AU2013202570A1

AU2013202570A1 - Tissue scaffold

Info

Publication number: AU2013202570A1
Application number: AU2013202570A
Authority: AU
Inventors: Christopher James POON
Original assignee: NEOPEC Pty Ltd
Current assignee: NEOPEC Pty Ltd
Priority date: 2012-03-23
Filing date: 2013-03-22
Publication date: 2013-10-10
Also published as: WO2013138864A1

Abstract

The present disclosure relates generally to the field of tissue engineering. The present disclosure teaches a tissue scaffold useful for promoting or facilitating the growth, development and differentiation of cells and tissues. The tissue scaffold may be used in vitro or in vivo in various tissue engineering applications and in other cell culture systems for nurturing and enriching inter alia adipose cells. The tissue scaffold is also useful as a base for creams, such as for use in the cosmetic and topical therapeutic industries and as an additive in foods.

Description

TISSUE SCAFFOLD FILING DATA [0001] This application is associated with and claims priority from US Provisional Patent 5 Application No. 61/614,720, filed on 23 March, 2012 entitled "Tissue scaffold", the entire contents of which, are incorporated herein by reference. BACKGROUND 10 FIELD [0002] The present disclosure relates generally to the field of tissue engineering. The present disclosure teaches a tissue scaffold useful for promoting or facilitating the growth, development and differentiation of cells and tissues. The tissue scaffold may be used in 15 vitro or in vivo in various tissue engineering applications and in other cell culture systems for nurturing and enriching inter alia adipose cells. The tissue scaffold is also useful as a base for creams, such as for use in the cosmetic and topical therapeutic industries and as an additive in foods. 20 DESCRIPTION OF THE PRIOR ART [0003] Bibliographic details of references in the subject specification are also listed at the end of the specification. 25 [0004] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in any country. [0005] Congenital, traumatic or post surgical deformities such as mastectomy often require 30 restoration of contour usually involving adipose tissue. Not only does adipose tissue act as a reservoir for lipids, it also provides insulation and physical protection to the -2 underlying tissue. Being vascularized, it makes an excellent graft bed for other tissues and can be used in complex reconstructive scenarios for which no appropriate donor tissue exists. Adipose tissue engineering has recently received much attention as it promises enhanced efficacy, reproducibility and predictability, compared with the contemporary 5 methods used to treat disfiguring contour imperfections. Autologous free fat grafting with processed lipo-aspirate has unpredictable results due to post-graft resorption with sometimes as little as 10% of the original fat volume retained (Karacal et al. (2007) JPlast Reconstr Aesthet Surg 60(3):300-303; Boschert et al. (2002) Plast Reconstr Surg 109(2):761-765; Mikus et al. (1995) Laryngoscope 105(1):17-22). While the use of 10 vascularized fat flaps generally has more favourable results, complications such as flap failure, infections, and pulmonary embolisms exist, along with morbidity relating to the donor site (Blondeel et al. (1997) Br JPlast Surg 50(5):322-330). [00061 Once removed from their native environments, stem cells differentiate less 15 efficiently (Chaubey and Burg (2008) Journal of Bioactive and Compatible Polymers 23(1):20-37; Chen et al. (2007) Stem Cells 25(3):553-561). Therefore, when designing a suitable replacement 3-dimensional scaffold for thick tissues such as fat, it is important to incorporate characteristics of the native cellular environment to maintain optimal tissue development. For adipose tissue engineering, the scaffold needs to induce 20 adipogenesis. Of particular importance is the regulatory and structural role of the native extracellular matrix (ECM) and associated factors (Boudreau and Weaver (2006) Cell 125(3):429-431). The mechanotransduction between the extracellular matrix (ECM) and cells play a critical role in the regulation of angiogenesis (Ingber and Folkman (1989) The Journal of Cell Biology 109(]):317-330) and in directing cells towards specific 25 differentiation pathways (Daniels and Solursh (1991) J Cell Sci 100(Pt 2):249-254). Not only does the ECM provide structural support for the cells, it is also a reservoir for tissue-specific growth factors and signalling molecules that an entirely synthetic scaffold lacks. 30 [0007] Chemical crosslinking agents are successful in creating scaffolds from soluble proteins, however, the introduction of artificial linkages risks converting an -3 otherwise native protein into something that may hinder cell infiltration and maturation (Jarman-Smith et al. Journal of Materials Science:Materials in Medicine 15(8):925-932). Naturally-derived scaffolds and hydrogels have been used for some years (Choi et al. (2009) J Control Release; Choi et al. (2009) Tissue Eng Part C Methods; Choi et al, 5 (2010) Tissue Eng Part C Methods 16(3):387-396; Sharma et al. (2010) FASEB 24(7):2364-2374; Uriel et al. (2008) Biomaterials 29(27):3712-3719; Uriel et al. (2009) Tissue Engineering Part C: Methods 15(3):309-321; Flynn et al. (2007) Biomaterials 28(26):3834-3 842; Flynn et al. (2009) JBiomed Mater Res A 89(4):929-941; Flynn et al. (2006) J Biomed Mater Res A 79(2):359-369; Flynn and Woodhouse (2008) 10 Organogenesis 4(4):228-235 and have shown potential in supporting cell growth whilst maintaining their volume. Sharma et al. (2016) supra demonstrated that adipocyte-derived ECM extract supported hepatocytes with a higher metabolic activity than with Matrigel, a commercially available ECM hydrogel, whereas the Flynn et al. publications reported that matrices prepared from a cellular placental ECM and hyaluronic acid, support the 15 differentiation of adipose-derived stem cells (ADSC). In another study, freeze-dried, injectable powders were prepared from human lipoaspirate (Choi et al. (2009) supra). This scaffolding material combined with ADSC resulted in well vascularized adipose tissue after implantation into nude mice. When prepared as a hydrogel, adipose-derived extracts have been shown to not only support the growth of seeded ADSC, but also showed signs of 20 inducing neoadipogenesis when implanted along the rat epigastric artery vascular pedicle (Uriel et al. (2008) supra). [0008] There is a clear need for an adipose-derived tissue scaffold which promotes cellular infiltration and has the potential to help form new vascularized adipose tissue once 25 implanted into a subject.

-4 SUMMARY [0009] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply 5 the inclusion of a stated element or integer or method step or group of elements or integers or method steps but not the exclusion of any other element or integer or method step or group of elements or integers or method steps. [0010] As used in the subject specification, the singular forms "a", "an" and "the" include 10 plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a matrix" includes a single matrix, as well as two or more matrices; reference to "an extract" includes a single extract, as well as two or more extracts; reference to "the disclosure" includes single and multiple aspects taught by the disclosure; and so forth. Aspects taught and enabled herein are encompassed by the term "invention". All such 15 aspects are enabled within the width of the claims which define the present invention. [0011] The present disclosure teaches a substantially cell-free adipose-derived material which is useful as a scaffold to support the growth and development of adipose tissue and differentiation of adipogenic stem cells. The scaffold is also useful to support the growth 20 and development of other cell types. The substantially cell-free formulation is derived from adipose tissue and comprises basement membrane components, collagen and growth factors. By "cell-free" does not necessarily mean that the material does not comprise cellular components such as nucleic acid molecules and proteins. Growth factors comprise one or more of, but not limited to, Activin A, TGF-p 1 and FGF-2. The collagen includes 25 but is not limited to Collagen J, IV and VL The material forms a gel at from about 20'C to about 50C including under physiological conditions (37-42 0 C), polymerizing from a viscous liquid at 4 0 C to form a gel under these conditions. [0012] The tissue material is also conveniently referred to herein as adipogenic matrix, 30 adipose basement membrane matrix, adipose-derived matrix (ADM), adipogenic hydrogel and adipose scaffold. The term "adipose-derived matrix" or ADM is conveniently used for -5 brevity with the understanding that it covers all forms of the preparation. In an embodiment, the ADM is in an injectable form. In another embodiment, the ADM is coated onto a solid phase material such as a biodegradable mesh, a surgical implant or a bead. The ADM may also be referred to as a "synthetic matrix" or "synthetic scaffold". 5 [0013] The ADM enabled herein has a variety of uses such as in tissue engineering to facilitate the generation of large amounts of tissue for tissue repair, augmentation and/or replacement therapy. The ADM is also useful as a scaffold for engineered tissues such as fat, It is also useful as a means to enrich and nurture appropriate pre-adipogenic cells from 10 appropriate stem cell locations. As a research tool, ADM is useful in the study of cell growth, development and differentiation of preadipocytes. In the cosmetic and food industries, ADM is useful as a base for creams and as food additives as well as therapeutically as cellular repair compositions. When used in tissue engineering in a subject, the ADM may be derived from adipose tissue heterologous or autologous to the 15 subject being treated. [0014] Hence, enabled herein is a tissue scaffold comprising a substantially cell-free extract of adipose tissue, basement membrane proteins, collagen and growth factors, wherein the tissue scaffold gels at a temperature of from about 20'C to about 50'C and is 20 adipogenic. [00151 Further provided is an ADM comprising a substantially cell-free extract of adipose tissue, basement membrane proteins, collagen and growth factors, wherein the ADM gels at a temperature of from about 20C to about 50C and is adipogenic. 25 [00161 The ADM may be used alone or in conjunction or association with an implantable medical device, collapsible bag or biodegradable mesh. [00171 Abbreviations used herein are defined in Table 1. 30 -6 TABLE 1 Abbreviations ADM Adipose-derived matrix ADSC Adipose-derived stem cells DMMB assay 1,9-Dimethylmethylene blue assay ECM Extracellular matrix ELISA Enzyme-linked Immunosorbant assay FGF-2 Fibroblast growth factor-2 GAG Glycosaminoglycan IL-la Interleukin- 1 alpha IL-l p Interleukin-1 beta MCP-1 Monocyte chemotactic protein-i PBS Phosphate buffered saline PDGF-Ap Platelet-derived growth factor-Ap TGF-p 1 Transforming growth factor-beta 1 TNFa Tumor necrosis factor alpha VEGF Vascular endothelial growth factor -7 BRIEF DESCRIPTION OF THE FIGURES [0018] Figure 1 is a photographical representation showing adipose-derived matrix (ADM) at different stages of gelation. Adipose-derived matrix (-3 mg/mL) sets within 15 5 min at 374C. [00191 Figure 2 is a photographical representation showing protein analysis of adipose derived matrix. Undiluted samples of crude ADM (~3 mg/mL) were stored at 4 0 C protected from the light for up to 63 days. Upon completion of the timecourse, 2.5 ptg of 10 each sample was resolved on a 4-12% w/v bis-tris gradient gel and silver-stained (A). Porcine (p) and human (hu) ADM was compared with Matrigel (MG) and Myogel (Myo). Samples were resolved on a 4-12% w/v bis-tris gradient gel and silver-stained. Arrows indicate apparent differences between the porcine and human samples (B). 15 100201 Figure 3 is a photographical representation of Western blot analysis of adipose derived matrix. Matrigel, Myogel and adipose-derived matrix (ADM) was resolved on 4 12% w/v bis-tris gradient gels and probed for actin, myosin, fibronectin, laminin and Collagens I, IV and VI. 20 [0021] Figure 4 is a graphical representation showing quantitation of the adipogenic effect of ADM (hydrogel) on human ADSC. Human ADSC (ADSC m) were supplemented with Zuk's adipogenic medium (Zuk m) as a positive control, with ADM (Gel A), or with both ADM and adipogenic Zuk's medium (Y) for 2 weeks. After incubating for 2 weeks, cells were stained with oil red 0 and the pmportion of positively stained cells was calculated 25 from at least 12 randomly selected fields from each culture using InagJ. Error bars = SEM. [0022] Figures 5A through F are photographic representations showing differentiation of ADSC. Human ADSC were supplemented with different adipose-derived matrix preparations and compared with a cell only control (A) and adipogenic medium control 30 (B). Cells were supplemented with 1 mg/mL (C) or 2.5 mg/mL of concentrated porcine ADM soluble fraction (D). In another experiment, cells were supplemented with 2 mg/mL ---- ~ 8- --- -8 (E), or 4 mg/mL soluble human ADM fraction (F). Lipid was visualized by oil red 0 staining. Scale bars = 100 pm. [00231 Figures 6A through D are photographic representations of 8 week rat 5 subcutaneous implants. Matrigel (A), Matrigel + FGF-2 (B), ADM (C) and ADM + ADSC (D) was implanted into the backs of Sprague-Dawley rats for 8 weeks, harvested and stained with haematoxylin-eosin. Scale bars = 200 pm.

-9 DETAILED DESCRIPTION [0024] The present disclosure teaches an adipose-derived formulation in the form of a tissue scaffold which is adipogenic and which is useful, inter alia, for adipogenesis 5 applications and other applications relating to tissue engineering, augmentation, repair and research. The tissue scaffold also has applications in the topical therapeutic, cosmetic and food industries. One form of the material comprises solubilized, extracted basement membrane material derived from adipose tissue. When used in a subject, the ADM may be derived from adipose tissue from a different subject (heterologous) or from the same 10 subject (autologous) to the subject ultimately treated. The tissue material is referred to variously as adipogenic matrix, adipose basement membrane matrix, adipose-derived matrix (ADM), adipogenic hydrogel and adipose scaffold. These terms are used interchangeably throughout the specification but are encompassed by the term "adipose derived matrix" or "ADM". In an embodiment, the ADM is in an injectable form. The 15 ADM may also be coated on a solid support or solid phase such as a biodegradable mesh, surgical implant or bead. The ADM may also be referred to as a "synthetic matrix" or "synthetic scaffold". [0025] The ADM is prepared from adipose tissue using a decellurization, protein 20 extraction and gelation process. Any source of adipose tissue may be used including adipose tissue in subcutaneous locations, organ adipose tissue, biopsied adipose tissue, biopsied adiposed tissue and the like. Subcutaneous adipose tissue is particularly convenient. The adipose tissue may be heterologous or autologous to a subject in which it is ultimately used. 25 [00261 Generally, the steps in the generation of ADM comprise decellurizing adipose tissue, collecting the tissue extract and subjecting it to homogenization and a protein extraction, followed by a concentration step. At 4"C, the resulting ADM is a viscous liquid, polymerizing into a gel at from about 37 0 C to about 42 0 C, which is regarded herein 30 as under physiological conditions. However, an ADM is contemplated herein which is self-gelling from 20-50'C such as 37"C.

- 10 [0027] The ADM may be maintained frozen at -20C or as a viscous liquid at about 44C. If maintained in a frozen state, it is thawed prior to use. 5 [00281 The ADM taught herein generally comprises one or more of basement membrane proteins, collagen and growth factors. The collagen but is not limited to includes Collagen I, Collagen IV and Collagen VI. Growth factors include Activin A, transforming growth factor beta 1 (TGF-p1), Fibroblast growth factor-2 (FGF-2) and trace amounts of platelet derived growth factor-Abeta (PDGF-Ap). ADM has a range of utilities including the 10 engineering and study of, inter alia, tissues comprising but not limited to adipose, material. It also provides a basis for an in vitro bioassay for adipogenic potential of source material, i.e. fat and precursor cells for fat from various sites. The preparations further have applications in the topical therapeutic, cosmetic and food industries. 15 [00291 Accordingly, tissue scaffold is provided comprising a substantially cell-free extract of adipose tissue, basement membrane proteins, collagen and growth factors, wherein the tissue scaffold gels under conditions of from about 20"C to about 50C and is adipogenic. [00301 Another aspect enabled herein is an ADM comprising a substantially cell-free 20 extract of adipose tissue, basement membrane proteins, collagen and growth factors, wherein the ADM gels at a temperature of from about 20'C to about 50 0 C and is adipogenic. [0031] In an embodiment, provided is a tissue scaffold comprising a substantially cell-free 25 extract of adipose tissue which gels under physiological conditions. By "cell-free" does not mean free of cellular components such as nucleic acids and proteins but rather substantially free of whole live cells. The term "substantially cell-free" also does not exclude the possibility for a low number of cells being present. By "low number" means a number which does not cause an adverse reaction if transplanted to a subject. Reference to 30 "low number" includes "trace cells".

- 11 [0032] By "physiological conditions" includes approximately from about 37 0 C to about 42"C. [00331 In an embodiment, the tissue material comprises Collagen I, IV and VI, Activin A, 5 TGF-p 1 and FGF2. In an embodiment, the formulation further comprises trace amounts of PDGF-Ap. In an embodiment, the formulation further comprises heparan sulfate proteoglycan (glycosaminoglycans). [0034] The source of the tissue material may be from any animal including a mammal 10 such as, but not limited to, a human, non-human primate (eg. gorilla, marmoset or orang outan), livestock animal (eg. cow, sheep, pig, horse, donkey, goat, camel), laboratory test animal (eg. mouse, rat, rabbit, guinea pigs, hamster) or companion animal (eg. dog, cat). The present disclosure further teaches avian sources such as chickens, ducks, geese, turkeys and other poultry or game birds, reptilian sources such as snakes and lizards and 15 amphibians sources such as frogs and toads. [0035j The instant disclosure is particularly instructional on generating ADM from human and porcine adipose tissue. The adipose tissue may also be derived from the subject whom is ultimately treated. Such an ADM would be autologous to the subject. ADM prepared 20 from a different subject or different animal is heterologous to be the subject. [0036j Enabled herein is a composition of matter comprising an adipose preparation from an animal, the composition comprising: 25 (i) a cell-free extract of adipose tissue; and (ii) components selected from the list comprising basement membrane protein, collagen and growth factors; 30 wherein the composition is self-gelling at from about 37"C to about 42C and wherein Activin A, TGF-p1 and FGF-2 are present, each in an amount of from about 10 pg/mg to -12 about 300 pg/mg material. In an embodiment, the composition of matter is in injectable form, [0037] Reference to the above components such as protein, collagen and growth factors is 5 not to be restricted solely to those components. In other words, the preparation may contain other components not recited. As indicated above, a trace number of cells may still be present in the "cell-free" adipose material. [0038] The components of the tissue material may be totally derived from adipose tissue or 10 additional factors such as, but not limited to, additional gelling agents (such as salts solutes and/or sugars), cytokines, antibiotics, growth enhancers, gene expression enhancers, proliferation inhibitors and/or stem cell differentiation facilitators may be added during preparation. 15 [0039] The tissue material may, in one embodiment, be considered as a composition which facilitates cell culture, cellular differentiation, de-differentiation or growth in vitro or in vivo. As taught herein, the ADM is adipogenic including the differentiation of adipocyte stem cells into adipose cells. The ADM is also a useful as a scaffold to support growth and differentiation of a range of cell types including stem cells. 20 [0040i Reference to "substantially cell-free" material includes from no intact cells to a low "trace" cell number. Conveniently, enzyme-based decellurization is employed such as using dispase or a related enzyme. The material contains cellular material. 25 [0041] Taught herein is a cell culture composition useful in facilitating growth of adipose cells or effecting a change in cell or tissue morphology wherein the cell culture composition comprises a substantially cell-free adipose tissue extract, basement membrane protein, collagen and growth factors which cell culture composition polymerizes into a gel at from about 37C to about 42C and is adipogenic. 30 H ar\Intenvoven\NRPorthUDCCARk296_1.doe-15o3/213 - 13 [00421 Reference to a "growth factor" includes a cytokine. A "cytokine" includes single or multiple cytokines selected from Activin A, TGF-p1, FGF-2 and PDGF-AD. Additional cytokines may be present or included. Additional extracellular matrix material may also be present or added. 5 [0043] The polymerization generally occurs at temperatures from about 20'C to about 50C such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50'C. Fluctuating temperatures may also be employed. By physiological conditions is meant approximately 37"C to 42C. 10 [00441 The term "gel" is used in its broadest sense and includes a semi-liquid, semi-rigid material, flexible material, dense liquid, cream, solid support or combination thereof including a material suitable for use as a food additive. At 4 0 C, the ADM is a viscous liquid which is not considered a gel. 15 [00451 In an embodiment, the growth factors are present in amounts as follows: Activn A, TGF-p1 and FGF2: from about 10 pg/ml to about 300 pg/ml such as 10, 20, 30, 40, 50, 60, 70, 80, 90, 20 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 pg/ml. PDGF-Ap: from about 0.3 pg/mg to about 4 pg/mg such as 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 25 1.1, 1.2, 1,3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0 pg/mg. [0046] Amounts may vary depending on the source of the adipose tissue. 30 [0047] In an embodiment, VEGF and IL-I a are not detectable.

Haaar\In.ta oven\NRPortbIDCC\AAR14229306_1.doc-15031201 - 14 [0048] The present disclosure is instructional on an adipose-derived matrix (ADM), the material derived from human, non-human primate, livestock animal, companion animal, avian, reptile or amphibian adipose tissue, wherein the ADM is cell-free and comprises growth factors selected from the list comprising, 10 to 300 pg/mg each of Activin A, 5 TGF-p1 and FGF-2 and from about 1.0 pg/mg to about 4 pg/mg PDGF-Ap, the ADM further comprising basement membrane protein and collagen and wherein the ADM is adipogenic. [0049] The ADM is in a gel form at least under physiological conditions. However, the 10 present disclosure enables gelling from 20'C to 50'C including from about 37 0 C to about 42 0 C. The tissue material may be in the gel form or it may be in a "precursor" form which is polymerizable to a gel form. Conveniently, the cell-free tissue material, when in precursor form, is reconstitutable to a gel or matrix form. Conveniently, the matrix form of the reconstituted precursor is referred to herein as "adipose-derived matrix" or ADM. 15 The ADM disclosed herein, either in gel form or precursor form may also be made into or incorporated into beads, biodegradable and/or tissue-compatible cellular scaffolds, sponges, creams and the like. The term "gel" includes a substantially cell-free preparation which has similar "gelling" characteristics to a gel. 20 10050] The ADM is useful in the promotion of cell growth and differentiation of a variety of cells and to effect a change in cell or tissue morphology. Epithelial cells, endothelial cells, neural cells and stem cells are amenable for growth and differentiation by ADM. It also aids in cell adhesion and in the growth, development, differentiation and/or proliferation of cells selected from, but not limited to, neurons, hepatocytes, Sertoli cells, 25 hair follicles, thyroid cells and the like. In an embodiment, the ADM is adipogenic. 10051J Cells may be cultured in vitro on the ADM and then returned to the animal including human subjects from which they originated or in immune suppressed or histocompatible animals. In this context, an "animal" includes a human, non-human 30 mammal, livestock animal, companion animal or avian, reptilian or amphibian species.

Hnaarxnerwoven\NRPotb1DCC\AAR\4229306_1,do-151/032013 -15 Likewise, the ADM may be used alone in vivo to promote cell growth or tissue growth at particular sites or in chambers or other scaffolds implanted in the body. [0052] The following is a description of how ADM is generated. Frozen adipose tissue is 5 shaved into pieces and homogenized with an equal volume of phosphate-buffered saline (PBS) until it reaches a smooth consistency. After centrifugation the tissue is treated with a dispase or other suitable enzyme to aid with decellularization. The tissue is then centrifuged and excess enzyme removed. This is followed by washing with a salt buffer followed by centrifugation to remove the buffer. The uppermost layer of lipid is removed 10 before subsequent washing after each step. The tissue is then extracted with an equal volume of a urea buffer. After incubation, visible lumps of solidified lipid are removed and the extract (Extract 1) recovered by centrifugation and dialyzed against a tris-buffered saline. 15 [0053] An equal volume of guanidine-HC buffer is then added to the remaining tissue and homogenized. This extract is mixed and then dialyzed against a buffer to produce an extract made up of a solid and liquid component. The liquid extract (Extract 2) is recovered by centrifugation and combined with Extract 1. Residual lipid is removed from the extract via filtration and high speed centrifugation before being concentrated up to four 20 times and stored at 4 0 C. [0054] The remaining pellet containing unextracted protein is combined with an equal volume of a pepsin or similar compound and mixed at room temperature until dissolved. After incubation, any undigested protein and residual lipid is removed by centrifugation 25 and the remaining digest is dialyzed against a buffer to inactivate the pepsin. The neutralized digest is combined with the concentrated liquid extract to form Adipose Derived Matrix (ADM). The ADM is sterilized by dialysis against chloroform before being dialyzed at 4 0 C with urea overnight, followed by dialysis in a chilled buffer. The ADM (-3 mg/mL) is stored long-term at -20'C or for up to 2 months at 4 0 C. 30 MIUMLW ~ u-1DO U\AK ID~_.dC -Dl/UjIZUIS -16 10055] When required, a sample of the ADM is retrieved and incubated at from about 20 to about 50"C such as around 37-42 0 C where the material polymerizes into a gel-like form. Additives to assist gelling can also be added, including but not restricted to plasma. 5 [0056] ADM disclosed herein may be packaged for sale in a pre-matrix form or in a matrix form and may come with instructions on how to use. Additional components may be in the package or kit and are included prior to use or admixed at the time of use. [0057] As indicated above, the ADM is suited to the culture of a variety of cells including 10 but not limited to adipocytes. [0058] The ADM is also useful in an in vitro bioassay for adipogenic potential of source material. The preparation also has capability in in vitro bioassays for the differentiation of other basement membrane-response cell types such as epithelial, neuronal and endothelial 15 cells and cells from pathogenic states such as cancer and diabetes. [0059] According to this aspect, the ADM is subjected to an in vitro assay to determine constituent components including cells and/or molecule components which induce adipogenesis. The assay includes coating a surface of a recepticle with a layer or multiple 20 layers of potential adipogenic components to be tested such as an extract of adipose tissue, seeding cells with a potential to undergo adipogenic differentiation and then screening for adipogenesis. Alternatively, ADM is coated onto the surface and other compounds added and then the system is screened for enhanced or reduced adipogenesis. In another embodiment, cells with a potential to undergo adipogenesis differentiation are maintained 25 in a suspension culture and the media supplemented with a potential adipogenesis component to be tested such as an extract or fraction of adipose tissue. A cell suspension allows for the rapid isolation of cells from the culturing media to determine if they have undergone differentiation. Alternatively, the assay includes the generation of a three dimensional scaffold comprising a potential adipogenic component or extract, seeding cells 30 with a potential to undergo adipogenic differentiation and then screening for adipogenesis. Yet another alternative includes adding ADM to pre-gelled cultures. The assay system -17 taught by the present disclosure may also provide or select or develop optimized populations of pre-adipocytes for use in tissue engineering. In an embodiment, the ADM is coated to a solid phase such as beads, the surface of a receptacle, a dipstick and the like including a range of polymers and then used in an adipogenic assay. 5 [0060] Accordingly, the present disclosure is instructional for an in vitro assay for adipogenesis-modulating components, extracts, or cell systems, the assay comprising screening an adipose preparation to identify a group of cells having a propensity for adipocytic differentiation, generating or obtaining a potential adipogenesis-modulating 10 component or extract or cell system, seeding onto the component or extract the group of cells having a propensity for adipocytic differentiation, incubating the cells for a time sufficient for adipogenesis to occur and then screening the cells for adipocytic differentiation. A "cell system" in this context has the same meaning as a cell-based preparation. 15 [0061] In an embodiment the potential adipogenesis-modulating component or extract or cell system promotes adipogenesis. In another embodiment, the adipogenesis-modulating component or extract or cell system induces cellular differentiation. 20 [00621 By "modulating" is meant increasing or decreasing, either directly or indirectly the level of adipogenesis. [0063] A layer of potential adipogenesis-modulating component or extract or cell system may be obtained or generated. Alternatively, a three-dimensional support matrix 25 comprising the potential adipogenesis-modulating component or extract or cell system may be obtained or generated. In an embodiment, the cells having a propensity to undergo adipocytic differentiation may be maintained in a suspension culture and the media is supplemented with the potential adipogenesis modulating component or extract. Reference to a "layer" includes two or more layers. The present method extends to adding potential 30 adipogenesis promoting agents to the ADM.

H iaarInten vn\RPortbxLDCC AR 4 229306I.doc-15/03/2013 - 18 [00641 Yet another aspect of the present invention provides a method of generating donor vascularized tissue suitable for transplantation into a recipient, the method comprising creating a vascular pedicle comprising a functional circulatory system and having tissue or tissue extract or a component thereof impregnated, attached or otherwise associated with 5 the vascular pedicle; associating the vascular pedicle within and/or on a support matrix; seeding the support matrix with isolated cells or pieces of tissue identified using an in vitro assay as promoting adipogenesis or cells for some other useful endpoint; implanting the support matrix containing the vascular pedicle into a recipient at a site where the functional circulatory system is anastomosized to a local artery or vein; and leaving the support 10 matrix at the implantation site for a period sufficient to allow the growth of vascularized new tissue wherein the impregnated material or seeding material is selected on a particular basis, for example, that it promotes adipogenesis when determined by the assay comprising screening a tissue or tissue extract to identify a group of cells having a propensity for adipogenic differentiation, generating or obtaining potential adipogenesis promoting 15 component or extract, seeding onto said component or extract a group of cells having a propensity for adipocytic differentiation, incubating the cells for a time sufficient for adipogenesis to occur and then screening said cells for adipocytic differentiation. [0065] In an embodiment, the vascular pedicle comprises attached fat or other adipose 20 tissue or tissue comprising pre-adipocytes, adipocytes, myoblasts and fibroblasts, cardiomyocytes, keratinocytes, endothelial cells, smooth muscle cells, chondrocytes, pericytes, bone marrow-derived stromal precursor cells, embryonic, mesenchymal or haematopoietic stem cells, Schwann cells and other cells of the peripheral and central nervous system, olfactory cells, hepatocytes and other liver cells, mesangial and other 25 kidney cells, pancreatic islet p-cells and ductal cells, thyroid cells, cells of other endocrine organs and spheroids of aforementioned cells. All these cells are tested in vitro for their potential to grow/survive on the matrix or capacity to differentiate into other useful tissues e.g. adipogenic potential, prior to selection. The presence of the attached tissue on the vascular pedicle further facilitates the growth of new fat tissue in or around the support 30 matrix. In an alternative embodiment, tissue extract or a recombinant, synthetic or purified component of the tissue is associated with the vascular pedicle. For example, these tH:uarurelnRonr4cr)L~CtAAK\42293U6_L.doc-fLW3/2U13 -19 components and extracts are derived from ADM and screened in vitro for adipogenic potential. [0066] The ADM is allowed to set and cells capable of adipocytic differentiation plated 5 over the monolayer of matrix in the presence of complete media (such as DMEM containing FCS) or differentiation media (complete media supplemented with tp.M dexamethasone, insulin, indomethacin and IBMX). Adipogenesis is observed over a period of from a few days to 14 days. Examples of suitable adipocytic cells include 3T3 Ll cells or preadipocytic cells isolated by standard procedures. In an alterantive 10 embodiment, the ADM is added pre-gelled to cultures. [0067] The present disclosure further enables an in vitro assay for adipogenesis-promoting components or extracts, the assay comprising generating or obtaining a layer of potential adipogenesis extract from ADM on the surface of a receptacle, seeding onto the layer a 15 group of cells having a propensity for adipocytic differentiation incubating the cells for a time sufficient for adipogenesis to occur and then screening the cells for adipocytic differentiation. [0068] In another embodiment, the assay is conducted on a three-dimensional support 20 matrix, which may be constructed substantially from the ADM or comprise a scaffold that is coated with the ADM, in respect of which three dimensional cell culturing techniques known to the person skilled in the art are carried out, for example the spinner flask technique (Mueller-Klieser, (1986) J Cancer Res Clin Oncol 13: 101-122), the liquid overlay technique (Yuhas, et al. (1977) Cancer Res 37: 3639-3643). Another example of a 25 three-dimensional cell culture technique is a rotating culture vessel specifically engineered to randomize the gravity vector by rotating a fluid-filled culture vessel about a horizontal axis while suspending cells and cell aggregates with minimum fluid shear. These devices have been described in U.S. Patent Nos 5,153,131; 5,153, 132; 5,153, 133; 5, 153, 034, and 5,155,035. 30 -20 [0069f An advantage of the three-dimensional matrix is that it sustains active proliferation of cells in culture for longer periods of time than will monolayer systems. This may be in part due to the increased area of the three dimensional matrix which results in a prolonged periods of active proliferation of cells. The matrix provides the support, growth factors 5 and regulatory factors necessary to sustain long-term active proliferation of cells in culture. The growth of the cells in the presence of the support may be further enhanced by adding proteins, glycoproteins, glycosaminoglycans, a cellular matrix and other materials to the support itself or by coating the support with these materials. The three-dimensionality of the matrix allows for the formation of microenvironments conducive to cellular maturation 10 and migration. When grown in this three-dimensional system, the proliferating cells mature and segregate properly to form components of adult tissues analogous to counterparts in vivo. [00701 In order for the three dimensional structures to be able to maintain the activity of 15 living cells three dimensional matrices should demonstrate appropriate spatial and compositional properties. Such matrices include hydrogels, or porous matrices such as fibre-based or sponge-like matrices. Common materials used in three-dimensional matrices are natural polymers or "biomatrices", synthetic polymers and inorganic composites. In an embodiment, where the method contemplates the use of the three 20 dimensional matrices for an in vitro assay, the biocompatibility of the matrix is not particularly important. In an embodiment, material is used which is biodegradable. [0071] Examples of biomatrices are those extracted from or resembling ADM or having a cell-free system comprising same. 25 [0072] The present disclosure is instructional on the use of ADM in the manufacture of a cell growth promoting composition. The ADM is also useful for the selective purification of specific cell types (e.g. preadipocytes) for complex cell mixtures based on selected growth and morphological characteristics specific to the adipose tissue preparation. 30 -21 EXAMPLES 10073] Aspects disclosed herein are further described by the following non-limiting Examples. 5 Methods Preparation of adipogenic hydrogels [00741 Frozen porcine subcutaneous adipose tissue was shaved into 1-2 g pieces and 10 homogenized with an equal volume of phosphate-buffered saline (PBS) until it reached a smooth consistency. After centrifugation (3000 G, 4'C, 10 min) the tissue was treated with 2 U/mL dispase II (Roche, Australia) 30 min in a shaking 37 0 C incubator to help with decellularization. The tissue was then centrifuged (3000 G, 4'C, 10 min) and excess dispase removed. This was followed by washes with 2 x volumes of salt buffer (3.4 M 15 NaCl, 50 mM tris-HCl pH 7.4, 2 mM NEM, 8 mM EDTA). After 5 min mixing, the tissue was centrifuged (3000 G, 4 0 C, 10 min) and the buffer removed. The uppermost layer of lipid was removed before subsequent washing after each step. This washing step was repeated until the majority of visible lipid had been removed. The tissue was then extracted with an equal volume of urea buffer (2 M urea, 50 mM tris-HCl pH 7.4) for 24 hr at 4 0 C. 20 After incubation, visible lumps of solidified lipid were removed and the extract (Extract 1) recovered by centrifugation (3000 G, 44C, 10 min) and dialyzed against tris-buffered saline (TBS - 50 mM tris-HCl pH 7.4, 150 mM NACl) at 4 0 C. [0075] An equal volume of guanidine-HC1 buffer (4 M GuHC, 50 mM tris-HC pH 7.4, 5 25 mM dithiothreitol [DTT]) was then added to the remaining tissue and homogenized. This extract was mixed for at least 18 hr at 4C and then dialyzed against TBS at 4 0 C to produce an extract made up of a solid and liquid component. The liquid extract (Extract 2) was recovered by centrifugation (3000G, 4C, 10 min) and combined with Extract 1. Residual lipid was removed from the extract via 0.2pim filtration and high speed 30 centrifugation (10,000 G, 4 0 C, 10 min) before being concentrated up to four times and stored at 4C.

- 22 [0076] The remaining pellet containing unextracted protein was combined with an equal volume of 1% w/v pepsin (Sigma-Aldrich, Australia), 0.5 M acetic acid, and mixed at room temperature until dissolved. After incubation, any undigested protein and residual 5 lipid was removed by centrifugation (10,000 G, 4 0 C, 10 min) and the remaining digest was dialyzed against TBS at 4 0 C to inactivate the pepsin. The neutralized digest was combined with the concentrated liquid extract to form AdiposeDerived Matrix (ADM). The ADM was sterilized by dialysis against chloroform to a final concentration of 0.5% v/v at 4 0 C before being dialyzed at 4 0 C with 8 M urea overnight, followed by dialysis in chilled TBS 10 or DMEM. The ADM (-3 mg/mL) was stored long-term at -20'C or for up to 2 months at 4 0 C. [0077] Human ADM was prepared with the following modifications: After the tissue had been extracted for a second time with 4 M GuHCl, the soluble fraction (Extract 2) was 15 collected by centrifugation and combined with Extract 1, then filtered and dialyzed in TBS, and concentrated up to four times. The leftover 4M GuHCl-extracted tissue was rinsed twice with two volumes of 70% v/v ethanol and then incubated for 30 min at 37 0 C in four volumes of 70% v/v ethanol to help remove excess lipid. After incubation, the tissue was washed three times with two volumes of water. After removing the water, the tissue. was 20 pepsin-digested with 0.5% w/v pepsin, 0.5 M acetic acid at room temperature until dissolved. The ADM was then produced as described above. SDS-PAGE and Western Blot Analysis [0078i All SDS-PAGE was performed with NuPAGE Novex Bis-Tris 4-12% w/v gradient 25 gels (Invitrogen, Australia). Polyclonal actin, myosin, and pan-laminin antibodies were purchased from Sigma-Aldrich, Australia. Polyclonal fibronectin, Collagen I, Collagen IV, and Collagen VI antibodies were produced by Abcam (supplied by Sapphire Bioscience). All samples were reduced with DTT (20 mM) except when probed for Collagen I, IV, and VI. To visualize resolved proteins, gels were stained with 0.1% w/v coomaisse brilliant 30 blue R-250 and/or 0.05% w/v silver nitrate. For Western blot analysis, resolved proteins were transferred to PVDF membrane and blocked with 3% BSA for 1 hr at room - 23 temperature. Primary antibodies were used at 1:500 in PBS/0.1% v/v Tween-20 and the membranes probed for at least 1 hr at room temperature followed by washing with PBS/0.1% v/v Tween-20. Fluorescent secondary antibodies: Alexa Fluor 680 goat anti mouse or anti-rabbit (Invitrogen, Australia) were used at 1:10,000 in PBS/0. 1% v/v 5 Tween-20. After a final wash, membranes were scanned with an Odyssey Infrared Imaging System (Li-Cor Biosystems) using the 700 nm channel. Protein Concentration Assays [0079] The protein concentration of adipose extracts was measured with a bicinchoninic 10 acid (BCA) assay (Thermo Fisher Scientific, Australia) using bovine serum albumin (BSA) as a protein standard. The assay was performed following manufacturer's instructions and absorbance was measured with a 96 well plate reader at 450 nm. Samples were diluted 1:10 in TBS for the BCA assay. For SD S-PAGE, protein concentrations were also estimated by absorbance at 280 nm prior to electrophoresis. 15 1,9-Dimethylmethylene blue assay [0080] The glycosaminoglycan concentration of adipose extracts was measured using the 1,9-dimethylmethylene blue (DMMB) dye-binding assay against chondroitin sulphate standards(Farndale et al. (1986) Biochim Biophys Acta 883(2):173-177). To reduce 20 interference in protein-rich samples, concentrated ADM was proteinase K-digested prior to glycosaminoglycan (GAG) measurements at 595 nm with a reference wavelength of 655 mn. Enzyme-Linked Immunosorbant Assay 25 [0081] Quantikine ELISA kits (R&D Systems, USA) were used to determine the concentration of Activin A, transforming growth factor-01 (TGF-p 1), vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF-2), platelet-derived growth factor AB (PDGF-Ap), interleukin-la (IL-la), interleukin-la (IL-la), tumor necrosis factor-a (TNF-a), and monocyte chemotactic protein-1 (MCP-1) in ADM or conditioned media. 30 Each assay was performed according to the manufacturer's instructions and samples assayed in duplicate. Briefly, protein standards and samples were added to precoated wells - 24 and allowed to incubate for at least an hour at room temperature or overnight at 4 0 C. After washing, the conjugated antibody was added to each well and the plate incubated for 2 hrs at room temperature before being washed and developed for 30 min. The reaction was stopped and absorbance measured at 450 nm. 5 Protein Stability [00821 ADM (3 mg/mL) was dispensed into polypropylene tubes (8 x 50 PL) and stored protected from light at 4 0 C for 0, 3, 7, 14, 21, 28, 35, and 63 days. At each time point, one tube was stored at -20'C until all samples had been collected. Upon completion of the time 10 course, 2.5-5 pg protein of each sample was resolved on a 4-12% w/v SDS-PAGE gradient gel and silver-stained. Cell Culture [00831 Human subcutaneous adipose stromal vascular fraction and primary mouse 15 macrophage cells were cultured in DMEM (Invitrogen, Australia) supplemented with 10% v/v fetal calf serum (CSL, Australia), 100 U/mL penicillin G / streptomycin sulphate, and 2.92 pg/mL L-glutamine, and incubated in a humidified 37 0 C incubator with 5% v/v C02. When required, cells were also cultured with adipogenic medium consisting of dexamethasone (1 piM), insulin (10 WM), isobutyl-methylxanthine (0.5 mM), indomethacin 20 (200 piM), antibiotics, and 10% v/v fetal calf serum in DMEM (Zuk et al. (2001) Tiss. Eng. 7(2):211-228). ADM was tested on cells either by overlaying 80% confluent cells with ~ 100 - 200 pL / cm 2 of ADM and allowing it to set at 37*C for 15 min before adding medium, by adding a water-dialyzed ADM to the medium, or by dissolving the soluble ADM fraction in medium. Cultures were incubated for up to 2 weeks at 37 0 C. ADM was 25 carefully removed from the cultures before the cells were fixed for 10 min with 4% formaldehyde and stained with oil red 0 (Sigma-Aldrich). [0084] The macrophage response to Matrigel, porcine and human ADM was measured in vitro. ADM (100 gL) was placed into a 24 well plate. Primary macrophages at passage 2 30 were seeded at a density of 5 x 104 cells/well with 1 ml of DMEM supplemented with 10% v/v fetal calf serum, 100 U/mL penicillin G / streptomycin sulphate, and 2.92 pg/mL L- H:\amnutenvcvnNR'rtbl\DCCiAAR\42293O%_Ldoc-1M/20JI3 - 25 glutamine, and incubated in a humidified 37 0 C incubator with air containing 5% v/v C02 (n=3). Media was aspirated daily for 4 days and stored at -20'C for ELISA analysis of inflammatory markers including TNFoc, IL-1, MCP-1I and TGF-p 1. 5 Adipocyte Quantitation [00851 ADSC were seeded into a 12 well plate and cultured under four conditions (n=3): cell only control, with adipogenic medium (Zuk et at. (2001) supra), with porcine ADM, and with porcine ADM supplemented with adipogenic medium. After incubation with ADM for 2 weeks, photographs were taken of the cells from at least 12 random fields 10 (chosen from triplicate wells) (Olympus CK40 microscope, photographed with an Olympus C5050 camera at -100x magnification). Cells were counted using ImageJ (Rasband et al. (1997-2009) http://rsb.info.nih.gov/i/, US National Institutes of Health, Abramoff et al (2004) Biophotonics International 11(7):36-42) with the cell counter plugin, and the proportion of oil red 0-stained cells determined. 15 Implant Preparation [0086] ADM preparations tested in vivo included porcine ADM and porcine ADM with rat ADSC. Matrigel (BD Biosciences, USA) supplemented with FGF-2 (PeproTech) was the positive control and GFR Matrigel without FGF-2 was the negative control. All hydrogel 20 preparations were implanted within silicone (Silastic, Dow Corning) rings, the use of which allowed the in vivo support of gels as 3-dimensional masses and aided in sample setting and handling during insertion and subsequent location and removal. Segments were cut from a length of tubing with internal diameters of 6.35 and 9.53 mm respectively, into 4 mm tall rings with a maximum volume of 120 pL. Liquid gels were pipetted into 70% 25 v/v ethanol-sterilized rings at 4C under sterile conditions and left to solidify at 37 0 C for 15 minutes prior to subcutaneous implantation. Gels requiring supplementation with either FGF-2 (1 pg/mL) or rat ADSC (2.5x10 4 cells per implant) were prepared immediately before pipetting into rings.

H:\aarntenvoven\NR ribIDCCQARW4229306_.doc-15/3/2013 -26 In Vivo Testing [0087] Implants were placed subcutaneously in the backs of 300-450 g male Sprague Dawley rats, removed at 2 and 8 weeks (n=4 per time point), and prepared for paraffin histology as previously described (Hamid et al. (2010) Biomaterials 31(25):6454-6567). 5 Samples were fixed and mounted in paraffin and 5 pm sections stained with haematoxylin and eosin for analysis.

-27 EXAMPLE 1 Elation [00881 A thermosersitive hydrogel was prepared from subcutaneous adipose tissue using a 5 heavily modified method based on one used to produce the skeletal muscle product, Myogel (Abberton et al. (2008) Cells Tissues Organs 188(4):347-358), and incorporating an enzymatic decellularization step (Figure 1). This gel displayed sol-gel properties similar to those of commercial purified collagen gels, remaining a viscous liquid at 4C and polymerizing once incubated at 37 0 C. Complete gelation was reached after -15 min. 10 The gelation of ADM did not appear to be significantly affected by multiple rounds of freeze-thawing, however, syneresis occurs if the hydrogel is maintained at room temperature for extended periods. This effect is less pronounced with higher concentrations of ADM or if the gel is undisturbed. 15 EXAMPLE 2 Analysis of Growth Factors [0089] The concentration of various growth factors was determined by ELISA in a representative batch of porcine ADM (-3 mg/ml) and results expressed as pg/mg of total 20 protein Table 2). All of the growth factors assayed were of a relatively low concentration. VEGF and IL-1a was unable to be detected and there was also very little PDGF-Ap detected (I pg/mg). Activin A, TGF- pl, and FGF-2 were measured at concentrations of 31-45 pg/mg.

1-1atUnter vw V "i<N Dlf W i O fIL UAUJ i . -1 LO1DV-J/2UL3 -28 EXAMPLE 3 ADM Stability [00901 A crude batch of undiluted ADM (-3 mg/mL) was stored at 4 0 C for up to 63 days 5 to determine its stability without additional protease inhibitors. At the times indicated (Figure 2A), samples were stored at -20'C until the completion of the time course. Silver staining revealed no appreciable protein degradation after 63 days. EXAMPLE 4 10 Comparison of ADM with other ECM products [0091] ADMs were prepared from porcine and human subcutaneous fat and compared with Matrigel and Myogel. The silver stain (Figure 2B) shows quite similar protein profiles for human and porcine products with a majority of the major bands being shared 15 across the two species. The human ADM has additional proteins in the 10-30 kDa range not present in the porcine ADM to any extent. A more intensely stained 60 kDa band was present in the porcine ADM (indicated in Figure 2B with arrows). A comparison of the adipose-derived matrices used in Figures 2A and 2B show differences in proteins in the 20-75 kDa range. The earlier ADM batch used for the time course relied solely on the 20 collagen fragments released by dispase digestion for gelation, whereas the more recent batch of ADM used in Figure 2B used a combination of dispase and pepsin to produce a gel with superior gelling capacity. The additional bands seen in Figure 2A are made up primarily of dispase-cleavage products, namely Collagen IV and fibronectin (Stenn et al. (1989) J Invest. Deraintol 93(2):287-290), while retaining the full complement of soluble 25 extracted proteins. The DMMB assay showed the GAG concentration to be twice as high in extracts prepared from pig (-20 gg/mg protein) than in human tissue (~12 pLg/mg protein).

H:\aaTntcn veiNRPmtbI)C.'IAARi42293U6_Ldoc-5/20fl2 -29 EXAMPLE 5 Western Blot Analysis [0092] Matrigel, Myoge and ADM, were probed for actin, myosin, fibronectin, laminin 5 and Collagens I, IV and VI (Figure 3). Four major bands immunoreactive for actin were present in ADM with bands of approximately 30, 37, 42 and 45 kDa. The 45 kDa product was also present in Myogel, along with a larger 100 kDa species. Myosin was detected in both Myogel and ADM. A single immunoreactive 260 kDa protein was detected in Myogel, ADM also contained this protein as well as a smaller 60 kDa product. Firbonectin 10 was detected in Matrigel and ADM, with ADM showing slightly more intense binding. Laminin (pan-laminin alblcl) was detected in all samples tested. Matrigel and ADM showed intense binding for this protein - Matrigel showed larger laminin forms in the 60 >500 kDa range mainly in excess of 100 kDa, whereas ADM showed binding over a wider range from 40 kDa - >500 kDa with a majority between 60-200 kDa. ADM showed 15 positive immunoreactivity for Collagens I, IV and VI, and displayed the most intense labeling for Collagen I with multiple products from ~130 kDa in size. However, Matrigel, while also showing positive binding to all three collagens, was found to be rich in Collagen IV with multiple products starting from 120 kDa, and moderate binding for collagen I with 140 and 150 kDa products. Myogel showed very little binding for Collagen VI and 20 moderate binding for Collagen I and IV (Figure 3).

- 30 EXAMPLE 6 Primary mouse macrophage response to ADM [0093] Primary mouse macrophage cultures were supplemented with Matrigel or ADM for 5 4 days. Media was collected daily and a selection of growth factors were analyzed by ELISA (Table 3). In all cases, insignificant amounts of IL-10 was present at any timepoint, with either Matrigel or ADM. During the first 2 days barely detectable concentrations of IL- 10 was found which then dropped to zero by day 3. The addition of Matrigel or ADM did not appear to influence the IL-10 levels. 10 [0094J In the cell only control, MCP-1 expression remained relatively low throughout the culture period. Exposure to Matrigel also returned a similar expression profile. However, exposure to either porcine or human ADM resulted in higher expression during the first day (-100 - 200 pg/mL) which returned to baseline levels by day 3. MCP-1 expression 15 appeared higher in response to porcine ADM than human ADM. TGF-pl expression in macrophages did not appear to be influenced by the addition of any supplement. Expression remained relatively high throughout the culture period (-500 - 1000 pg/mL). TNF-a levels in both the cell only control and with Matrigel were at almost undetectable levels. The addition of either porcine or human ADM resulted in a dramatic upregulation 20 of TNF-a in the first day (-300 pg/nL) and quickly reached control levels by day 3 (-10 20 pg/mL).

-31 EXAMPLE 7 Adipogenic activity [0095] ADM was tested for its potential to differentiate human ADSC into adipocytes. 5 ADSC appeared to be tolerant of the tris present in the ADM, showing no signs of cytotoxicity. The results (Figure 4) show that adipogenic medium and ADM hydrogenl) at the standard -3 mg/mL concentration, were both able to induce adipogenesis to a similar degree, and that the combination of both ADM and adipogenic medium made no significant change in the proportion of adipocytes after the 2 week incubation period. 10 Routine testing of different ADM batches on ADSC, have consistently shown that ADM is adipogenic, displaying adipogenic activity comparable or better than what would be seen if adipogenic medium (Zuk et al. (2001) supra) was used. [0096] Further in vitro analysis of porcine and human ADMs was performed to determine 15 which fraction contained this adipogenic activity. The collagenous fractions were removed from both porcine and human ADM so that they consisted primarily of concentrated soluble protein. In Figures 5C,D, ADSC were exposed to increasing amounts of porcine ADM (soluble). Within 24 hrs, cells grown in culture medium containing up to 1 mg/mL of concentrated ADM had stopped proliferating and cells exposed to 2.5 mg/mL of ADM 20 showed signs of lipid accumulation. After 1 week, close to 100% of cells supplemented with either I or 2.5 mg/mL of ADM had differentiated to adipocytes (Figures 5C,D). Cells exposed to higher amounts of ADM (5 mg/mL) showed even greater degrees of adipogenesis, with many detaching after 24 hrs. 25 [0097] Human ADM (soluble) returned similar results. After 1 week, cells were fixed and stained with oil red 0. Signs of adipogenesis were observed at all of the concentrations tested, but at 2 and 4 mg/nL, adipocytes appeared more mature with larger accumulations of lipid (Figures 5E,F). Tests of both porcine and human concentrated soluble ADM extracts confirm that the adipogenic activity is isolated to the soluble fraction, and that the use of comparatively 30 more soluble fraction than the levels present in the ADM hydrogen results in close to 100% differentiation compared with -50% seen with the hydrogel (Figure 4).

- 32 EXAMPLE 8 In vivo testing of ADM 5 [0098] Implants were removed for histological assessment after 2 and 8 weeks in vivo. Subcutaneous implants generally showed little remodeling after 2 weeks, with a majority of the ADM still present. Low levels of lymphocyte infiltration were observed in some Matrigel and ADM samples, and signs of angiogenesis were observed in only the Matrigel implants. After 2 weeks the gel was very clearly still present within the ring, though only a 10 very few adipocytes were observed at this point. The majority of the analysis was done on the 8 week implants: [00991 Matrigel alone (negative control): Variable amounts of gel remained and were of a similar appearance to that originally inserted (Figure 6A). In 2 of 4 samples, a minimal 15 number of non-contiguous adipocytes were observed. [00100] Matrigel supplemented with FGF-2 (positive control): was generally observed to maintain its original volume and appearance, with clear gel-tissue interfaces observed at the top and bottom of the ring space (Figure 6B). Cellular infiltration was 20 greater than with Matrigel alone, and small areas of lymphocyte inflammation were present in some samples. In contrast to Matrigel alone, all implants showed at least some adipogenesis, and in most, conversion of gel to adipose was well advanced with greater than 50% of gel replaced by adipocytes. Individual adipocytes were separated by varying amounts of gel. 25 [0100] For ADM 3 of the 4 silicone rings were greater than 50% filled with adipocytes. Adipocytes were seen scattered within the gel with adipose tissue adjacent to it, which was clearly distinct from the tissue infiltrating from outside the ring. Small blood vessels were seen throughout the developing tissue. One of the four silicone ring showed comparatively 30 less fat but the gel seemed to persist with isolated adipocytes dispersed throughout the gel (Figure 6C).

H:\aar\IenvovenNRPvrtb\DCCAAR422906ILado-15/03/20I3 - - 33 [0101] ADM with rat ADSC: All 4 implants showed a remarkable adipogenic response. There was still some gel persisting in small amounts between the adipocytes but most of it had been replaced with new adipose tissue. Substantial vascularization was also seen 5 within the rings (Figure 6D). EXAMPLE 9 Histological assessment 10 [0102] ADM showed little remodeling after 2 weeks, however, by 8 weeks, the majority of the gel had been resorbed and replaced with vascularized adipose tissue; this effect was enhanced by the addition of ADSC. Residual ADM could clearly be identified within new adipose tissue at 8 weeks. ADM alone is adipogenic and does not require the addition of FGF-2. 15 EXAMPLE 10 Utility [0103] The ADM enabled herein allows for adipose tissue to be replaced using a non 20 invasive procedure. The ADM has a use inter alia in reconstructive surgery and helps avoid complications associated with the implantation of synthetic materials. The foregoing examples show that the ADM provides all the components necessary to produce an extract capable of forming a gel under physiological conditions without the need for additional crosslinking agents. 25 [0104] When determining potential scaffolding material, the metabolic activity and physical properties of the target tissue are important. Adipose tissue is a highly metabolically active endocrine organ with an average Youngs modulus of 1.9 kPa (Samani et al. (2003) Phys. Med. Biol. 48(14):2183-2198), and requires significant vasculature in 30 order to maintain healthy function. Daily turnover rate for adipocytes is as high as 5% (Rigamonti et al. (2011) PLOS One 6(3):e17637). Currently available synthetic scaffolds -34 may not be suitable for a highly vascularized, metabolically active tissue like adipose, as they lack the proteins necessary to initiate many important biochemical processes such as cell recognition signaling and protease-mediated cellular invasion (Rosso et al. (2004) J Cell. PhysioL 199(2):174-180; Rosso et al. (2005) J Cell. Physiol. 203(3):465-470). For 5 adipose tissue engineering, a biological component may be required to supply the proteins required to allow angiogenesis and adipogenesis to proceed at optimal rates. The ADM enabled herein addresses short comings of current synthetic scaffolds. [01051 The production of ADM is divided into three components: decellularization, 10 protein extraction, and gelation. If the ADM is to be suitable for clinical applications, it needs to be free of nuclear material to keep immunoreactivity to a minimum. The next requirement is to extract intact proteins from the source tissue. By keeping the soluble proteins in their native state, any biological activity is also retained. Finally gelation of the matrix. A timecourse analysis of crude batches of ADM showed no significant degradation 15 after 63 days storage at 40C. It is possible that the high concentration of collagen present in the extract helps to stabilise the extract. Timecourse analysis of hydrogels at lower concentrations of I mg/mL showed signs of degradation after one month at 4 0 C. At a concentration of 3 mg/mL, 3 to 4 mm adipose-derived hydrogels are able to withstand some physical manipulation without tearing and given that no exogenous crosslinking 20 agents are used, the gel appears to be surprisingly resilient and adequate for the applications presented these examples. [0106] In an embodiment enzymatic decellularization using dispase was used. Dispase has the advantage of cleaving only fibronectin and Collagen IV (Stein et al. (1989) supra) 25 and does not require extended incubation times. After 30 min digestion, >90% decellularization was achieved, as determined by haematoxylin-staining. Achieving complete decellularization may not be feasible with a dispase-only approach as longer incubation times have too great an effect on the consistency of the tissue, making it more difficult to process. However, the little remaining cellular material in the final product does 30 not appear to result in any significant reactions when implanted.

H:aanten ven\NRPotbl\DCC\.AAR\4229306_1.doc-15/03/2013 - 35 [0107] ADM forms solid gels when prepared at concentrations above 1 mg/mL. The ADM has gelation properties similar to purified Collagen 1. Like all uncrosslinked collagens, ADM displays signs of syneresis, slowly releasing its soluble proteins over a course of a few days but more so during the first few hours. 5 [01081 By extracting the soluble proteins from the tissue prior to solubilization of the collagen scaffold, the majority of proteins remain intact and maintain their activity as demonstrated by the adipogenic activity of the gel. This is important for a successful scaffold as it then retains the ability to function as it would in its native environment. 10 [0109] Production of ADM from human adipose tissue required more stringent washing steps in order to compensate for increased volume of lipid present in the tissue. However, the increased amount of washing with hypertonic buffer increased the loss of some soluble proteins. This was particularly evident with VEGF as no detectable levels of this growth 15 factor have been found in ELISA of ADMs. The low levels of some growth factors, especially VEGF, may result from extensive washing. The most common isoforms are freely secreted from the cell, while a small proportion remains bound to the cell surface (Heuz et al. (2001) Mol Endocrinol 15(21):2197-2210; Rosenbaum-Dekel et al. (2005) Biochem Bipphys Res Commun 332(]):271-278; Tee and Jaffe (2001) Biochem J 259(Pt 20 1):219-226). The loss of VEGF during processing is, therefore, difficult to avoid; the fatty nature of the tissue requires extensive washing be performed in order to remove the lipid. If necessary, the loss of growth factors could be reduced by purifying the proteins from the washing solutions discarded during processing, however this would add greatly to the production time. For applications such as tissue repair following tumor resection, where 25 the risk of tumor regrowth and metastasis is high, exposure to growth factors should be kept at a minimum. In these cases, low growth factor-containing hydrogels such as ADM which promote the gradual ingrowth of tissue will have significant value. [0110] All of the cytokines assayed in ADM were at concentrations falling well below 30 physiological levels (Czarkowska-Paczek et al. (2006) J Physiol Pharmacol 57(2):189 197; Gudewill et al. (1992) European Archieves of Psychiatry and Clinical Neuroscience H:\arIntenvoven\NRPorteDCCAR\A4229306_1.doc-1532013 -36 242(1):53-56; Jung et al. (2010) Mediators of Inflammation; Loria et al. (1998) European Journal of Endocrinology 139(5):487-492) with the highest being for FGF-2 at -45 pg / mg of total protein and slightly lower for Activin A and TGF. No IL-la was detected. Growth factors at such low concentrations may not have any significant effect on cells, 5 which is an advantage in some clinical applications. For example, the members of the TGF family which includes Activin A are important regulators of adipogenesis, promoting the proliferation of adipose progenitors and at the same time, inhibiting their differentiation to adipocytes(Tsurutani et al. (2011) Biochem Biophys Res Commun 407(1):68-73; Hirai et al. (2005) Molecular and Cellular Endorincology 232(1-2):21-26; Zaragosi et al. (2010) 10 Diabetes; Zamani and Brown (2010) Endocr Rev 32(3):387-403). Low concentrations of these growth factors may be advantageous. The low levels of inflammatory cytokines is reassuring, as it indicates the healthy state of the source tissue and the addition of exogenous inflammatory factors to a tissue engineered construct may increase the inflammatory response from the body. Despite the low concentrations of cytokines in 15 ADM, in vitro exposure to macrophages results in an upregulation of MCP-1 and TNF-a which quickly returns to baseline levels after 48 hours. By recruiting macrophages to the target site, it appears that ADM is capable of initiating the early stages of the wound healing response and may play an important role in initiating adipogenesis (Kelly et al. (2006) Tissue Eng 12(7):2041-2047; Stillaert et al. (2007) Tissue Eng 13(9):2291-2300). 20 The subsequent downregulation of MCP-1 and TNF-a. by day 3 indicates implantation of this material is unlikely to result in a persistent inflammatory response. [0111] ADM was compared with Zuk's adipogenic medium, the "gold standard" for adipocytic differentiation (Zuk et al. (2001) supra) using human ADSC. The results 25 demonstrated that ADM hydogel at -3 mg/mL was able to induce the same level of adipogenesis as adipogenic medium. The combination of ADM and adipogenic medium added to the culture did not significantly increase the level of adipogenesis observed over media alone and indicates that when used at this concentration, ADM may activate similar pathways as adipogenic medium, or perhaps inhibit its activity. From routine screening of 30 different ADM batches, the adipogenesis of ADSC is a dose-dependent response to the amount of soluble protein present in the ADM. Assays with relatively higher Hiaarmerwove, R(Portb1DCCAARkI2293O6_doc1/32013 -37 concentrations of ADM soluble fractions result in close to 100% of cells differentiating to adipocytes compared with the 50% seen with ADM hydrogel. The lesser amount of the soluble adipogenic factor combined with its gradual diffusion out of the hydrogel may explain this difference in differentiation efficiency. In either case, the 50 to 100% 5 differentiation into adipocytes is far greater than the 1 to 6.5% preadipocytes reported to be present in human adipose tissue (Pettersson et al. (1985) Metabolism 34(9):808-812; Pettersson et al. (1984) Acta Medica Scandinavica 215(5):447-45 1). This indicates that ADM's soluble component is responsible for differentiating a large proportion of the resident stem cell population into adipocytes as opposed to merely supporting the 10 development of the existing preadipocytes. [01121 After implantation into rat subcutaneous tissue, ADM shows no signs of angiogenesis after 2 weeks in vivo, however, there are signs of angiogenesis after 8 weeks, indicating that ADM is indirectly angiogenic in that it allows for the invasion and 15 proliferation of endothelial cells following the early stages of ADM remodelling. Subcutaneous implants also showed significant signs of adipogenesis with levels approaching those seen in Matrigel controls after 8 weeks. Both ADM implants, with and without ADSC, showed a remarkable adipogenic response after 8 weeks of implantation. The rat ADSC added to the ADM implants survived and differentiated into fat, and 20 recruited new precursors as observed in the implants containing ADM without any additives. There was some physical evidence of the gel persisting in the rings to at least 8 weeks showing the potential of ongoing neoadipogenesis and confirning that the gel, as it undergoes conversion to adipose tissue, does not undergo major contraction and maintains the original space. This subcutaneous model of testing the gels allows only a qualitative 25 analysis of the results, but it is valuable as a screening tool to test various gels for future in vivo models. The ADM was not in contact with any major blood vessels nor did the gel require any supplementation or modification in order to produce adipose tissue in vivo. The ADM has a potential to be used as an injectable matrix for adipose tissue engineering as well as a control mechanism for adipogenesis. 30 H aantmyoven\NRPortbl\DCC\AAR\42293%_L do-15!03/2013 -38 [01131 Despite the lack of VEGF and some other soluble proteins, the adipogenic activity of ADM is retained after multiple salt washes. This is particularly evident with the human ADM as its adipogenic activity is similar to porcine ADM despite the extra processing. 5 [0114] Human and porcine subcutaneous adipose tissue can be processed to form ADM containing high levels of ECM proteins and basement membrane components. This decellularized matrix is able to induce the adipogenic differentiation of ADSC both in vivo and in vitro. On its own, the ADM induced significant differentiation of the resident cells to form fat, thus moving towards the establishment of a three-dimensional biological 10 scaffold for clinical applications with wide ranging applications in the tissue engineering and plastic and reconstructive surgery. TABLE2 Concentration of various growth factors as determined by ELISA 15 pgfm protein Activin A 37 TGF-p1 31 VEGF Not Detected FGF-2 45 PDGF-AB I IL-Ja Not detected [01151 Porcine adipose-derived matrix (-3 mg/mL) was analyzed for a variety of growth factors using Quantikine quantitative sandwich ELISA kits. The concentration of Activin 20 A, TGF-p1, VEGF, FGF-2, PDGF-Ap and IL-la were measured and expressed as pg/mg of total protein.

H:\aamr Jwvn\NRWortbl\DCC\AARW42293 06_1T.doc-15/503/2013 -39 TABLE3 Macroph age response to ADM IL-1I Average protein (pg/niL) Days Cells Matrigel ADM ADM 1 3.0 4.1 2.3 13 2 L2 0.4 18 L7 3 02 ND ND 0.0 4 0.2 ND ND ND MCP- Average protein (pg/nL.) Days Cells Matrigel ADM ADM (p) (hi) 1 63.0 66.5 202.8 129.6 2 38.0 353 100.2 47.3 3 32.8 173 537 34.3 4 68.4 20 1 44.6 59.3 TGF-p1 Average protein (pg/rnL) Days Cells Matrigel ADM ADNM (p) (h) 1 923.4 927.3 729.5 959 2 1055.2 817.0 727.1 895.0 3 714.4 6965 532.0 975. 4 885.2 833.3 616.8 1010.7 TNF-a Average l)rotein (pg/nL) Days Cells Matrigel ADMI ADMtI (p) (h) 1 20.7 13.9 374.5 288.0 2 6-6 11.0 68.4 42.5 3 3.3 3.1 19.7 12.3 4 5-5 6.1 7.3 10.2 5 [01161 Primary mouse macrophages were exposed to porcine (p) ADM, human (h) ADM, or Matrigeni for 4 days. Unsupplemented cells served as controls. Media was collected at each day and a selection of growth factors were assayed for by ELSA. Tests were done in duplicate and the results averaged. Concentrations below the threshold of the ELISA are 10 marked as Not Detected (ND).

-40 [0117] Those skilled in the art will appreciate that aspects of aspects described herein are susceptible to variations and modifications other than those specifically described. It is to be understood that these aspects include all such variations and modifications. These aspects also include all of the steps, features, compositions and compounds referred to or 5 indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features.

Hear\renoven\NRPortbDCCVAR4229306_ I.doc15/03/2013 -41 BIBLIOGRAPHY Abberton et al. (2008) Cells Tissues Organs 188(4):347-358 Abramoff et al (2004) Biophotonics International 1](7):36-42 Blondeel et al. (1997) Br JPlast Surg 50(5):322-330 Boschert et al. (2002) Plast Reconstr Surg 109(2):761-765 Boudreau and Weaver (2006) Cell 125(3):429-431 Chaubey and Burg (2008) Journal of Bioactive and Compatible Polymers 23(]):20-37 Chen et al. (2007) Stem Cells 25(3):553-561 Choi et al. (2009) J Control Release Choi et al. (2009) Tissue Eng Part C Methods Choi et al. (2010) Tissue Eng Part C Methods 16(3):387-396 Czarkowska-Paczek et al. (2006) JPhysiol Pharmacol 57(2):189-197 Daniels and Solursh (1991) JCell Sci 100(Pt 2):249-254 Famdale et al. (1986) Biochim Biophys Acta 883(2):173-177 Flynn and Woodhouse (2008) Organogenesis 4(4):228-235 iHaarrntenyvyenNR rtDI\DCC\AAR4229306 l.doe-15103/2013 -42 Flynn et al. (2006) JBiomed Mater Res A 79(2):359-369 Flynn et al. (2007) Biomaterials 28(26):3834-3842 Flynn et al. (2009) JBiomed Mater Res A 89(4):929-941 Gudewill et al. (1992) European Archieves of Psychiatry and Clinical Neuroscience 242(J):53-56 Hamid et al. (2010) Biomaterials 3](25):6454-6567 Heuz et al. (2001) Mol Endocrinol 15(21):2197-2210 Hirai et al. (2005) Molecular and Cellular Endorincology 232(1-2):21-26 Ingber and Folkman (1989) The Journal of Cell Biology 109(]):317-330 Inoue et al., (1983) J Cell Biol, 97:1524-1539 Jarman-Smith et al. Journal of Materials Science:Materials in Medicine 15(8):925-932 Jung et al. (2010) Mediators of Inflammation Karacal et al. (2007) JPlast .Reconstr Aesthet Surg 60(3):300-303 Kelly et al. (2006) Tissue Eng 12(7):2041-2047 Kleinman et al., (1982) Biochem 21:8188-6193 Loria et al. (1998) European Journal of Endocrinology 139(5):487-492) H baninrentvenve\Rortbl\DCC\AARk229306 1.doc-103/2O13 -43 Mikus et al. (1995) Laryngoscope 105():17-22) Mueller-Klieser, (1986) JCancer Res Clin Oncol 13: 101-122 Pettersson et al. (1984) Acta Medica Scandinavica 215(5):447-451 Pettersson et al. (1985) Metabolism 34(9):808-812 Rasband et al. (1997-2009) http://rsb.info.nih.gov/i/ US National Institutes of Health Rigamonti et al> (2011) PLOS One 6(3):e 17637 Rosenbaum-Dekel et al. (2005) Biochem Bipphys Res Commun 332(1):271-278 Rosso et al. (2004) J Cell. Physiol. 199(2):174-180 Rosso et al. (2005) J Cell. Physiol. 203(3):465-470 Samani et al. (2003) Phys. Med Biol. 48(14):2183-2198 Sharma et al. (2010) FASEB 24(7):2364-2374 Stenn et al. (1989) J Invest. Deramtol 93(2):287-290 Stillaert et al. (2007) Tissue Eng 13(9):2291-2300 Tee and Jaffe (2001) Biochem J259(Pt 1):219-226 Terranova et al., (1980) Cell 22:719-726 - 44 Tsurutani et al. (2011) Biochem Biophys Res Commun 407(]):68-73 Uriel et al. (2008) Biomaterials 29(27):3712-3719 Uriel et al. (2009) Tissue Engineering Part C: Methods 15(3):309-321 Young et al. (2010) In Press, Uncorrected Proof - Injectable hydrogel scaffold from decellularized human lpoaspirate, Acta Biomaterialia Yuhas, et al. (1977) Cancer Res 37: 3639-3643 Zamani and Brown (2010) Endocr Rev 32(3):387-403 Zaragosi et al. (2010) Diabetes; Zuk et al. (2001) Tissue Eng 7(2):211-228

Claims

1. A tissue scaffold comprising a substantially cell-free extract of adipose tissue, basement membrane proteins, collagen and growth factors, wherein the tissue scaffold gels at a temperature of from about 20"C to about 50 C and is adipogenic.

2. The scaffold of Claim 1 wherein the scaffold gels at from about 37 0 C to about 42 0 C.

3. The scaffold of Claim 2 wherein the scaffold is injectable form.

4. The scaffold of Claim 1 or 2 or 3 wherein the adipose tissue is subcutaneous adipose tissue.

5. The scaffold of Claim 4 wherein the collagen is selected from Collagen I, IV and V.

6. The scaffold of Claim 1 wherein the adipose tissue is from a human, non-human, primate, livestock animal, laboratory test animal, companion animal, avian species, reptile or amphibian.

7. The scaffold of Claim 6 wherein the adipose tissue is from a human or pig.

8. The scaffold of Claim 1 or 6 wherein the adipose tissue is autologous to a subject treated with the scaffold.

9. The scaffold of Claim I further comprising one or more exogenous cytokines, antibiotics, growth enhancers, gene expression enhancers, proliferation inhibitors and/or stem cell differentiation facilitators. -46

10. The scaffold of Claim 1 wherein the scaffold comprises Activin A, TFG- 1 and FGF-2 each at from about 10 pg/mg to about 300 pg/mg of scaffold material.

11. The scaffold of Claim 1 wherein the scaffold comprises PDGF-Ap at from about 0.3 pg/mg to about 4 pg/mg.

12. Use of the scaffold of any one of Claims I to 11 in the manufacture of a bioassay to identify the adipogenic potential of material.

13. Use of the scaffold of any one of Claims 1 to 11 in the manufacture of an in vitro assay for adipogenesis-modulating components, extracts, or cell systems, said assay comprising screening an adipose tissue preparation to identify a group of cells having a propensity for adipocyte differentiation, generating or obtaining a potential adipogenesis modulating-component or extract or cell system, seeding onto said component or extract said group of cells having a propensity for adipocytic differentiation, incubating said cells for a time sufficient for adipogenesis to occur and then screening said cells for adipocytic differentiation.

14. Use of the scaffold of any one of Claims 1 to 11 in the repair, augmentation or replacement of tissue in a subject.

15. Use of Claim 14 wherein the scaffold is autologous to the subject.

16. Use of Claim 14 wherein the scaffold is heterologous to the subject.

17. A method of generating donor vascularized tissue suitable for transplantation into a recipient, said method comprising creating a vascular pedicle comprising a functional circulatory system and having tissue or tissue extract or a component thereof impregnated, attached or otherwise associated with the vascular pedicle; associating the vascular pedicle within and/or on a support matrix; seeding the support matrix with isolated cells or pieces of tissue identified using an in vitro assay as promoting adipogenesis or cells some other - 47 useful endpoint implanting the support matrix containing the vascular pedicle into a recipient at a site where the functional circulatory system is anastomosized to a local artery or vein; and leaving the support matrix at the implantation site for a period sufficient to allow the growth of vascularized new tissue wherein the impregnated material or seeding material is selected on the basis that it promotes adipogenesis when determined by the assay comprising screening a tissue or tissue extract to identify a group of cells having a propensity for adipogenic differentiation, generating or obtaining potential adipogenesis promoting component or extract, seeding onto said component or extract a group of cells having a propensity for adipocytic differentiation, incubating said cells for a time sufficient for adipogenesis to occur and then screening said cells for adipocytic differentiation.

18. The method of Claim 17 wherein the vascular pedicle comprises attached fat or other adipose tissue or tissue comprising pre-adipocytes, adipocytes, myoblasts, fibroblasts, cardiomyocytes, keratinocytes, endothelial cells, smooth muscle cells, chondrocytes, pericytes, bone marrow-derived stromal precursor cells, embryonic, mesenchymal or haematopoietic stem cells, Schwann cells and other cells of the peripheral and central nervous system, olfactory cells, hepatocytes and other liver cells, mesangial and other kidney cells, pancreatic islet p-cells and ductal cells, thyroid cells, cells of other endocrine organs and spheroids of aforementioned cells.

19. The method of Claim 18 wherein the vascular pedicle comprises an in vitro assay for adipogenesis promoting components or extracts, said assay comprising generating or obtaining a layer of potential adipogenesis extract from muscle matrix on the surface of a receptacle, seeding onto said layer a group of cells having a propensity for adipocytic differentiation incubating said cells for a time sufficient for adipogenesis to occur and then screening said cells for adipocytic differentiation.