WO2013138864A1 - Echafaudage de tissu - Google Patents

Echafaudage de tissu Download PDF

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Publication number
WO2013138864A1
WO2013138864A1 PCT/AU2013/000291 AU2013000291W WO2013138864A1 WO 2013138864 A1 WO2013138864 A1 WO 2013138864A1 AU 2013000291 W AU2013000291 W AU 2013000291W WO 2013138864 A1 WO2013138864 A1 WO 2013138864A1
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Prior art keywords
cells
tissue
scaffold
adm
adipogenesis
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PCT/AU2013/000291
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English (en)
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Christopher James POON
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Neopec Pty Ltd
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Priority to AU2013202570A priority Critical patent/AU2013202570A1/en
Publication of WO2013138864A1 publication Critical patent/WO2013138864A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3633—Extracellular matrix [ECM]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
    • A61L27/3645—Connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L28/00—Materials for colostomy devices
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/06—Flowable or injectable implant compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/34—Materials or treatment for tissue regeneration for soft tissue reconstruction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/15—Transforming growth factor beta (TGF-β)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

Definitions

  • 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 OF THE PRIOR ART
  • adipose tissue act as a reservoir for lipids, it also provides insulation and physical protection to the 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 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.
  • ECM extracellular matrix
  • 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 differentiation of adipose-derived stem cells (ADSC).
  • ADSC adipose-derived stem cells
  • 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.
  • adipose-derived extracts 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 inducing neoadipogenesis when implanted along the rat epigastric artery vascular pedicle (Uriel et al. (2008) supra).
  • 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 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.
  • 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- ⁇ ⁇ and FGF-2.
  • the collagen includes but is not limited to Collagen I. IV and VI.
  • the material forms a gel at from about 20°C to about 50°C including under physiological conditions (37-42°C), polymerizing from a viscous liquid at 4°C to form a gel under these conditions.
  • the tissue material is also conveniently referred to herein as adipogenic matrix, adipose basement membrane matrix, adipose-derived matrix (ADM), adipogenic hydrogel and adipose scaffold.
  • ADM adipose-derived matrix
  • ADM is conveniently used for brevity with the understanding that it covers all forms of the preparation.
  • the ADM is in an injectable form.
  • 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”.
  • 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 appropriate stem cell locations.
  • ADM is useful in the study of cell growth, development and differentiation of preadipocytes.
  • ADM is useful as a base for creams and as food additives as well as therapeutically as cellular repair compositions.
  • the ADM may be derived from adipose tissue heterologous or autologous to the subject being treated.
  • 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.
  • 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 20°C to about 50°C and is adipogenic.
  • the ADM may be used alone or in conjunction or association with an implantable medical device, collapsible bag or biodegradable mesh.
  • FGF-2 Fibroblast growth factor-2
  • TNFa Tumor necrosis factor alpha
  • 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 min at 37°C.
  • 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°C protected from the light for up to 63 days.
  • 2.5 ⁇ g of 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- 1 2% w/v bis-tris gradient gel and silver-stained. Arrows indicate apparent di fferences between the porcine and human samples (B).
  • 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.
  • Figure 4 is a graphical representation showing quantitation of the adipogenic effect of ADM (hydrogel) on human ADSC.
  • FIGS 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 (B).
  • Cells were supplemented with 1 mg/mL (C) or 2.5 mg/mL of concentrated porcine ADM soluble fraction (D).
  • C cell only control
  • D adipogenic medium control
  • E 2 mg/mL
  • E 2 mg/mL
  • F 4 mg/mL soluble human ADM fraction
  • FIGS 6A through D are photographic representations of 8 week rat subcutaneous implants.
  • Matrigel A
  • Matrigel + FGF-2 B
  • ADM C
  • ADM + ADSC D
  • 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".
  • the ADM is in an injectable form.
  • the 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”.
  • the ADM is prepared from adipose tissue using a decellurization, protein 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.
  • 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.
  • the resulting ADM is a viscous liquid, polymerizing into a gel at from about 37°C to about 42°C, which is regarded herein as under physiological conditions.
  • an ADM is contemplated herein which is self-gelling from 20-50°C such as 37°C.
  • the ADM may be maintained frozen at -20°C or as a viscous liquid at about 4°C. If maintained in a frozen state, it is thawed prior to use.
  • 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- ⁇ ⁇ ), Fibroblast growth factor-2 (FGF-2) and trace amounts of platelet- derived growth factor-Abeta (PDGF- ⁇ ).
  • ADM has a range of utilities including the 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.
  • tissue scaffold 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 50"C and is adipogenic.
  • a tissue scaffold comprising a substantially cell-free extract of adipose tissue which gels under physiological conditions.
  • cell-free does not mean free of cellular components such as nucleic acids and proteins but rather substantially free of whole live cells.
  • substantially cell-free also does not exclude the possibility for a low number of cells being present.
  • low number means a number which does not cause an adverse reaction if transplanted to a subject. Reference to “low number” includes “trace cells”.
  • physiological conditions includes approximately from about 37°C to about 42°C.
  • the tissue material comprises Collagen I, IV and VI, Activin A, TGF- ⁇ and FGF2.
  • the formulation further comprises trace amounts of PDGF- ⁇ .
  • the formulation further comprises heparan sulfate proteoglycan (glycosarninoglycans).
  • the source of the tissue material may be from any animal including a mammal 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).
  • livestock animal eg. cow, sheep, pig, horse, donkey, goat, camel
  • laboratory test animal eg. mouse, rat, rabbit, guinea pigs, hamster
  • 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 amphibians sources such as frogs and toads.
  • 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 from a different subject or different animal is heterologous to be the subject.
  • composition of matter comprising an adipose preparation from an animal, the composition comprising: (i) a cell-free extract of adipose tissue; and
  • composition of matter is in injectable form.
  • the components of the tissue material may be totally derived from adipose tissue or 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.
  • additional gelling agents such as salts solutes and/or sugars
  • cytokines antibiotics
  • growth enhancers such as antibiotics
  • gene expression enhancers such as cytokines
  • proliferation inhibitors and/or stem cell differentiation facilitators may be added during preparation.
  • 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.
  • 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.
  • 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.
  • 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 37°C to about 42°C and is adipogenic.
  • Reference to a "growth factor” includes a cytokine.
  • a "cytokine” includes single or multiple cytokines selected from Activin A, TGF- ⁇ ⁇ , FGF-2 and PDGF- ⁇ . Additional cytokines may be present or included. Additional extracellular matrix material may also be present or added.
  • the polymerization generally occurs at temperatures from about 20°C to about 50°C 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 42°C.
  • 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.
  • the ADM is a viscous liquid which is not considered a gel.
  • the growth factors are present in amounts as follows:
  • Amounts may vary depending on the source of the adipose tissue.
  • VEGF and IL- l a are not detectable.
  • 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, TGF- ⁇ ⁇ and FGF-2 and from about 1.0 pg/mg to about 4 pg/mg PDGF- ⁇ , the ADM further comprising basement membrane protein and collagen and wherein the ADM is adipogenic,
  • the ADM is in a gel form at least under physiological conditions. However, the present disclosure enables gelling from 20°C to 50°C including from about 37°C to about 42°C.
  • the tissue material may be in the gel form or it may be in a "precursor" form which is polymcrizable to a gel form.
  • the cell-free tissue material when in precursor form, is reconstitutable to a gel or matrix form.
  • the matrix form of the reconstituted precursor is referred to herein as "adipose-derived matrix" or ADM.
  • the ADM disclosed herein 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.
  • 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.
  • the ADM is adipogenic.
  • ADM 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.
  • an "animal” includes a human, non-human mammal, livestock animal, companion animal or avian, reptilian or amphibian species.
  • 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.
  • ADM phosphate-buffered saline
  • Extract 1 After incubation, visible lumps of solidified lipid are removed and the extract (Extract 1 ) recovered by centrifugation and dialyzed against a tris-buffered saline.
  • An equal volume of guanidine-HCl 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 times and stored at 4°C.
  • 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 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).
  • ADM Adipose- Derived Matrix
  • the ADM is sterilized by dialysis against chloroform before being dialyzed at 4°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°C.
  • 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.
  • the ADM is suited to the culture of a variety of cells including but not limited to adipocytes.
  • 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 cells and cells from pathogenic states such as cancer and diabetes.
  • 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 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.
  • ADM is coated onto the surface and other compounds added and then the system is screened for enhanced or reduced adipogenesis.
  • cells with a potential to undergo adipogenesis differentiation are maintained 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.
  • the assay includes the generation of a three dimensional scaffold comprising a potential adipogenic component or extract, seeding cells 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 taught by the present disclosure may also provide or select or develop optimized populations of pre-adipocytes for use in tissue engineering.
  • 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.
  • 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 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.
  • the potential adipogenesis-modulating component or extract or cell system promotes adipogenesis.
  • the adipogenesis-modulating component or extract or cell system induces cellular differentiation.
  • modulating is meant increasing or decreasing, either directly or indirectly the level of adipogenesis.
  • a layer of potential adipogenesis-modulating component or extract or cell system may be obtained or generated.
  • a three-dimensional support matrix comprising the potential adipogenesis-modulating component or extract or cell system may be obtained or generated.
  • 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 adipogenesis promoting agents to the ADM.
  • 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 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 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
  • the vascular pedicle comprises attached fat or other adipose 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 kidney cells, pancreatic islet ⁇ -cells and ductal cells, thyroid cells, cells of other endocrine organs and spheroids of aforementioned cells.
  • tissue extract or a recombinant, synthetic or purified component of the tissue is associated with the vascular pedicle.
  • these components and extracts are derived from ADM and screened in vitro for adipogenic potential.
  • the ADM is allowed to set and cells capable of adipocytic differentiation plated over the monolayer of matrix in the presence of complete media (such as DMEM containing FCS) or differentiation media (complete media supplemented with ⁇ dexamethasone, insulin, indomethacin and IBMX).
  • complete media such as DMEM containing FCS
  • differentiation media complete media supplemented with ⁇ dexamethasone, insulin, indomethacin and IBMX.
  • Adipogenesis is observed over a period of from a few days to 14 days.
  • suitable adipocytic cells include 3T3- Ll cells or preadipocytic cells isolated by standard procedures.
  • the ADM is added pre-gelled to cultures.
  • 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 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.
  • the assay is conducted on a three-dimensional support 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- lieser, ( 1986) J Cancer Res Clin Oncol 13: 101 - 122), the liquid- overlay technique (Yuhas, et al. ( 1977) Cancer Res 37: 3639-3643).
  • a 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.
  • 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 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 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.
  • three dimensional matrices In order for the three dimensional structures to be able to maintain the activity of 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.
  • the biocompatibility of the matrix is not particularly important.
  • material is used which is biodegradable.
  • biomatrices are those extracted from or resembling ADM or having a cell-free system comprising same.
  • 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.
  • [0074J Frozen porcine subcutaneous adipose tissue was shaved into 1 -2 g pieces and 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 IJ/m L dispase II (Roche. Australia) 30 min in a shaking 37°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 NaCl, 50 mM tris-HCl pH 7.4, 2 mM NEM, 8 mM EDTA).
  • salt buffer 3.4 M NaCl, 50 mM tris-HCl pH 7.4, 2 mM NEM, 8 mM EDTA.
  • tissue was centrifuged (3000 G, 4°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°C. After incubation, visible lumps of solidified lipid were removed and the extract (Extract 1 ) recovered by centrifugation (3000 G, 4 Q C, 10 min) and dialyzed against tris-buffered saline (TBS - 50 mM tris-HCl pH 7.4, 150 mM NACl) at 4°C.
  • 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 lipid was removed by centrifugation ( 10,000 G, 4°C, 10 min) and the remaining digest was dialyzed against TBS at 4°C to inactivate the pepsin. The neutralized digest was combined with the concentrated liquid extract to form AdiposeDerived Matrix (ADM).
  • ADM AdiposeDerived Matrix
  • the ADM was sterilized by dialysis against chloroform to a final concentration of 0.5% v/v at 4°C before being dialyzed at 4°C with 8 M urea overnight, followed by dialysis in chilled TBS or DMEM.
  • the ADM (-3 mg/mL) was stored long-term at -20°C or for up to 2 months at 4°C.
  • 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 collected by centrifugation and combined with Extract 1 , then filtered and dialyzed in TBS, and concentrated up to four times. The leftover 4M GuHCT-extracted tissue was rinsed twice with two volumes of 70% v/v ethanol and then incubated for 30 min at 37°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 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.
  • the protein concentration of adipose extracts was measured with a bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Australia) using bovine serum albumin (BSA) as a protein standard.
  • BCA bicinchoninic acid
  • BSA bovine serum albumin
  • 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 SDS-PAGE, protein concentrations were also estimated by absorbance at 280 nm prior to electrophoresis.
  • 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): ⁇ Ti- ⁇ l1).
  • DMMB 1 ,9-dimethylmethylene blue
  • ADM proteinase K-digested prior to glycosaminoglycan (GAG) measurements at 595 nm with a reference wavelength of 655 nm.
  • Quantikine ELISA kits were used to determine the concentration of Activin A, transforming growth factor-01 (TGF- ⁇ ⁇ ), vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF-2), platelet-derived growth factor- AB (PDGF- ⁇ ), interleukin- la (IL- la), interleukin- l a (IL- l a), tumor necrosis factor-a (TNF-a), and monocyte chemotactic protein- 1 (MCP-1 ) in ADM or conditioned media. Each assay was performed according to the manufacturer's instructions and samples assayed in duplicate.
  • TGF- ⁇ ⁇ transforming growth factor-01
  • VEGF vascular endothelial growth factor
  • FGF-2 fibroblast growth factor 2
  • PDGF- ⁇ platelet-derived growth factor- AB
  • IL- la interleukin- la
  • IL- l a interleukin- l a
  • TNF-a tumor necrosis
  • protein standards and samples were added to precoated wells and allowed to incubate for at least an hour at room temperature or overnight at 4°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.
  • ADM (3 mg/mL) was dispensed into polypropylene tubes (8 x 50 ⁇ ) and stored protected from light at 4°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 course. 2.5-5 ⁇ g protein of each sample was resolved on a 4- 12% w/v SDS-PAGE gradient gel and silver-stained.
  • ADM was tested on cells either by overlaying 80% confluent cells with ⁇ 100 - 200 ⁇ I 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°C. ADM was carefully removed from the cultures before the cells were fixed for 10 min with 4% formaldehyde and stained with oil red O (Sigma-Aldrich).
  • 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 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 ⁇ .
  • Liquid gels were pipetted into 70% v/v ethanol-sterilized rings at 4°C under sterile conditions and left to solidify at 37°C for 15 minutes prior to subcutaneous implantation. Gels requiring supplementation with either FGF-2 ( 1 ⁇ g/mL) or rat ADSC (2.5x 10 4 cells per implant) were prepared immediately before pipetting into rings. ln Vivo Testing
  • thermosersitive hydrogel was prepared from subcutaneous adipose tissue using a heavily modified method based on one used to produce the skeletal muscle product, Myogel (Abberton et al. (2008) Cells Tissues Organs J 88 (4) 47 -35%), 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 4°C and polymerizing once incubated at 37°C. Complete gelation was reached after -15 min.
  • 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.
  • 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 protein Table 2). All of the growth factors assayed were of a relatively low concentration. VEGF and IL- l oc was unable to be detected and there was also very little PDGF- ⁇ detected ( 1 pg/mg). Activin A, TGF- ⁇ ⁇ , and FGF-2 were measured at concentrations of 31 -45 pg/mg.
  • Matrigel, Myoge and ADM were probed for actin, myosin, fibronectin, laminin 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 was detected in Matrigel and ADM, with ADM showing slightly more intense binding.
  • Laminin (pan-laminin al bl c l ) 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 1 00 kDa, whereas ADM showed binding over a wider range from 40 kDa - >500 kDa with a majority between 60-200 kDa.
  • ADM showed positive immunoreactivity for Collagens I, IV and VI, and displayed the most intense labeling for Collagen 1 with multiple products from -130 kDa in size.
  • 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 moderate binding for Collagen I and IV (Figure 3).
  • ADM was tested for its potential to differentiate human ADSC into adipocytes.
  • 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 (hydrogel) at the standard ⁇ 3 mg/mL concentration, were both able to induce adipogenesis to a similardegree, 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.
  • 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 el al. (2001 ) supra) was used.
  • 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 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.
  • 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.
  • the ADM enabled herein allows for adipose tissue to be replaced using a non- 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.
  • 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):2 l S3-2 ⁇ 9$), and requires significant vasculature in order to maintain healthy function.
  • Daily turnover rate for adipocytes is as high as 5% (Rigamonti et al. (201 1 ) PLOS One 6(3):e l l637).
  • ADM ADM is divided into three components: decellularization, 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 after 63 days storage at 4°C. 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 1 mg/mL showed signs of degradation after one month at 4°C.
  • 3 to 4 mm adipose-derived hydrogels are able to withstand some physical manipulation without tearing and given that no exogenous crosslinking agents are used, the gel appears to be surprisingly resilient and adequate for the applications presented these examples.
  • Dispase has the advantage of cleaving only fibronectin and Collagen IV (Stenn et al. (1989) supra) 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 not appear to result in any significant reactions when implanted.
  • ADM forms solid gels when prepared at concentrations above 1 mg/mL. The ADM has gelation properties similar to purified Collagen I. 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.
  • 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. (201 1 ) Biochem Biophys Res Commun 407(1 :68-73; Hirai et al (2005) Molecular and Cellular Endorincolog ⁇ ' 232(1 -2):2 ⁇ -26; Zaragosi et al. (2010) Diabetes; Zamani and Brown (2010) Endocr Rev 52(5 ⁇ :387-403).
  • 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.
  • 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.
  • 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 75 ⁇ :2291 -2300).
  • 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.
  • ADM was compared with Zuk's adipogenic medium, the "gold standard" for adipocytic differentiation (Zuk et al. (2001 ) supra) using human ADSC.
  • ADM 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 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 recruited new precursors as observed in the implants containing ADM without any additives.
  • 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 A, TGF- ⁇ ⁇ , VEGF, FGF-2, PDGF- ⁇ and IL- l were measured and expressed as pg/mg of total protein. TABLE 3

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Abstract

La présente invention concerne de manière générale le domaine de l'ingénierie des tissus. La présente invention concerne un échafaudage de tissu utile pour promouvoir ou faciliter la croissance, le développement et la différenciation de cellules et de tissus. L'échafaudage de tissu peut être utilisé in vitro ou in vivo dans diverses applications de l'ingénierie des tissus et dans d'autres systèmes de culture de cellule pour nourrir et enrichir entre autres les cellules adipeuses. L'échafaudage de tissu est aussi utile comme base pour des crèmes, comme pour l'utilisation dans les industries cosmétiques et thérapeutiques topiques et comme un additif dans les aliments.
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CN114984320A (zh) * 2022-05-31 2022-09-02 南开大学 一种脱细胞基质组织纸的制备方法及应用

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CN110402284B (zh) * 2016-12-16 2024-03-08 伊维瓦医疗有限公司 用于基底膜支架的薄膜插入
CN114984320A (zh) * 2022-05-31 2022-09-02 南开大学 一种脱细胞基质组织纸的制备方法及应用

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