to 200 mg/kg per day, such as 0.1 mg/kg to 100 mg/kg per day, such as 0.01 mg/kg to 10 mg/kg per day, such as 0.01 mg/kg to 2 mg/kg per day, including 0.02 mg/kg to 2 mg/kg per day. In other embodiments, methods include using the subject adipose tissue biocomposites deliver a dosage of 0.01 to 100 mg/kg four times per day (QID), such as 0.01 to 50 mg/kg QID, such as 0.01 mg/kg to 10 mg/kg QID, such as 0.01 mg/kg to 2 mg/kg QID, such as 0.01 to 0.2 mg/kg QID. In other embodiments, methods include using the subject adipose tissue biocomposites to deliver a dosage of 0.01 mg/kg to 50 mg/kg three times per day (TID), such as 0.01 mg/kg to 10 mg/kg TID, such as 0.01 mg/kg to 2 mg/kg TID, and including as 0.01 mg/kg to 0.2 mg/kg TID. In yet other embodiments, methods include using the subject adipose tissue biocomposites to deliver a dosage of may range from 0.01 mg/kg to100 mg/kg two times per day (BID), such as 0.01 mg/kg to 10 mg/kg BID, such as 0.01 mg/kg to 2 mg/kg BID, including 0.01 mg/kg to 0.2 mg/kg BID.

KITS

Aspects of the invention further include kits, where kits include one or more components for preparing and using the subject adipose tissue biocomposites. In certain embodiments, kits include a three-dimensional mold for contacting the adipose tissue component with sources of thrombin, fibrinogen and plasminogen. As discussed above, the three-dimensional mold may be any suitable container for contacting the adipose tissue component with the source of thrombin, fibrinogen and plasminogen and may be in the shape of a circle, oval, half-circle, crescent-shaped, star-shaped, square, triangle, rhomboid, pentagon, hexagon, heptagon, octagon, rectangle or other suitable polygon or may be a tube, such as test tube, a centrifuge tube, conical bottom tube as well as tubing (e.g., to form a thin tubular adipose tissue biocomposite). For example, the three-dimensional mold may be a container having the shape of a cube, cone, half sphere, star, triangular prism, rectangular prism, hexagonal prism or other suitable polyhedron or may be in the shape of a tube. The mold may include one or more ports for inputting the adipose tissue component as well as the source of thrombin, fibrinogen and plasminogen. Any suitable port configuration may be employed, where examples of ports include channels, orifices, channels having a check valve, a Luer taper fitting, a port with a breakable seal (e.g., single use ports) among other types of ports. In some embodiments, the port is configured to connect to a syringe. In other embodiments, the port is configured to facilitate access for a needle into the cavity of the container to aspirate, mix and remove components from the container. In certain embodiments, the port is configured with a Luer taper fitting, such as a Luer-Lok or a Luer-slip.

In some embodiments, kits include one or more syringes for inputting

components of the adipose tissue biocomposite into the mold. Where desired, syringes may be configured with a Luer taper fitting, such as a Luer-Lok or a Luer-slip for connection to the three-dimensional mold or may be configured with a conduit (e.g., tubing) to fluidly connect one or more syringes to three-dimensional mold. Syringes, as well as conduits when present, may also include one or more valves such as a stop-cock valve for controlling the rate of inputting a component of the adipose tissue biocomposite into the three-dimensional mold.

In some instances, the kits can include one or more additional components (e.g., buffers, water, solvent etc.). In some instances, the kits may further include a sample collection device, e.g., blood collection device such as an evacuated blood collection tube, needle, syringe, pipette, tourniquet, etc. as desired.

Kits may also include, in certain embodiments, one or more devices for preparing (e.g., harvesting) the components of the adipose tissue biocomposite, such as the adipose tissue component, thrombin, fibrinogen and plasminogen. In some

embodiments, kits include a device for preparing the adipose tissue component from lipoaspirate from a subject. In certain instances, the adipose tissue component is prepared from lipoaspirate from a subject using a collection and centrifugation container such as described in International Patent Application No. PCT/US2013/000036 published as WO2013/122683 on August 22, 2013 as well as United States Provisional Patent Application No. 62/002,052 filed on May 22, 2014, the disclosures of which is herein incorporated by reference. In other embodiments, kits include a blood collection device for obtaining a whole blood sample. In yet other embodiments, kits include a plasma preparation device for obtaining plasma (e.g., platelet-rich and platelet-poor) for use as a source of one or more of thrombin, fibrinogen and plasminogen. In certain instances, the source of thrombin, fibrinogen and plasminogen is prepared using a centrifugation container such as described in co-pending United States Patent

Application Serial No. 13/199,129 filed on August 19, 201 1 , United States Patent Application Serial No. 13/199,1 1 1 filed on August 19, 201 1 , United States Patent Application Serial No. 13/199,1 19 filed on August 19, 201 1 as well as United States

Provisional Patent Application No. 62/069,783 filed on October 28, 2014, the disclosures of which are herein incorporated by reference. In certain embodiments, kits include one or more devices for preparing a source of thrombin. In some instances, the subject kits may include devices for preparing thrombin such as those described in International Patent Application No. PCT/US2013/061756 published as WO2014/052496 on April 3, 2014, the disclosure of which is herein incorporated by reference.

The various components of the kits may be present in separate containers, or some or all of them may be pre-combined. For example, in some instances, one or more components of the kit, e.g., the three-dimensional mold, syringes, lipoaspirate preparation device, plasma preparation container and thrombin preparation device are present in a sealed pouch, e.g., a sterile foil pouch or envelope.

In addition to the above components, in certain instances the subject kits may further include instructions for assembling the subject kit components as well as for practicing methods for preparing an adipose tissue biocomposite as described herein. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like. Yet another form of these instructions is a computer readable medium, e.g., diskette, compact disk (CD), portable flash drive, and the like, on which the information has been recorded. Yet another form of these instructions that may be present is a website address which may be used via the internet to access the information at a removed site. UTILITY

The subject adipose tissue biocomposites and methods find use in a variety of applications where it is desirable to separate two tissues, such as in a body site during wound repair from trauma or surgery or in the treatment or prevention of fistulas, skin excisions, skin ulcers and burns. In certain embodiments, the present disclosure finds use in preventing adhesion formation at a wound site during tissue repair at a body site. In some embodiments, the present disclosure provides for wound healing while promoting one or more of hemostasis, reduced time for wound closure, reduced postsurgical wound complications, reduced scarring as we as enhanced cosmetic appearance of the wound subsequent to healing. Embodiments also find use where a subject would benefit from delivery of an active agent. Likewise, adipose tissue biocomposites of interest also find use in any application where a bioactive agent would benefit from a tunable biocompatible and biodegradable delivery vehicle which could be used to stabilize or provide site specific delivery of the bioactive agent.

In certain examples, the subject adipose tissue biocomposites find use during the repair of tissue at a wound site in reducing or altogether preventing adhesions formation between adjacent tissue. In another example, adipose tissue biocomposites find use in delivery of growth factors (e.g., tissue growth factors), hemostatics, pharmaceuticals or other active agents used to treat and ailment where delivery to a site of administration can be made using a biocompatible delivery vehicle such the biocomposites described herein. As described above, treatment is meant that at least an amelioration of the symptoms associated with the condition afflicting the subject is achieved, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., symptom, associated with the condition being treated. As such, treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g., terminated, such that the subject no longer suffers from the condition, or at least the symptoms that characterize the condition. The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. EXPERIMENTAL

Efforts have been made to ensure accuracy with respect to numbers used, but some experimental error and deviation should, of course, be allowed for. Example 1

To prepare a two-layer adipose tissue biocomposite, a 1 -to-2 ratio by volume of adipose tissue fragments having tissue plasminogen activator and viable stem cells was mixed with plasma having added thrombin. 20 mL of adipose tissue from lipoaspirate as mixed in a syringe with 40 mL of plasma three times using two 60 mL syringes connected by a stopcock and then adding 1 mL of thrombin to achieve a 1 -5 unit/mL thrombin containing plasma. Mixing was continued and cast into a mold and left for polymerization of fibrin and crosslinking with Factor XIII for more than 5 minutes.

Polymerization was observed after 30 seconds after casting the composition into the mold.

Example 2

A two-layer adipose tissue biocomposite was prepared by mixing a 1 -to-2 ratio by volume of adipose tissue fragments having tissue plasminogen activator and viable stem cells was mixed with plasma having added thrombin. 20 mL of adipose tissue from lipoaspirate as mixed in a syringe with 40 mL of plasma three times using two 60 mL syringes connected by a stopcock and then adding 8 mL of thrombin to the plasma. Mixing was continued and cast into a mold and left for polymerization of fibrin and crosslinking with Factor XIII for more than 5 minutes. FIG. 7 A illustrates the molded 7- inch long and 1 .5-inch wide adipose tissue biocomposite and shows tensile strength significant enough to support its own weight. To further remove any excess plasma from the prepared adipose tissue biocomposite, the molded 7-inch long and 1 .5-inch wide adipose tissue biocomposite was blotted using sterile gauze (see FIG. 7B). The adipose tissue biocomposites can be cut into two or more sheets. FIG. 7C depicts cutting the 7- inch long and 1 .5-inch wide adipose tissue biocomposite into two separate sheets. FIG. 7D depicts the two separate sheets of adipose tissue biocomposite in culture on a petri dish. The adipose tissue biocomposite demonstrates strong mechanical properties sufficient for manual handling. FIG. 7E illustrates an example of the adipose tissue biocomposite being rolled and twisted. Example 3

A molded adipose tissue biocomposite was prepared with recombinant thrombin with fibrinogen and an adipose tissue component. In this experiment, a 5000 IU vial of Recothrom (Zymogenetics, Lot ZAH1301 A) was used as the source of thrombin. The recombinant thrombin was reconstituted with 5 mL of 10% CaCI₂ to make a 1000 U/mL thrombin composition. 200 μΐ of the diluted thrombin was added to 9.8 mL PBS to make a 20 U/mL thrombin solution. 2 mL of the adipose tissue component with less than 20% water content per 5 mL of plasma was mixed well with the 20 U/mL thrombin solution. The thrombin solution, adipose tissue component and plasma was mixed and discharged into a mold where gelling of the composite took place within 30 seconds. The biocomposite gel was removed from the mold and stored at room temperature. The biocomposite gel was observed to undergo fibrinolysis within 8 hour to 24 hours due to thrombin stimulated production of tissue plasminogen activator (TPA) from endothelial cells in the adipose tissue and the TPA activating the plasminogen in the plasma to form plasmin. In this example, the gel fragments were floating as free adipose tissue fragments instead as a result of fibrinolysis in the biocomposite gel.

Example 4

A sprayable adipose tissue biocomposite composition was prepared with recombinant thrombin with fibrinogen and an adipose tissue component. In this experiment, a 5000 IU vial of Recothrom (Zymogenetics, Lot ZAH1301 A) was used as the source of thrombin. The recombinant thrombin was reconstituted with 5 mL of 10% CaCI₂ to make a 1000 U/mL thrombin composition. 5 mL of the diluted thrombin was added to 5 mL PBS to make a 500 U/mL thrombin solution. 2 mL of the adipose tissue component with less than 20% water content per 5 mL of plasma was mixed well with the 500 U/mL thrombin solution. 1 mL of the 500 U/mL thrombin solution was added to 4 mL of plasma as a source of fibrinogen. Concurrently thrombin was combined with and adipose tissue component and plasma and then discharged with compressed gas prior to polymerization of the fibrin so as to create a spray of adipose tissue, fibrinogen and thrombin mixture. The spray was used to achieve coating of surface with adipose tissue being held in place by fibrin resulting from the polymerization reaction. Example 5

A molded adipose tissue biocomposite was prepared with recombinant thrombin with fibrinogen and an adipose tissue component. In this experiment, a 5000 IU vial of Recothrom (Zymogenetics, Lot ZAH1301 A) was used as the source of thrombin. The recombinant thrombin was reconstituted with 5 mL of 10% CaCI₂ to make a 1000 U/mL thrombin composition. 200 μΙ_ of the diluted thrombin was added to 9.8 mL PBS to make a 20 U/mL thrombin solution. 2 mL of the adipose tissue component with less than 20% water content per 5 mL of plasma was mixed well with the 20 U/mL thrombin solution. 1 mL of the 20 U/mL thrombin per 5 mL of plasma (source of fibrinogen) were mixed together. The thrombin solution, adipose tissue component and plasma was mixed and discharged into a mold where gelling of the composite took place within 30 seconds. The gel was removed from the mold and plasma and water removed from the gel by gently blotting with sterile gauze and stored at room temperature. The adipose tissue biocomposite did not undergo fibrinolysis with 24 hours. The removal of plasma proteins and excess water provided stronger tensile strength (e.g., for manipulation of the biocomposite by the surgeon). The adipose tissue in fibrin could be placed into culture and viable adipocytes observed for more than a month. Viable mesenchymal stem cells were observed to be continually released from the adipose tissue component during cell culture of the adipose tissue biocomposite.

Example 6

A molded adipose tissue biocomposite in a syringe was prepared with recombinant thrombin with fibrinogen and an adipose tissue component. In this experiment, a 5000 IU vial of Recothrom (Zymogenetics, Lot ZAH1301 A) was used as the source of thrombin. The recombinant thrombin was reconstituted with 5 mL of 10% CaCI₂ to make a 1000 U/mL thrombin composition. 200 μΐ of the diluted thrombin was added to 9.8 mL PBS to make a 20 U/mL thrombin solution. 2 mL of the adipose tissue component with less than 20% water content per 5 mL of plasma was mixed well in a 6- mL syringe with the 20 U/mL thrombin solution. 1 mL of the 20 U/mL thrombin solution per 5 mL of plasma (source of fibrinogen) was added to the 60 mL syringe. The thrombin, adipose tissue component and plasma was mixed thoroughly in the 60 mL syringe and then left undisturbed for 5 minutes during which time the gelling of the adipose tissue biocomposite composite took place. The adipose tissue biocomposite was applied from the syringe through a needle by depressing the plunger of the 60 mL syringe.

Notwithstanding the appended clauses, the disclosure set forth herein is also defined by the following clauses:

1 . An adipose tissue biocomposite comprising an adipose tissue component in a fibrin network, wherein the fibrin network is configured to break down after a period of time to release the adipose tissue component from the biocomposite.

2. The adipose tissue biocomposite according to clause 1 , wherein the adipose tissue component comprises adipose tissue, viable stem cells and tissue plasminogen activator.

3. The adipose tissue biocomposite according to clause 1 , wherein the fibrin network comprises thrombin and fibrin.

4. The adipose tissue biocomposite according to clause 3, wherein ratio of fibrin to adipose tissue in the adipose tissue biocomposite is from 1 :1 to 10:1 .

5. The adipose tissue biocomposite according to clause 4, wherein the ratio of fibrin to adipose tissue in the adipose tissue biocomposite is from 1 :1 to 5:1 .

6. The adipose tissue biocomposite according to clause 4, wherein the ratio of fibrin to adipose tissue in the adipose tissue biocomposite is 2:1

7. The adipose tissue biocomposite according to clause 3, wherein the fibrin network further comprises plasminogen.

9. The adipose tissue biocomposite according to any of clauses 1 -8, wherein the adipose tissue biocomposite further comprises a second layer comprising thrombin and fibrin.

10. The adipose tissue biocomposite according to clause 9, wherein the first layer comprises the adipose tissue component and the second layer comprises the fibrin network.

1 1 . The adipose tissue biocomposite according to any of clauses 9-10, wherein the first layer has a thickness of from 1 mm to 50 mm.

12. The adipose tissue biocomposite according to clause 1 1 , wherein the first layer has a thickness of from 5 mm to 25 mm.

13. The adipose tissue biocomposite according to any of clauses 9-12, wherein the second layer has a thickness of from 1 mm to 50 mm. 14. The adipose tissue biocomposite according to clause 1 1 , wherein the second layer has a thickness of from 5 mm to 25 mm.

15. The adipose tissue biocomposite according to any of clauses 1 -14, wherein the fibrin network has a crosslink density of from 1 x10^"15 moles/cm³ to 1 x10 ³ moles/cm³. 16. The adipose tissue biocomposite according to clause 15, wherein the fibrin network has a crosslink density of from 1 x10 ^s moles/cm³ to 1 x10 ³ moles/cm³.

17. The adipose tissue biocomposite according to any of clauses 1 -16 wherein the adipose tissue biocomposite has a shape selected from the group consisting of circle, oval, half-circle, crescent-shaped, star-shaped, square, triangle, rhomboid, pentagon, hexagon, heptagon, octagon or rectangle.

18. The adipose tissue biocomposite according to any of clauses 1 -16 wherein the adipose tissue biocomposite has a shape selected from the group consisting of a cube, cone, half sphere, star, triangular prism, rectangular prism, hexagonal prism, a polyhedron, a cylinder and a tube.

19. The adipose tissue biocomposite according to any of clauses 1 -18, wherein the adipose tissue biocomposite has a surface area of from 0.1 to 100 cm².

20. The adipose tissue biocomposite according to clause 19, wherein the adipose tissue biocomposite has a surface area of from 5 to 50 cm².

21 . The adipose tissue biocomposite according to any of clauses 1 -18, wherein the adipose tissue biocomposite has a volume of 0.1 to 100 cm³.

22. The adipose tissue biocomposite according to clause 19, wherein the adipose tissue biocomposite has a volume of from 5 to 50 cm³.

23. The adipose tissue biocomposite according to any of clauses 1 -22, further comprising a bioactive agent.

24. The adipose tissue biocomposite according to clause 23, wherein the bioactive agent is selected from the group consisting of interferon, interleukin, erythropoietin, granulocyte-colony stimulating factor (GCSF), stem cell factor (SCI:), leptin (OB protein), interferon (alpha, beta, gamma), antibiotics such as vancomycin, gentamicin

ciprofloxacin, amoxycillin, lactobacillus, cefotaxime, levofloxacin, cefipime,

mebendazole, ampicillin, lactobacillus, cloxacillin, norfloxacin, tinidazole, cefpodoxime, proxctil, azithromycin, gatifloxacin, roxithromycin, cephalosporin, anti-thrombogenics, aspirin, ticlopidine, sulfinpyrazone, heparin, warfarin, growth factors, differentiation factors, hepatocyte stimulating factor, plasmacytoma growth factor, glial derived neurotrophic factor (GDNF), neurotrophic factor 3 (NT3), fibroblast growth factor (FGF), transforming growth factor (TGF), platelet transforming growth factor, milk growth factor, endothelial growth factors, endothelial cell-derived growth factors (ECDGF), alpha- endothelial growth factors, beta-endothelial growth factor, neurotrophic growth factor, nerve growth factor (NGF), vascular endothelial growth factor (VEGF), 4-1 BB receptor (4- IBBR), TRAIL (TNF-related apoptosis inducing ligand), artemin (GFRalpha3-RET ligand), BCA-I (B cell-attracting chemokinel), B lymphocyte chemoattractant (BLC), B cell maturation protein (BCMA), brain-derived neurotrophic factor (BDNF), bone growth factor such as osteoprotegerin (OPG), bone-derived growth factor, thrombopoietin, megakaryocyte derived growth factor (MDGF), keratinocyte growth factor (KGF), platelet-derived growth factor (PDGF), ciliary neurotrophic factor (CNTF), neurotrophin 4 (NT4), granulocyte colony-stimulating factor (GCSF), macrophage colony-stimulating factor (mCSF), bone morphogenetic protein 2 (BMP2), BRAK, C-IO, Cardiotrophin 1 (CTI), CCR8, anti- inflammatory: paracetamol, salsalate, diflunisal, mefenamic acid, diclofenac, piroxicam, ketoprofen, dipyrone, acetyl sal icy lie acid, anti-cancer drugs such as aliteretinoin, altertamine, anastrozole, azathioprine, bicalutarnide, busulfan, capecitabine, carboplatin, cisplatin, cyclophosphamide, cytarabine, doxorubicin, epirubicin, etoposide, exemestane, vincristine, vinorelbine, hormones, thyroid stimulating hormone (TSH), sex hormone binding globulin (SHBG), prolactin, luteotropic hormone (LTH), lactogenic hormone, parathyroid hormone (PTH), melanin concentrating hormone (MCH), luteinizing hormone (LHb), growth hormone (HGH), follicle stimulating hormone (FSHb), haloperidol, indomethacin, doxorubicin, epirubicin, amphotericin B, Taxol, cyclophosphamide, cisplatin, methotrexate, pyrene, amphotericin B, anti- dyskinesia agents, Alzheimer vaccine, antiparkinson agents, ions, edetic acid, nutrients, glucocorticoids, heparin, anticoagulation agents, antivirus agents, anti-HIV agents, polyamine, histamine and derivatives thereof, cystineamine and derivatives thereof, diphenhydramine and derivatives, orphenadrine and derivatives, muscarinic antagonist, phenoxybenzamine and derivatives thereof, protein A, streptavidin, amino acid, beta- galactosidase, methylene blue, protein kinases, beta-amyloid, lipopolysaccharides, eukaryotic initiation factor-4G, tumor necrosis factor (TNF), tumor necrosis factor-binding protein (TNF -bp), interleukin- 1 (to 18) receptor antagonist (IL-lra), granulocyte macrophage colony stimulating factor (GM-CSF), novel erythropoiesis stimulating protein (NESP), thrombopoietin, tissue plasminogen activator (TPA), urokinase, streptokinase, kallikrein, insulin, steroid, acetaminophen, analgesics, antitumor preparations, anti-cancer preparations, anti-proliferative preparations or pro-apoptotic preparations.

25. The adipose tissue biocomposite according to any of clauses 1 -24, wherein the fibrin network is configured to break down after a period of from 6 hours to 72 hours to release the adipose tissue component from the biocomposite.

26. The adipose tissue biocomposite according to any of clauses 1 -24, wherein the fibrin network is configured to break down after a period of from 8 hours to 48 hours to release the adipose tissue component from the biocomposite.

27. The adipose tissue biocomposite according to any of clauses 1 -24, wherein the fibrin network is configured to break down after a period of from 6 hours to 24 hours to release the adipose tissue component from the biocomposite.

28. A method for preparing an adipose tissue biocomposite comprising an adipose tissue component in a fibrin network, the method comprising contacting an adipose tissue component with thrombin and fibrinogen in a manner sufficient to produce the adipose tissue biocomposite, wherein the fibrin network is configured to break down after a period of time to release the adipose tissue component from the biocomposite.

29. The method according to clause 28, wherein the adipose tissue component is contacted with thrombin and fibrinogen by applying the adipose tissue component and thrombin to a body site of a subject.

30. The method according to clause 29, wherein the body site is a wound site.

31 . The method according to any of clauses 29-30 wherein the body site is the abdomen.

32. The method according to any of clauses 29-30, wherein the body site comprises an organ tissue selected from the group consisting of integumentary tissue, oral tissue, respiratory tissue, gastrointestinal tissue, cardiovascular tissue, endocrine tissue, genitourinary tissue, muscular tissue, nervous tissue and soft skeletal tissue.

33. The method according to clause 28, wherein the adipose tissue component is contacted with thrombin and fibrinogen by mixing the adipose tissue component with thrombin and fibrinogen in a three-dimensional mold.

34. The method according to clause 33, wherein the three-dimensional mold is a container having a mold shape selected from the group consisting of circle, oval, half- circle, crescent-shaped, star-shaped, square, triangle, rhomboid, pentagon, hexagon, heptagon, octagon or rectangle. 35. The method according to clause 33, wherein the three-dimensional mold comprises a petri dish, a syringe, tubing or a rectangular box.

36. The method according to clause 35, wherein the three-dimensional mold further comprises one or more ports.

37. The method according to clause 36, wherein the one or more ports is a luer-lock or luer taper.

38. The method according to clause any of clauses 28-37, wherein ratio by volume of fibrinogen and thrombin to the adipose tissue component in the adipose tissue biocomposite is from 1 :1 to 10:1 .

39. The method according to clause any of clauses 28-37, wherein ratio by volume of fibrinogen and thrombin to the adipose tissue component in the adipose tissue biocomposite is from 1 :1 to 5:1 .

40. The method according to clause any of clauses 28-37, wherein ratio by volume of fibrinogen and thrombin to the adipose tissue component in the adipose tissue biocomposite is from is 2:1 .

41 . The method according to clause 28, comprising preparing a layer of adipose tissue biocomposite comprising an adipose tissue component in a fibrin network, the method comprising contacting an adipose tissue component with thrombin and fibrinogen in a manner sufficient to produce a layer of the adipose tissue biocomposite, wherein the fibrin network is configured to dissolve after a period of time to release the adipose tissue component from the biocomposite.

42. The method according to clause 41 , wherein the method further comprises preparing a second layer comprising fibrin and thrombin on the adipose tissue biocomposite.

43. The method according to clause 42, wherein the method comprises preparing a first layer comprising the adipose tissue component and a second layer comprising the fibrin network.

44. The method according to clause 43, wherein the method comprises:

contacting the adipose tissue component with a composition comprising thrombin and fibrinogen; and

allowing the adipose tissue component to float to the top of the thrombin and fibrinogen composition. 45. The method according to clause 44, wherein the adipose tissue component is allowed to float to the top of the thrombin and fibrinogen composition for 5 minutes or more.

46. The method according to clause 44, wherein the adipose tissue component is allowed to float to the top of the thrombin and fibrinogen composition for 15 minutes or more.

47. The method according to clause 44, wherein the adipose tissue component is allowed to float to the top of the thrombin and fibrinogen composition for 30 minutes or more.

48. The method according to any of clauses 44-47, wherein the thrombin and fibrinogen composition is configured to polymerize into the fibrin network while the adipose tissue component floats to the top of the thrombin and fibrinogen composition.

49. The method according to clause 48, wherein 5% or less of the fibrin network is formed while the adipose tissue component floats to the top of the thrombin and fibrinogen composition.

50. The method according to clause 48, wherein 15% or less of the fibrin network is formed while the adipose tissue component floats to the top of the thrombin and fibrinogen composition.

51 . The method according to clause 48, wherein 25% or less of the fibrin network is formed while the adipose tissue component floats to the top of the thrombin and fibrinogen composition.

52. The method according to any of clauses 44-51 , further comprising removing unpolymerized thrombin and fibrinogen composition from the adipose tissue biocomposite.

53. The method according to clause 52, wherein removing comprises blotting with a sterile wipe.

54. The method according to clause 53, wherein the sterile wipe is gauze.

55. The method according to any of clauses 28-54, wherein the adipose tissue biocomposite has a surface area of from 5 to 50 cm².

56. The method according to any of clauses 28-54, wherein the adipose tissue biocomposite has a volume of 0.1 to 100 cm³.

57. The method according to any of clauses 28-56, further comprising performing lipoplasty on a subject to harvest lipoaspirate from the subject. 58. The method according to clause 57, further comprising harvesting the adipose tissue component from the lipoaspirate.

59. The method according to any of clauses 57-58, wherein lipoplasty is selected from suction assisted lipoplasty (SAL), ultra-sound assisted lipoplasty (USAL), power assisted lipoplasty (PAL), syringe assisted lipoplasty (SAL), laser assisted lipoplasty (LAL) and water jet assisted lipoplasty (WJAL).

60. The method according to any of clauses 28-58, wherein the source of thrombin is selected from autologous thrombin serum, autologous thrombin serum supplemented with ethanol, allogeneic thrombin serum, allogeneic thrombin serum supplemented with ethanol, bovine thrombin, recombinant thrombin, and human thrombin derived from pooled plasma.

61 . The method according to clause 60, wherein the thrombin source is autologous thrombin.

62. The method according to any of clauses 28-61 , wherein the source of fibrinogen is autologous whole blood anti-coagulated with a calcium-chelating agent, platelet rich plasma with its associated growth factors, autologous plasma, autologous platelet rich plasma, plasma and collagen mixture.

63. The method according to clause 62, wherein the fibrinogen source is autologous whole blood.

64. The method according to clause 62, wherein the fibrinogen source is autologous platelet rich plasma.

65. The method according to any of clauses 28-61 , wherein the source of plasminogen is autologous whole blood anti-coagulated with a calcium-chelating agent, platelet rich plasma with its associated growth factors, autologous plasma, autologous platelet rich plasma, plasma and collagen mixture.

66. The method according to clause 65, wherein the plasminogen source is autologous whole blood.

66. The method according to clause 65, wherein the plasminogen source is autologous platelet rich plasma.

67. A method comprising applying an adipose tissue biocomposite to a body site of a subject, wherein the adipose tissue biocomposite comprises an adipose tissue component in a fibrin network that is configured to break down at the body site after a period of time to release the adipose tissue component from the biocomposite.

68. The method according to clause 67, wherein the body site is a wound site. 69. The method according to clause 68, wherein the wound site is a wound from trauma to the body.

70. The method according to clause 68, wherein the wound site is from a surgical operation.

71 . The method according to any of clauses 67-70, wherein the body site is the abdomen.

72. The method according to any of clauses 67-70, wherein the body site comprises organ tissue selected from the group consisting of integumentary tissue, oral tissue, respiratory tissue, gastrointestinal tissue, cardiovascular tissue, endocrine tissue, genitourinary tissue, muscular tissue, nervous tissue and soft skeletal tissue.

73. The method according to any of clauses 67-72, wherein applying the adipose tissue biocomposite prevents tissue adhesion at the body site.

74. The method according to any of clauses 67-72, wherein applying the adipose tissue biocomposite is for treating or preventing tissue scarring at the body site.

75. The method according to any of clauses 67-72, wherein applying the adipose tissue biocomposite promotes hemostasis.

76. The method according to any of clauses 67-72, wherein applying the adipose tissue biocomposite is for treating a skin excision at the body site.

78. The method according to any of clauses 67-72, wherein applying the adipose tissue biocomposite reduces the time for wound healing at the body site.

79. The method according to any of clauses 67-72, wherein applying the adipose tissue biocomposite is for treating a burn at the body site.

80 The method according to any of clauses 67-79, wherein the adipose tissue component comprises adipose tissue, viable stem cells and tissue plasminogen activator.

81 . The method according to any of clauses 67-79, wherein the fibrin network comprises thrombin and fibrin.

82. The method according to any of clauses 67-79, wherein ratio of fibrin to adipose tissue in the adipose tissue biocomposite is from 1 :1 to 10:1 .

83. The method according to any of clauses 67-79, wherein the ratio of fibrin to adipose tissue in the adipose tissue biocomposite is from 1 :1 to 5:1 .

84. The method according to any of clauses 67-79, wherein the ratio of fibrin to adipose tissue in the adipose tissue biocomposite is 2:1 85. The method according to any of clauses 67-79, wherein the fibrin network further comprises plasminogen.

86. The method according to any of clauses 67-79, wherein the adipose tissue biocomposite further comprises a second layer comprising thrombin and fibrin.

87. The method according to clause 86, wherein the first layer comprises the adipose tissue component and the second layer comprises the fibrin network.

88. The method according to any of clauses 86-87, wherein the first layer has a thickness of from 1 mm to 50 mm.

89. The method according to any of clauses 88, wherein the first layer has a thickness of from 5 mm to 25 mm.

90. The method according to any of clauses 87-89, wherein the second layer has a thickness of from 1 mm to 50 mm.

91 . The method according to clause 90, wherein the second layer has a thickness of from 5 mm to 25 mm.

92. The method according to any of clauses 67-91 , wherein the fibrin network has a crosslink density of from 1 x10^"15 moles/cm³ to 1 x10 ³ moles/cm³.

93. The method according to clause 92, wherein the fibrin network has a crosslink density of from 1 x10 ^s moles/cm³ to 1 x10 ³ moles/cm³.

94. The method according to any of clauses 67-93, wherein the adipose tissue biocomposite has a shape selected from the group consisting of circle, oval, half-circle, crescent-shaped, star-shaped, square, triangle, rhomboid, pentagon, hexagon, heptagon, octagon or rectangle.

95. The method according to any of clauses 67-93, wherein the adipose tissue biocomposite has a shape selected from the group consisting of a cube, cone, half sphere, star, triangular prism, rectangular prism, hexagonal prism, a polyhedron, a cylinder and a tube.

96. The method according to any of clauses 67-93, wherein the adipose tissue biocomposite has a surface area of from 0.1 to 100 cm².

97. The method according to clause 96, wherein the adipose tissue biocomposite has a surface area of from 5 to 50 cm².

98. The method according to any of clauses 67-97, wherein the adipose tissue biocomposite has a volume of 0.1 to 100 cm³.

99. The method according to clause 98, wherein the adipose tissue biocomposite has a volume of from 5 to 50 cm³. 100. The method according to any of clauses 67-99, further delivering a bioactive agent to the body site from the adipose tissue biocomposite.

101 . The method according to clause 100, wherein the bioactive agent is selected from the group consisting of interferon, interleukin, erythropoietin, granulocyte-colony stimulating factor (GCSF), stem cell factor (SCI:), leptin (OB protein), interferon (alpha, beta, gamma), antibiotics such as vancomycin, gentamicin ciprofloxacin, amoxycillin, lactobacillus, cefotaxime, levofloxacin, cefipime, mebendazole, ampicillin, lactobacillus, cloxacillin, norfloxacin, tinidazole, cefpodoxime, proxctil, azithromycin, gatifloxacin, roxithromycin, cephalosporin, anti-thrombogenics, aspirin, ticlopidine, sulfinpyrazone, heparin, warfarin, growth factors, differentiation factors, hepatocyte stimulating factor, plasmacytoma growth factor, glial derived neurotrophic factor (GDNF), neurotrophic factor 3 (NT3), fibroblast growth factor (FGF), transforming growth factor (TGF), platelet transforming growth factor, milk growth factor, endothelial growth factors, endothelial cell-derived growth factors (ECDGF), alpha-endothelial growth factors, beta-endothelial growth factor, neurotrophic growth factor, nerve growth factor (NGF), vascular endothelial growth factor (VEGF), 4-1 BB receptor (4- IBBR), TRAIL (TNF-related apoptosis inducing ligand), artemin (GFRalpha3-RET ligand), BCA-I (B cell-attracting chemokinel), B lymphocyte chemoattractant (BLC), B cell maturation protein (BCMA), brain-derived neurotrophic factor (BDNF), bone growth factor such as osteoprotegerin (OPG), bone-derived growth factor, thrombopoietin, megakaryocyte derived growth factor (MDGF), keratinocyte growth factor (KGF), platelet-derived growth factor (PDGF), ciliary neurotrophic factor (CNTF), neurotrophin 4 (NT4), granulocyte colony-stimulating factor (GCSF), macrophage colony-stimulating factor (mCSF), bone morphogenetic protein 2 (BMP2), BRAK, C-IO, Cardiotrophin 1 (CTI), CCR8, anti- inflammatory:

paracetamol, salsalate, diflunisal, mefenamic acid, diclofenac, piroxicam, ketoprofen, dipyrone, acetylsalicylic acid, anti-cancer drugs such as aliteretinoin, altertamine, anastrozole, azathioprine, bicalutarnide, busulfan, capecitabine, carboplatin, cisplatin, cyclophosphamide, cytarabine, doxorubicin, epirubicin, etoposide, exemestane, vincristine, vinorelbine, hormones, thyroid stimulating hormone (TSH), sex hormone binding globulin (SHBG), prolactin, luteotropic hormone (LTH), lactogenic hormone, parathyroid hormone (PTH), melanin concentrating hormone (MCH), luteinizing hormone (LHb), growth hormone (HGH), follicle stimulating hormone (FSHb), haloperidol, indomethacin, doxorubicin, epirubicin, amphotericin B, Taxol, cyclophosphamide, cisplatin, methotrexate, pyrene, amphotericin B, anti- dyskinesia agents, Alzheimer vaccine, antiparkinson agents, ions, edetic acid, nutrients, glucocorticoids, heparin, anticoagulation agents, antivirus agents, anti-HIV agents, polyamine, histamine and derivatives thereof, cystineamine and derivatives thereof, diphenhydramine and derivatives, orphenadrine and derivatives, muscarinic antagonist, phenoxybenzamine and derivatives thereof, protein A, streptavidin, amino acid, beta-galactosidase, methylene blue, protein kinases, beta-amyloid, lipopolysaccharides, eukaryotic initiation factor-4G, tumor necrosis factor (TNF), tumor necrosis factor-binding protein (TNF -bp), interleukin- 1 (to 18) receptor antagonist (IL-lra), granulocyte macrophage colony stimulating factor (GM-CSF), novel erythropoiesis stimulating protein (NESP), thrombopoietin, tissue plasminogen activator (TPA), urokinase, streptokinase, kallikrein, insulin, steroid, acetaminophen, analgesics, antitumor preparations, anti-cancer preparations, anti-proliferative preparations or pro-apoptotic preparations.

102. The method according to any of clauses 67-101 , wherein the fibrin network is configured to break down at the body site after a period of from 6 hours to 72 hours to release the adipose tissue component from the biocomposite.

103. The method according to any of clauses 67-101 , wherein the fibrin network is configured to break down at the body site after a period of from 8 hours to 48 hours to release the adipose tissue component from the biocomposite.

104. The method according to any of clauses 67-101 , wherein the fibrin network is configured to break down at the body site after a period of from 6 hours to 24 hours to release the adipose tissue component from the biocomposite.

105. A kit comprising:

a container;

a syringe configured to be coupled into fluid communication with the container; a lipoaspirator for harvesting an adipose tissue component; and

a vessel configured for preparing platelet rich plasma.

106. The kit according to clause 105, wherein the container is a three-dimensional mold having a mold shape selected from the group consisting of circle, oval, half-circle, crescent-shaped, star-shaped, square, triangle, rhomboid, pentagon, hexagon, heptagon, octagon or rectangle.

107. The kit according to clause 106, wherein the three-dimensional mold comprises a petri dish, a syringe, tubing or a rectangular box.

108. The kit according to any of clauses 105-107, wherein the three-dimensional mold further comprises one or more fluid ports. 109. The kit according to clause 108, wherein the one or more ports is a luer-lock or luer taper.

1 10. The kit according to any of clauses 108-109, wherein the three-dimensional mold comprises 2 or more fluid ports.

1 1 1 . The kit according to any of clauses 108-1 10, wherein each fluid port is connected to a conduit.

1 12. The kit according to clause 1 1 1 , wherein the conduit further comprises a stopcock valve.

1 13. The kit according to any of clauses 105-1 12, further comprising a syringe.

1 14. The kit according to any of clauses 105-1 13, further comprises a source of thrombin.

1 15. The kit according to clause 1 14, wherein the source of thrombin is a thrombin vessel configured to prepare thrombin from a fibrin gel.

1 16. The kit according to clause 1 15, wherein the fibrin gel comprises calcium.

1 17. A method for inhibiting adhesion of apposing internal body tissue layers by means of a tissue graft, said method comprising the steps of:

a) harvesting a plurality of tissue fragments from a donor;

b) contacting said plurality of tissue fragments with a source of thrombin, fibrinogen ; and

c) positioning said thrombin, fibrinogen and plasminogen contacted tissue fragments in, on or in proximity to a body site at risk of forming adhesions wherein fibrinolysis occurs proximate to said tissue fragments.

1 18. The method of clause 1 17, wherein said plurality of tissue fragments comprises autologous adipose tissue fragments contained in lipoaspirate.

1 19. The method of clause 1 17, wherein said tissue fragments contact a fibrinogen and thrombin sufficient to form a fibrin hydrogel containing said plasminogen.

120. The method of clause 1 17, wherein said inhibition of adhesion formation includes at least one of the effects of: providing a passive physical barrier between the tissue layers and actively promoting fibrinolysis by conversion of plasminogen to plasmin by adipose tissue derived tissue activation factor.

121 . The method of clause 1 17, wherein said adipose tissue fragments are derived using lipoplasty methods selected from a group consisting of: suction assisted lipoplasty (SAL), ultra-sound assisted lipoplasty (USAL), power assisted lipoplasty (PAL), syringe assisted lipoplasty (SAL), laser assisted lipoplasty (LAL) and water jet assisted lipoplasty (WJAL).

122. The method of clause 1 17, wherein said thrombin source is selected from a group consisting of: autologous thrombin serum, autologous thrombin serum

supplemented with ethanol, allogeneic thrombin serum, allogeneic serum supplemented with ethanol, bovine thrombin, recombinant thrombin, and human thrombin derived from pooled plasma and any protein that enzymatically converts fibrinogen to fibrin and wherein fibrinogen and plasminogen are derived from whole blood or a whole blood fraction including single donor plasma or pooled plasma.

123. The method of clause 1 17, wherein said concentration of thrombin source is sufficient to cause the adipose tissue fragments to increase its baseline secretion of tissue plasminogen activator by at least two fold.

124. The method of clause 1 17, wherein said adipose tissue fragments constitute 5% to 95% of the total volume of said adipose tissue biocomposite graft.

125. The method of clause 1 17, wherein said body has undergone a surgical procedure selected from a group consisting of: cosmetic, therapeutic, childbirth, exploratory, and diagnostic procedures.

126. A method for inhibiting adhesion of apposing internal body tissue layers by means of a tissue graft, said method comprising the steps of:

a) harvesting said plurality of tissue fragments from a body;

b) contacting said plurality of tissue fragments with fibrin and

plasminogen,

c) and positioning said fibrin and plasminogen contacted tissue fragments in, on or in proximity to a body site at risk of forming adhesions.

127. The method of clause 126 wherein applying said tissue fragments are derived from adipose tissue fragments.

128. The method of clause 126 wherein said tissue fragments are contacted fibrin as a result of mixing a fibrinogen proteolytic enzyme source and a fibrinogen source.

129. The method of clause 126 wherein said fibrin, plasminogen and tissue fragments are contacted in a mold to confer a desired geometry to the tissue graft prior to applying to said body site.

130. The method of clause 126, wherein said three dimensional mold cavity is of a size, shape and dimension to control the structure of said adipose tissue biocomposite graft. 131 . The method of clause 126, wherein said inhibition of adhesion formation of said tissue graft includes at least one of the effects of: fibrinolysis, physical barrier, extracellular matrix remodeling or cell seeding of site with tissue derived cells.

132. The method of clause 126, wherein said tissue fragments are derived using lipoplasty methods selected from a group consisting of: suction assisted lipoplasty

(SAL), ultra-sound assisted lipoplasty (USAL), power assisted lipoplasty (PAL), syringe assisted lipoplasty (SAL), laser assisted lipoplasty (LAL) and water jet assisted lipoplasty (WJAL).

133. The method of clause 126, wherein said thrombin source is selected from a group consisting of: autologous thrombin serum, autologous thrombin serum

supplemented with ethanol, allogeneic thrombin serum, allogeneic thrombin serum supplemented with ethanol, bovine thrombin, recombinant thrombin, and human thrombin derived from pooled plasma.

134. The method of clause 126, wherein said fibrinogen and plasminogen source is selected from a group consisting of: autologous whole blood anti-coagulated with a calcium-chelating agent, plasma anti-coagulated with a calcium-chelating agent, platelet rich plasma with its associated growth factors, autologous plasma, autologous platelet rich plasma, plasma and collagen mixture, purified allogeneic fibrinogen or combinations thereof.

135. The method of claim 126, further comprising controlling the structure of said adipose tissue biocomposite graft by controlling the relative percentage of the graft volume derived from adipose tissue fragments and controlling concentrations of said fibrinogen source and said thrombin source.

136. The method of clause 126, wherein said concentration of thrombin source is 0.1 to 5000 units/gram of said adipose tissue biocomposite graft.

137. The method of clause 126 wherein said concentration of fibrinogen source is 0.1 to 100 mg/gram of said adipose tissue biocomposite graft.

138. The method of clause 126, wherein said adipose tissue fragments constitute 1 % to 99% of the total volume of said adipose tissue biocomposite graft.

139. The method of clause 126, wherein said medical application is selected from a group consisting of: cosmetic, therapeutic and surgical procedures.

140. A method for inhibiting adhesion of apposing internal human body tissue layers by means of an adipose tissue graft with a wound-healing promoter, the method comprising the steps of: a) performing lipoplasty to derive a plurality of adipose tissue fragments from a donor;

b) harvesting said plurality of adipose tissue fragments from said donor, said adipose tissue fragments containing at least one viable stem cell;

c) contacting said plurality of adipose tissue fragments with appropriate concentrations of a thrombin source, a fibrinogen source, a plasminogen source and said wound healing promoter to achieve an appropriate gelling reaction and conversion of at least some fraction of said plasminogen to plasmin; and

d) applying the mixture of said adipose tissue fragments, said thrombin source, said fibrinogen source, said plasminogen source and said wound-healing promoter to a body site at risk of adhesion formation in the donor so as to inhibit the formation of an adhesion.

141 . The method of clause 140, wherein said wound-healing promoter is selected from a group consisting of: nutrients, vitamins, electrolytes, protease inhibitors, lipophilic antibiotics, hydrophilic antibiotics, germicides, fibrinolytic agents, corticosteroids, autologous cells, cytokines, hormones, anti-inflammatory drugs and combinations thereof.

142. The method of any of clauses 140-141 , wherein step (d) further comprises: applying said mixture of said adipose tissue fragments, said thrombin source, said wound healing promoter and said fibrinogen source to the wound site in the form of: a liquid biocomposite, a molded gel biocomposite and gel biocomposite fragments.

143. The method of clause 140, wherein said liquid biocomposite is prepared by mixing said adipose tissue fragments, said thrombin source and said fibrinogen source in liquid form to conform to said wound site in donor's body.

144. The method of clause 140, wherein preparation of said molded gel biocomposite comprises:

a) injecting the mixture of said adipose tissue fragments, said thrombin source and said fibrinogen source into a three dimensional mold cavity to achieve the gelling reaction and to confer a three dimensional shape to said adipose tissue biocomposite graft;

b) removing said adipose tissue biocomposite graft from said three dimensional mold cavity; and c) applying said adipose tissue biocomposite graft to a body site at risk of developing an adhesion of the donor so as to inhibit the formation of an adhesion.

145. The method of clause 144 wherein said three dimensional mold cavity is of a size, shape and dimension to control the structure of said adipose tissue biocomposite graft.

146. The method of clause 140, wherein said promotion of wound healing includes at least one of the effects of: promoting hemostasis, reducing time for wound closure, reducing post-surgical wound complications including adhesion prevention, and reducing scarring.

147. The method of clause 140, wherein said adipose tissue fragments are derived using lipoplasty methods selected from a group consisting of: suction assisted lipoplasty (SAL), ultra-sound assisted lipoplasty (USAL), power assisted lipoplasty (PAL), syringe assisted lipoplasty (SAL), laser assisted lipoplasty (LAL) and water jet assisted lipoplasty (WJAL).

148. The method of clause 140 wherein said thrombin source is selected from a group consisting of: autologous thrombin serum, autologous thrombin serum supplemented with ethanol, allogeneic thrombin serum, allogeneic serum supplemented with ethanol, bovine thrombin, recombinant thrombin, and human thrombin derived from pooled plasma.

150. The method of clause 140, wherein said fibrinogen and plasminogen source is selected from a group consisting of: autologous whole blood anti-coagulated with a calcium-chelating agent, platelet rich plasma with its associated growth factors, autologous plasma, autologous platelet rich plasma, plasma and collagen mixture, purified allogeneic fibrinogen and other naturally occurring adhesive glycoproteins to promote adhesion to collagen.

151 . The method of clause 140, further comprising controlling the structure of said biocomposite graft by controlling the relative percentage of the graft volume derived from adipose tissue fragments and controlling concentrations of said fibrinogen source and said thrombin source.

152. The method of clause 140, wherein said adipose tissue fragments constitute 5% to 95% of the total volume of said adipose tissue biocomposite graft.

153. The method of clause 140, wherein said medical application is selected from a group consisting of: cosmetic, therapeutic and surgical procedures. 154. A method for inhibiting adhesion of apposing internal body tissue layers by means of an adipose tissue graft, said method comprising the steps of:

a) performing lipoplasty to derive a plurality of adipose tissue fragments from a donor;

b) harvesting said plurality of adipose tissue fragments from said donor, said adipose tissue fragments containing;

c) contacting said plurality of adipose tissue fragments with appropriate concentrations of a thrombin source, a fibrinogen source and plasminogen source; and d) injecting the mixture of said adipose tissue fragments, said thrombin source and said fibrinogen source in the form of gel fragments to a site at risk of adhesion formation in the body of the donor so as to inhibit adhesion formation.

155. The method of clause 154, wherein said plurality of adipose tissue fragments comprises autologous adipose tissue fragments contained in lipoaspirate.

156. The method of clause 154, wherein the mixture of said plurality of adipose tissue fragments, said thrombin source and said fibrinogen source takes the structure of the application space and said space serves as a mold for the gelling reaction to occur and thereby creates a physical barrier between two opposing body sites.

157. The method of clause 154, wherein said promotion of wound healing includes at least one of the effects of: promoting hemostasis, reducing time for wound closure, reducing post-surgical wound complications, and reducing scarring.

158. The method of clause 154, wherein said thrombin source is selected from a group of consisting of: autologous thrombin serum, autologous thrombin serum supplemented with ethanol, allogeneic thrombin serum, allogeneic serum supplemented with ethanol, bovine thrombin, recombinant thrombin, and human thrombin derived from pooled plasma.

159. The method of clause 154, wherein said fibrinogen and plasminogen source is selected from a group consisting of: autologous whole blood anti-coagulated with a calcium-chelating agent, platelet rich plasma with its associated growth factors, autologous plasma, autologous platelet rich plasma, plasma and collagen mixture, purified allogeneic fibrinogen and other naturally occurring adhesive glycoproteins to promote adhesion to collagen.

160. The method of clause 154, wherein said adipose tissue fragments constitute 10% to 90% of the total volume of said adipose tissue biocomposite graft. 161 . A method for inhibiting adhesion of apposing internal human body tissue layers after a surgical procedure by means of positioning an adipose tissue graft between said opposing tissue layers.

162. A method for inhibiting adhesion of apposing internal human body tissue layers after a surgical procedure by means of positioning an adipose tissue graft between said opposing tissue layers wherein said adipose tissue graft inhibits adhesion formation by physically separating opposing tissue surfaces at risk of forming an adhesion site, by actively promoting fibrinolysis at the site of its placement and by a combination thereof.

163. The method of clause 162, wherein said adipose graft inhibits adhesion formation by actively promoting fibrinolysis by secreting tissue factors that convert plasminogen into plasmin.

164. A method for inhibiting adhesion of apposing internal human body tissue layers after a surgical procedure by means of positioning an activated adipose tissue graft between said opposing tissue layers wherein said cells contained in the adipose graft contacted with thrombin release tissue plasminogen activator.

165. The method of clause 164, wherein the concentration of fibrin in the scaffold is controlled by varying the amount of fibrinogen added prior to contacting with a thrombin source.

166. A method for treating a wound, injury or physical defect of a living body by means of a tissue graft, said method comprising the steps of:

a) harvesting a plurality of tissue fragments from a donor;

b) contacting said plurality of tissue fragments with thrombin,

fibrinogen and plasminogen,

c) said thrombin, fibrinogen and plasminogen contacted tissue fragments being positioned to be in, on or in proximity to a body site with a wound, injury or physical defect.

167. The method of 166, wherein the tissue fragments are derived from adipose tissue.

168. The method of clause 166, wherein the thrombin is derived from a human, animal or cell culture.

169. The method of clause 166, wherein the thrombin is defined as a protein capable of forming fibrin from fibrinogen.

170. A method for treating a wound, injury or physical defect of a living body by means of a tissue graft, said method comprising the steps of: a) harvesting a plurality of tissue fragments from a donor;

b) and contacting said plurality of tissue fragments with fibrin and plasminogen, wherein said fibrin contacted tissue fragments are positioned to be in, on or in proximity to a body site with a wound, injury or physical defect.

171 . The method of clause 170, wherein the tissue fragments are sourced from adipose tissue.

172. The method of clause 170, wherein the fibrin is sourced from a human, animal or cell culture.

173. The method of clause 170, wherein plasminogen is sourced from a human, animal or cell culture.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.

Claims

What is claimed is:

2. The adipose tissue biocomposite according to claim 1 , wherein the adipose tissue component comprises adipose tissue, viable stem cells and tissue plasminogen activator.

3. The adipose tissue biocomposite according to claim 1 , wherein the fibrin network comprises thrombin and fibrin.

4. The adipose tissue biocomposite according to claim 3, wherein the fibrin network further comprises plasminogen.

5. The adipose tissue biocomposite according to claim 1 , wherein the adipose tissue biocomposite further comprises a second layer comprising thrombin and fibrin.

6. A method for preparing an adipose tissue biocomposite comprising an adipose tissue component in a fibrin network, the method comprising contacting an adipose tissue component with thrombin and fibrinogen in a manner sufficient to produce the adipose tissue biocomposite,

wherein the fibrin network is configured to break down after a period of time to release the adipose tissue component from the biocomposite.

7. The method according to claim 6, wherein the adipose tissue component is contacted with thrombin and fibrinogen by one of:

applying the adipose tissue component and thrombin to a body site of a subject; or

mixing the adipose tissue component with thrombin and fibrinogen in a three- dimensional mold.

8. The method according to claim 6, further comprising forming a second layer comprising fibrin and thrombin on the adipose tissue biocomposite.

9. The method according to claim 6, further comprising harvesting the adipose tissue component from lipoaspirate from a subject.

10. The method according to claim 1 , wherein the adipose tissue component comprises adipose tissue, viable stem cells and tissue plasminogen activator.

1 1 . A method comprising applying an adipose tissue biocomposite to a body site of a subject, wherein the adipose tissue biocomposite comprises an adipose tissue component in a fibrin network that is configured to break down at the body site after a period of time to release the adipose tissue component from the biocomposite.

12. The method according to claim 1 1 , wherein the body site is a wound site.

13. The method according to claim 1 1 , wherein the adipose tissue biocomposite further comprises a second layer comprising thrombin and fibrin.

14. A kit comprising:

a container;

a vessel configured for preparing platelet rich plasma.

15. The kit according to claim 14, further comprising a source of thrombin.