WO2000015765A1 - Produits de construction ameliores de greffe de tissu sous-muqueux - Google Patents

Produits de construction ameliores de greffe de tissu sous-muqueux Download PDF

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Publication number
WO2000015765A1
WO2000015765A1 PCT/US1999/020996 US9920996W WO0015765A1 WO 2000015765 A1 WO2000015765 A1 WO 2000015765A1 US 9920996 W US9920996 W US 9920996W WO 0015765 A1 WO0015765 A1 WO 0015765A1
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Prior art keywords
submucosa
tissue
cells
vertebrate
intestinal
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PCT/US1999/020996
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English (en)
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WO2000015765A9 (fr
Inventor
Stephen F. Badylak
Kristina Lindberg
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Purdue Research Foundation
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Priority to CA002342589A priority Critical patent/CA2342589A1/fr
Priority to EP99945660A priority patent/EP1109888A4/fr
Priority to JP2000570292A priority patent/JP2002525043A/ja
Priority to AU58224/99A priority patent/AU766719B2/en
Publication of WO2000015765A1 publication Critical patent/WO2000015765A1/fr
Publication of WO2000015765A9 publication Critical patent/WO2000015765A9/fr

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    • 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
    • A61L27/38—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 containing added animal cells
    • A61L27/3804—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 containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3817—Cartilage-forming cells, e.g. pre-chondrocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/38—Stomach; Intestine; Goblet cells; Oral mucosa; Saliva
    • 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
    • A61L27/3629—Intestinal tissue, e.g. small intestinal submucosa
    • 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
    • A61L27/36—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
    • A61L27/3654—Cartilage, e.g. meniscus
    • 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
    • A61L27/38—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 containing added animal cells
    • A61L27/3804—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 containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3813—Epithelial cells, e.g. keratinocytes, urothelial cells
    • 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
    • A61L27/38—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 containing added animal cells
    • A61L27/3839—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 containing added animal cells characterised by the site of application in the body
    • A61L27/3843—Connective tissue
    • A61L27/3852—Cartilage, e.g. meniscus
    • 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
    • A61L27/38—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 containing added animal cells
    • A61L27/3839—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 containing added animal cells characterised by the site of application in the body
    • A61L27/3843—Connective tissue
    • A61L27/3865—Dental/periodontal tissues
    • 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
    • C12N5/0068—General culture methods using substrates
    • 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
    • C12N5/0655—Chondrocytes; Cartilage
    • 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
    • C12N5/0676—Pancreatic cells
    • 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
    • C12N5/0679—Cells of the gastro-intestinal tract
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • C—CHEMISTRY; METALLURGY
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
    • C12N2533/92—Amnion; Decellularised dermis or mucosa

Definitions

  • the present invention relates to intestinal tissue derived tissue grafts and their use in repairing damaged or diseased tissues. More particularly, this invention is directed to intestinal submucosal tissue grafts that have been seeded with a preselected population of cells to enhance the repair capabilities of the tissue graft construct.
  • the present invention is directed to vertebrate submucosa-derived collagenous matrices in combination with preselected cell population as tissue graft construct for the use in the repair of damaged or diseased tissues.
  • the collagenous matrices for use in accordance with the present invention comprise highly conserved collagens, glycoproteins, proteoglycans, and glycosaminoglycans in their natural configuration and natural concentration.
  • the extracellular collagenous matrix for use in this invention is derived from submucosal tissue of a warm-blooded vertebrate.
  • the submucosa is isolated from warm-blooded vertebrate tissues including the alimentary, respiratory, intestinal, urinary or genital tracts of warm-blooded vertebrates.
  • the preparation of intestinal submucosa is described and claimed in U.S. Patent No. 4,902,508, the disclosure of which is expressly incorporated herein by reference.
  • Urinary bladder submucosa and its preparation is described in U.S. Patent No. 5,554,389, the disclosure of which is expressly incorporated herein by reference.
  • Stomach submucosa has also been obtained and characterized using similar tissue processing techniques. Such is described in U.S. patent application No.
  • stomach submucosa is prepared from a segment of stomach in a procedure similar to the preparation of intestinal submucosa.
  • a segment of stomach tissue is first subjected to abrasion using a longitudinal wiping motion to remove the outer layers (particularly the smooth muscle layers) and the luminal portions of the tunica mucosa layers.
  • the resulting submucosa tissue has a thickness of about 100 to about 200 micrometers, and consists primarily (greater than 98%) of acellular, eosinophilic staining (H&E stain) extracellular matrix material.
  • H&E stain acellular, eosinophilic staining
  • Preferred submucosal tissues for use in accordance with this invention include intestinal submucosa, stomach submucosa, urinary bladder submucosa, and uterine submucosa.
  • Intestinal submucosal tissue is one preferred starting material, and more particularly intestinal submucosa delaminated from both the tunica muscularis and at least the tunica mucosa of warm-blooded vertebrate intestine.
  • submucosal tissue undergoes remodeling and induces the growth of endogenous tissues upon implantation into a host. It has been used successfully in vascular grafts, urinary bladder and hernia repair, replacement and repair of tendons and ligaments, and dermal grafts.
  • the preparation and use of submucosa as a tissue graft composition is described in U.S. Patent Nos. 4,902,508; 5,281,422; 5,275,826; 5,554,389; and other related U.S. patents.
  • the graft constructs When used in such applications the graft constructs appear not only to serve as a matrix for the regrowth of the tissues replaced by the graft constructs, but also promote or induce such regrowth of endogenous tissue.
  • Common events to this remodeling process include: widespread and very rapid neovascularization, proliferation of granulation mesenchymal cells, biodegradation/resorption of implanted intestinal submucosal tissue material, and lack of immune rejection.
  • the use of submucosal tissue in sheet form and fluidized forms for inducing the formation of endogenous tissues is described and claimed in U.S. Patent Nos. 5,281,422 and 5,275,826, the disclosures of which are expressly incorporated herein by reference.
  • Submucosal tissue can be obtained from various sources, including intestinal tissue harvested from animals raised for meat production, including, for example, pigs, cattle and sheep or other warm-blooded vertebrates. This tissue can be used in either its natural configuration or in a comminuted or partially digested fluidized form. Vertebrate submucosal tissue is a plentiful by-product of commercial meat production operations and is thus a low cost cell growth substrate, especially when the submucosal tissue is used in its native layer sheet configuration.
  • the submucosa tissue graft constructs prepared in accordance with the present invention are a substantially acellular matrix that provides a superior cell growth substrate resembling the matrix environment found in vivo. The natural composition and configuration of submucosal tissue provides a unique cell growth substrate that promotes the attachment and proliferation of cells.
  • compositions comprising submucosal tissue of the intestine of warm-blooded vertebrates can be used as tissue graft materials in sheet or fluidized form.
  • U.S. Patent No. 4,902,508 describes tissue graft compositions that are characterized by excellent mechanical properties, including high compliance, a high burst pressure point, and an effective porosity index. These properties allow such compositions to be used for vascular and connective tissue graft constructs. When used in such applications the preferred graft constructs serve as a matrix for the in vivo regrowth of the tissues replaced by the graft constructs.
  • U.S. Patent No. 5,275,826 describes use of fluidized forms of vertebrate submucosal tissues as injectable or implantable tissue grafts.
  • the present invention is directed to submucosa tissue graft constructs and a method of enhancing or expanding the functional properties of vertebrate submucosal tissues as an implantable or injectable tissue graft construct.
  • the improved tissue graft constructs are prepared by seeding the submucosal tissue in vitro with preselected or predetermined cell types prior to implanting or injecting the graft construct into the host.
  • the present invention is directed to an improved tissue graft construct comprising vertebrate submucosa delaminated from both the external smooth muscle layers and the luminal portions of the tunica mucosa.
  • the improvement comprises the addition of a preselected population of cells to the substantially acellular submucosa matrix.
  • the cells to be combined with the submucosa are selected based on the cell type of the intended tissue to be repaired.
  • the preselected cells comprise primary cells isolated from epithelial, endothethial or cartilage tissues.
  • the combination of the preselected population of cells with the submucosa matrix provides an improved tissue graft construct that shows surprising improved wound healing capabilities and better restoration of tissue function when compared to the use of either component alone as a therapeutic agent.
  • the composition comprising submucosa seeded with added cells can be cultured prior to the implantation of the construct into the affected region.
  • Intestinal submucosa is capable of supporting the proliferation and growth of a wide variety of cells, including primary cells that are normally difficult to culture in vitro.
  • the ability of submucosa to provide a substrate that supports the growth of such cells provides the opportunity to expand a population of cells prior to implantation into a host.
  • the submucosa is seeded with autologenous cells isolated from the patient to be treated.
  • a method and composition for supporting the proliferation and inducing tissue differentiation of eukaryotic cells cultured in vitro comprises the step of contacting eukaryotic cells, in vitro, with a vertebrate submucosa-derived collagenous matrix under conditions conducive to eukaryotic cell growth.
  • the term "contacting" as used herein with reference to cell culture is intended to include both direct and indirect contact, for example in fluid communication, of the submucosal tissue and the cultured cells.
  • condition conducive to eukaryotic cell growth refers to the environmental conditions, such as sterile technique, temperature and nutrient supply, that are considered optimal for eukaryotic cell growth under currently available cell culture procedures.
  • optimum cell culture conditions used for culturing eukaryotic cells depend somewhat on the particular cell type, cell growth conditions are generally well known in the art. However a number of differentiated cell types are still considered difficult to culture (i.e. islets of Langerhans, hepatocytes, chondrocytes, osteoblasts, etc.).
  • the collagenous matrix component of the present cell culture substrate is derived from vertebrate submucosa and comprises naturally associated extracellular matrix proteins, glycoproteins and other factors.
  • the collagenous matrix comprises intestinal submucosal tissue of a warm-blooded vertebrate.
  • the small intestine of warm-blooded vertebrates is a particularly preferred source of the cell culture substrate for use in this invention.
  • Suitable intestinal submucosal tissue typically comprises the tunica submucosa delaminated from the tunica muscularis and at least the luminal portion of the tunica mucosa.
  • the intestinal submucosal tissue comprises the tunica submucosa and basilar portions of the tunica mucosa including the lamina muscularis mucosa and the stratum compactum which layers are known to vary in thickness and in definition dependent on the source vertebrate species.
  • the preparation of submucosal tissue for use in accordance with this invention is described in U.S. Patent No. 4,902,508, the disclosure of which is expressly incorporated herein by reference.
  • a segment of vertebrate intestine preferably harvested from porcine, ovine or bovine species, but not excluding other species, is subjected to abrasion using a longitudinal wiping motion to remove the outer layers, comprising smooth muscle tissues, and the innermost layer, i.e., the luminal portion of the tunica mucosa.
  • the submucosal tissue is rinsed with saline and optionally sterilized; it can be stored in a hydrated or dehydrated state. Lyophilized or air dried submucosa tissue can be rehydrated and used in accordance with this invention without significant loss of its cell proliferative activity.
  • the submucosa component of the present invention can be sterilized, prior to the addition of the preselected cells, using conventional sterilization techniques including glutaraldehyde tanning, formaldehyde tanning at acidic pH, propylene oxide treatment, gas plasma sterilization, gamma radiation, electron beam, peracetic acid sterilization. Sterilization techniques which do not adversely affect the mechanical strength, structure, and biotropic properties of the submucosal tissue is preferred. For instance, strong gamma radiation may cause loss of strength of the sheets of submucosal tissue.
  • Preferred sterilization techniques include exposing the graft to peracetic acid, 1-4 Mrads gamma irradiation (more preferably 1-2.5 Mrads of gamma irradiation) or gas plasma sterilization; peracetic acid sterilization is the most preferred sterilization method.
  • the submucosal tissue is subjected to two or more sterilization processes. After the submucosal tissue is sterilized, for example by chemical treatment, the tissue may be wrapped in a plastic or foil wrap and sterilized again using electron beam or gamma irradiation sterilization techniques.
  • the submucosal tissue specified for use in accordance with this invention can also be in a fluidized form.
  • Submucosal tissue can be fluidized by comminuting the tissue and optionally subjecting it to protease digestion to form a homogenous solution.
  • the preparation of fluidized forms of submucosa tissue is described in U.S. Patent No. 5,275,826, the disclosure of which is expressly incorporated herein by reference.
  • Fluidized forms of submucosal tissue are prepared by comminuting submucosa tissue by tearing, cutting, grinding, or shearing the harvested submucosal tissue.
  • pieces of submucosal tissue can be comminuted by shearing in a high speed blender, or by grinding the submucosa in a frozen or freeze- dried state to produce a powder that can thereafter be hydrated with water or a buffered saline to form a submucosal fluid of liquid, gel or paste-like consistency.
  • the fluidized submucosa formulation can further be treated with a protease such as trypsin or pepsin at an acidic pH for a period of time sufficient to solubilize all or a major portion of the submucosal tissue components and optionally filtered to provide a homogenous solution of partially solubilized submucosa.
  • the viscosity of fluidized submucosa for use in accordance with this invention can be manipulated by controlling the concentration of the submucosa component and the degree of hydration.
  • the viscosity can be adjusted to a range of about 2 to about 300,000 cps at 25° C.
  • Higher viscosity formulations, for example, gels, can be prepared from the submucosa digest solutions by adjusting the pH of such solutions to about 6.0 to about 7.0.
  • compositions comprising submucosal tissue can be used for supporting growth or proliferation of eukaryotic cells in vitro.
  • Submucosal tissue can be used in accordance with this invention as a cell growth substrate in a variety of forms, including its native sheet-like configuration, as a gel matrix, as an addition for art-recognized cell/tissue culture media, or as coating for culture-ware to provide a more physiologically relevant substrate that supports and enhances the proliferation of cells in contact with the submucosal matrix.
  • the submucosal tissue provides surfaces for cell adhesion and also induces cell differentiation.
  • the submucosal tissue is preferably sterilized prior to use in cell culture applications, however nonsterile submucosal tissue can be used if antibiotics are included in the cell culture system.
  • cells are seeded directly onto sheets of vertebrate submucosal tissue under conditions conducive to eukaryotic cell proliferation.
  • the porous nature of submucosal tissue allows diffusion of cell nutrients throughout the submucosal matrix.
  • cells can be cultured on either the luminal or abluminal surface of the submucosal tissue.
  • the luminal surface is the submucosal surface facing the lumen of the organ source and typically adjacent to an inner mucosa layer in vivo
  • the abluminal surface is the submucosal surface facing away from the lumen of the organ and typically in contact with smooth muscle tissue in vivo.
  • Cells cultured on solid sheets of vertebrate submucosal tissue display a different growth pattern, and exhibit different interactions with the submucosal growth substrate, depending on which side of the submucosal sheet the cells are grown. Histological examination of tissue/cells cultured on intestinal submucosal tissue sheets in accordance with this invention reveals that cells that are seeded onto the abluminal surface not only grow/proliferate along the surface of the submucosal tissue, but they also more readily migrate into and proliferate within the submucosal tissue itself.
  • the luminal surface comprises a more dense matrix than the abluminal side and thus cells are less likely to penetrate the luminal side.
  • Cells that are seeded onto the luminal surface attach to the matrix but generally do not penetrate the surface. However certain cell types are capable of penetrating both the abluminal and luminal surfaces (eg squamous carcinoma cells and fibroblasts). In addition, certain cell types, such as fetal rat cells, when seeded on the luminal side proliferate to form a polylayer of cells. Cells of this polylayer can differentiate to perform functions characteristic of cells in vivo and indicative of their position in the polylayer.
  • cell growth substrates in accordance with the present invention are formed from fluidized forms of submucosal tissue.
  • the fluidized submucosal tissue can be gelled to form a solid or semi-solid matrix.
  • Eukaryotic cells can then be seeded directly on the surface of the matrix and cultured under conditions conducive to eukaryotic cell proliferation.
  • the cell growth substrate of the present invention can be combined with nutrients, including minerals, amino acids, sugars, peptides, proteins, or glycoproteins that facilitate cellular proliferation, such as laminin and fibronectin and growth factors such as epidermal growth factor, platelet-derived growth factor, transforming growth factor beta, or fibroblast growth factor.
  • fluidized or powder forms of submucosal tissue can be used to supplement standard eukaryotic culture media to enhance the standard media's capacity for sustaining and inducing the proliferation of cells cultured in vitro.
  • a cell culture composition for supporting growth in vitro of an eukaryotic cell population in combination with submucosal tissue of a warm-blooded vertebrate.
  • the composition comprises nutrients, and optionally growth factors required for optimal growth of the cultured cells.
  • the submucosa substrates of the present invention can be used with commercially available cell culture liquid media (both serum based and serum free).
  • proliferating cells can either be in direct contact with the submucosal tissue or they can simply be in fluid communication with the submucosal tissue.
  • the cell growth compositions of the present invention can be used to stimulate proliferation of undifferentiated stems cells as well as differentiated cells such as islets of Langerhans, hepatocytes and chondrocytes. Furthermore the described cell growth composition is believed to support the growth of differentiated cells while maintaining the differentiated state of such cells.
  • submucosal tissue is capable of inducing host tissue proliferation, remodeling and regeneration of appropriate tissue structures upon implantation in a number of micro environments in vivo (e.g., tendon, ligament, bone, articular cartilage, artery, and vein).
  • tissue in sheet form and fluidized forms for inducing the formation of endogenous tissues is described and claimed in U.S. Patent Nos. 5,281,422 and 5,275,826, the disclosures of which are expressly incorporated by reference.
  • the tissue replacement capabilities of graft compositions comprising submucosal tissue of warm-blooded vertebrates are further enhanced or expanded by seeding the tissue with various cell types, prior to implantation.
  • submucosal tissue may be seeded with endothelial cells or keratinocytes and used as a vascular graft or skin replacement, respectively.
  • the submucosal tissue is seeded with islet of langerhans cells for use as an auxiliary pancreas.
  • the submucosal tissue can be seeded with mesenchymal cells (stem cells) initially for expansion of the cell population and thereafter for implantation into a host.
  • Submucosal tissue can also serve as a delivery vehicle for introducing genetically modified cells to a specific location in a host.
  • the submucosal tissue for use in accordance with this embodiment can either be in a fluidized form or in its native solid form.
  • the graft composition can be subjected to conditions conducive to the proliferation of eukaryotic cells to further expand the population of the seeded cells prior to implantation of the graft into the host.
  • compositions comprising submucosal tissue and a proliferating cell population can be encapsulated in a biocompatible matrix for implantation into a host.
  • the encapsulating matrix can be configured to allow the diffusion of nutrients to the encapsulated cells while allowing the products of the encapsulated cells to diffuse from the encapsulated cells to the host cells.
  • Suitable biocompatible polymers for encapsulating living cells are known to those skilled in the art. For example a polylysine/alginate encapsulation process has been previously described by F. Lim and A. Sun (Science Vol. 210 pp. 908-910).
  • vertebrate submucosa itself could be used advantageously to encapsulate a proliferating cell population on a submucosal matrix in accordance with this invention for implantation as an artificial organ.
  • Submucosal tissue advantageously provides a physiological environment that supports the differentiation of cells cultured in vitro on the submucosal tissue.
  • cell culture substrates comprising submucosal tissue can be used in combination with standard cell culture techniques known to those of ordinary skill in the art, to produce tissue grafts, in vitro, for implantation into a host in need thereof.
  • the cells of such a tissue perform their proper natural function based on cell type and position within the submucosal tissue graft construct.
  • the method of forming a tissue graft in vitro comprises the steps of seeding eukaryotic cells onto a cell growth substrate comprising submucosal tissue of a warm-blooded vertebrate and culturing the cells in vitro under conditions conducive to proliferation of the eukaryotic cells.
  • a tissue graft construct wherein the cells of the tissue perform their proper natural function, allows the generation of tissue grafts from an initially small cell population that can be expanded in vitro prior to implantation.
  • an improved tissue graft construct comprises tunica submucosa delaminated from both the tunica muscularis and at least the luminal portion of the tunica mucosa of vertebrate intestinal tissue combined with a preselected population of cells.
  • the preselected population of cells includes connective tissue precursor cells.
  • Intestinal submucosa can induce the differentiation of precursor cells into cells that assist in the repair of damaged tissues.
  • submucosa seeded with a population of precursor cells can be implanted into a variety of different in vivo locations and the precursor cells will differentiate into the appropriate cell type for the environment.
  • vertebrate submucosa is combined with primary cells to form an improved vertebrate submucosa tissue graft construct.
  • the improved tissue graft construct comprises vertebrate submucosa delaminated from both the external smooth muscle layers and the luminal portions of the tunica mucosa and added primary cells. More particularly, in one embodiment the vertebrate submucosa comprises tunica submucosa delaminated from both the tunica muscularis and at least the luminal portion of the tunica mucosa of vertebrate intestinal tissue.
  • the improved graft construct of the present invention are implanted into an in vivo site in need of repair to enhance the repair of the endogenous tissues.
  • the primary cells can be selected from the group consisting of endothelial, keratinocytes, chondrocytes, epithelial and mesenchymal cells.
  • the submucosa will be in a solid form, however in an alternative embodiment the submucosa utilized is fluidized submucosa.
  • the submucosa can be fluidized by comminuting the tissue and/or digesting the submucosa with an enzyme for a period of time sufficient to solubilize the submucosa.
  • the improved tissue graft construct of the present invention comprises tunica submucosa delaminated from both the tunica muscularis and at least the luminal portion of the tunica mucosa of vertebrate intestinal tissue and a population of primary cells selected from the group consisting of endothelial cells, keratinocytes and mesenchymal cells.
  • the preselected cell type may include cells that have been genetically modified.
  • the cell may be modified by including genes that express proteins that enhance the repair of the damaged or diseased tissues.
  • the present invention further provides a method for enhancing the capabilities of a submucosa graft construct to repair articular cartilage defects.
  • the method comprises the step of seeding the vertebrate submucosa with chondrocytes prior to implanting or injecting the graft construct into a host.
  • a composition for the repair of articular cartilage defects comprises tunica submucosa delaminated from both the tunica muscularis and at least the luminal portion of the tunica mucosa of vertebrate intestinal tissue and added primary chondrocyte cells.
  • the present invention also provides a method for enhancing the capabilities of vertebrate submucosa graft construct to repair epithelial defects (such as periodontal structures or the esophagus), said method comprising the step of seeding the submucosa with primary epithelial cells prior to implanting or injecting the graft construct into a host.
  • the method of repairing these tissue can further comprising the step of subjecting the seeded graft construct to conditions conducive to the proliferation of the cells prior to implanting or injecting the graft material into the host.
  • a composition for the repair of periodontal structures or the esophagus comprises tunica submucosa delaminated from both the tunica muscularis and at least the luminal portion of the tunica mucosa of vertebrate intestinal tissue and added primary epithelial cells, and more particularly primary epithelial cells selected from the group consisting of primary gingiva epithelial cells and primary esophageal epithelial cells.
  • Example 1 Sterilization of Submucosal Tissue Because cell culture techniques must be performed under strict aseptic conditions, if antibiotics are not included in the culture system, the submucosa tissue must be prepared in a sterile manner for use as a cell culture substrate.
  • Example 2 Sterilization of Submucosal Tissue with Peracetic Acid Submucosal tissue is soaked in a peracetic acid/ethanol solution for 2 hours at room temperature using a ratio of 10: 1 (mis peracetic solution: grams submucosal tissue) or greater.
  • the peracetic acid/ethanol solution comprises 4% ethanol, 0.1% (volume: volume) peracetic acid and the remainder water.
  • the 0.1% peracetic acid component is a dilution of a 35% peracetic acid stock solution commercially available and defined as in table 1.
  • the submucosal tissue is shaken on a rotator while soaking in the peracetic acid solution.
  • the peracetic acid solution is poured off and replaced with an equivalent amount of lactated Ringer's solution or phosphate buffered saline (PBS) and soaked (with shaking) for 15 minutes.
  • PBS phosphate buffered saline
  • the submucosal tissue is subjected to four more cycles of washing with lactated Ringer's or PBS and then rinsed with sterile water for an additional 15 minutes.
  • composition % by weight
  • Small intestinal submucosa was harvested and prepared from freshly euthanatized pigs as described in U.S. Patent No. 4,902,508. Following sterilization via various techniques (gamma irradiation, peracetic acid, etc.), the submucosal tissue was clamped within a polypropylene frame to create a flat surface area (50 mm 2 ) for cell growth. The frame was submerged in culture medium to allow access of medium nutrients to both surfaces of the submucosal tissue. Various cell types were seeded (3 x 10 4 cells/submucosal tissue section) on the submucosal tissue and then placed in a 5% CO 2 , 95% air incubator at 37°C.
  • the seeded submucosal tissue was fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned (6 um).
  • Various histological and immunohisto chemical staining procedures were then applied to determine the cell growth characteristics.
  • the growth characteristics of the following cell lines have been studied using submucosal tissue as a growth substrate:
  • IMR90 Human fetal lung fibroblasts HT-29 Human colon adenocarcinoma, moderately well differentiated, grade II
  • Table 2 summarizes various cell types and the corresponding specific medium conditions used to culture on the submucosa derived cell culture substrates.
  • the medium chosen represents optimal or near optimal conditions for propagation of each cell type under standard cell culture conditions (i.e., plastic tissue culture flasks). All cell preparations were incubated at 37° C in a humidified atmosphere of 5%
  • Intestinal submucosal tissue as a growth substrate exhibits sidedness, that is, the cell/matrix interactions are different when the cells are cultured on the abluminal versus the luminal side of the intestinal submucosal tissue
  • selected cell types such as rat FR cells
  • the cells attach to the matrix surface and proliferate to form a cellular polylayer
  • FR cells when FR cells are seeded on the abluminal side, the cells not only grow along the surface but also migrate into the submucosal matrix
  • the stratum compactum of the luminal side of vertebrate intestinal submucosal tissue provides a dense connective tissue matrix and more readily supports monoiayer or polylayer formation of select cell types (i.e. endothelial and epithelial cells).
  • the abluminal side represents a more loose connective tissue structure that more readily supports migration of cells within the matrix
  • IMR-90 fibroblasts when seeded upon the abluminal or luminal sides of the intestinal submucosal tissue, quickly became adherent and proliferated throughout the matrix components. These cells illustrated their characteristic spindle shape and large vesicular nucleus within the extracellular matrix components. However, 3T3 fibroblasts showed minimal adherence and growth potential when seeded upon the intestinal submucosal tissue.
  • Endothelial cells formed a confluent monoiayer of spindle-shaped cells along the stratum compactum surface of the intestinal submucosal tissue within 3 days. At later times the monoiayer became more dense and some cells intercalated down into the matrix components. Interestingly, some endothelial cells that penetrated into the matrix components formed a lining along the lumen of structures representing original blood vessels of the native intestine.
  • Primary cell strains are cells that have been harvested from an organism and placed in culture. Subsequent passages of these cells (from 2-3 times) using standard in vitro cell culture techniques (to increase the number of cells) were frozen for later use. Each of the above listed cell strains was thawed, cultured in the presence of intestinal submucosal tissue and examined histologically. Each of the cultured cell strain populations proliferated and retained their differentiated appearance as determined by histological examination.
  • the human osteoblast cells continued to accumulate appatite crystals and respond to osteogenic stimuli such as hormones; rat cardiac muscle cells retained their contractile properties; porcine smooth muscle cells retained smooth muscle actin; and porcine endothelial cells made factor eight.
  • Example 4 Intestinal Submucosal Cell Culture Substrates as a Tumor Cell Growth Model System
  • SCC-12 human squamous cell carcinoma of the face known as SCC-12 (obtained from W. Greenlee, Purdue University) cultured in vitro were studied.
  • skin cells e.g., gamma-irradiated or mitomycin C- treated feeder layer of Swiss 3T3 mouse fibroblasts
  • a monoiayer of flattened cells is formed.
  • SCC-12 cells when seeded upon the abluminal surface of intestinal submucosal tissue, showed, upon histological examination active degradation of the submucosal matrix components and invasion of the intestinal submucosal tissue.
  • SCC-12 cells were seeded (3 x 10 4 cells/0.8 cm 2 of intestinal submucosal tissue) on either the abluminal or luminal surface of sterilized intestinal submucosal tissue and floated in growth medium consisting of DMEM containing 5% fetal calf serum, 4mM L-glutamine, and lmM sodium pyruvate. At timepoints representing 3, 7, 14, and 21 days, the growth characteristics were analyzed using standard histologic techniques.
  • the cells were strongly adherent and appeared to form a continuous layer (1-2 cells thick) along surface of the intestinal submucosal tissue. Morphologically, the cells were round and actively producing extracellular matrix products. After 7 days, a significant difference was noted in the cells' ability to invade the abluminal versus the luminal surface of the intestinal submucosal tissue. The layer of cells along the luminal surface of the intestinal submucosal tissue appeared to only increase in density. Alternatively, those cells seeded upon the abluminal surface, showed active degradation of the submucosal matrix components and penetration up to 30 um. At longer durations, there was an increasing number of cells at greater depths of penetration and a greater extent of intestinal submucosal tissue degradation. Although the SCC-12 cells actively invade intestinal submucosal tissue from both the abluminal and luminal surfaces, the observed invasion rate was greater when SCC-12 cells were placed on the abluminal side.
  • Example 5 Intestinal Submucosal Tissue Supports Cytodifferentiation FR Epithelial cells form a stratified polylayer when cultured on the luminal (stratum compactum) side of intestinal submucosal tissue. Cells adjacent to the intestinal submucosal tissue were columnar in shape and became progressively more flattened near the surface of the polylayer. After 14 days, structures resembling desmosomes were identified and the cellular layer stained positively for cytokeratin with a pan cytokeratin antibody. In addition, it appeared that the epithelial cells produced supporting matrix products (potentially basement membrane) as they do in vivo under normal healthy conditions. These findings suggest that the intestinal submucosal tissue supports natural epithelial cell maturation and differentiation processes. The observed stratification of FR cells grown on the luminal side
  • cytokeratin is a predominant intracellular structural protein produced by terminally differentiated epithelial cells known as keratinocytes.
  • Immunohisto chemistry was performed on the protease-digested, formalin-fixed, paraffin embedded sections of FR cells grown on intestinal submucosal tissue using an anti-pan cytokeratin (C2931, Sigma, St. Louis, MO) as the primary antibody.
  • Immunodetection was performed using the avidin-biotin complex (ABC) method and the Biogenex supersensitive StriAviGen kit (Vector Laboratories, Burlingame, CA). Tissue sections representing rat skin biopsies and HT29 cells grown on intestinal submucosal tissue were included in the analysis as positive and negative controls, respectively.
  • a goat anti-mouse antibody conjugated with fluorescein isothiocyanate was then applied to facilitate immunodetection.
  • the cell preparations were then analyzed on a EPICS Elite flow cytometer (Coulter Corp., Hialeah, FL) using 488 nm excitation produced by an air-cooled argon laser. Fluorescence emissions were measured at 525 nm with a bandpass filter. Untreated cells and cells treated only with GAM-FITC were also analyzed to establish background fluorescence levels. Table 3 represents the relative percentage of FITC fluorescence for each cell type following indirect immunofluorescence staining.
  • the common bile duct was cannulated under a stereomicroscope with a polyethylene catheter (PE-10 tubing; CMS; Houston, TX), through which approximately 3-4 mis of ice cold M-199 medium (commercially available from Gibco BRL) containing 0.7 mg/ml of collagenase P was injected slowly until whole pancreas was swollen.
  • the pancreas was excised and digested at 37° C for approximately 50 minutes in M-199 medium containing 100 ⁇ g/ml of penicillin G and 100 ⁇ g/ml of streptomycin (no additional collagenase).
  • Submucosal Tissue Islets of Langerhans were cultured on submucosal cell growth substrates at 37°C in an incubator supplemented with 5% CO and 95% air.
  • the islet cells were cultured in the presence of various forms of intestinal submucosal tissue using the following procedures:
  • Indirect Contact Intestinal submucosal tissue and the cultured cells are separated by a stainless steel mesh.
  • Solubilized intestinal submucosal tissue is added to the culture media
  • Cells are cultured on solubilized intestinal submucosa coated culture plate.
  • the coating was applied by placing 1 ml of solubilized intestinal submucosal tissue in a 35mm culture plate, heated at 37°C for 2 hours, removing the excess intestinal submucosal tissue fluid by aspiration and washing the coated plates once with culture media.
  • an intestinal submucosa membrane of approximately lxl cm was placed on top of stainless steel mesh with the stratum compactum side facing up. Isolated islets were then placed onto the membrane and continuously cultured in M-199 medium (commercially available from Gibco BRL) for 7 days. Cell proliferation was examined every second day under a stereomicro sco e and was compared with the control group (cultured in the absence of submucosa tissue).
  • Intestinal submucosal tissue derived cell culture substrates were sterilized by several different means: peracetic acid treatment or gamma irradiation.
  • Gamma irradiated and the native (no further treatment after isolation of the intestinal submucosal tissue) membranes can be used directly as cell culture substrates provided they have been sufficiently rehydrated with the culture media prior to the co-culture (native membranes must be cultured in the presence of antibiotics).
  • Peracetic acid sterilized membranes must first be washed to remove residual peracetic acid prior to culturing since peracetic acid residue may be toxic to the cells.
  • peracetic acid sterilized tissues were soaked in a large quality of medium for 24 hours followed by extensive washing with the same medium.
  • Solubilized forms of intestinal submucosal tissue were sterilized by dialyzing against 6.5% chloroform in either 0. IM acetic acid (AA-submucosa) or phosphate buffered saline (PBS-submucosa) for 2 hours at room temperature.
  • the exterior surface of the dialysis tubing is sterilized by rinsing the outside of the tubing with 70% alcohol prior to removal of the intestinal submucosal tissue.
  • the dialysis tubing has a molecular weight cut-off of 12,000-14,000; thus, proteins retained inside tubing are those with molecular weight greater than 14,000.
  • Islet cells can thus be isolated from pancreatic tissue and grown in vitro in contact with a cell growth substrate comprising intestinal submucosal tissue of a warm-blooded vertebrate under conditions conducive to the proliferation of the islet cells and without concurrent growth of fibroblasts. These islet cell culture compositions remain substantially free of fibroblast overgrowth.
  • Example 7 The ability of intestinal submucosa to augment wound healing and promote tissue remodeling has been previous disclosed. However, certain tissues repair slowly after tissue damage, or have been damaged to such a great extent (for example in burn patients) that submucosal tissue alone fails to provide the desired speed of recovery.
  • the combination of a preselected population of cells with the submucosa matrix has been found to enhance the repair capabilities of the submucosa tissue graft constructs.
  • the submucosa tissue is combined with primary cell cultures specific for damaged or diseased tissues to be repaired in the body.
  • the combination of the preselected population of cells with the submucosa matrix provides an improved tissue graft construct that shows surprising improved wound healing and subsequent better restoration of function when compared to the use of either component alone as a therapeutic agent.
  • Example 8 A large study recently completed in pigs shows that intestinal submucosa when used in combination with a split thickness skin graft (which essentially represents an autograft) results in improved "take" rates for the split thickness skin graft (STSG) when compared to use of the STSG alone.
  • This combination intestinal submucosa plus thin STSG fills a specific need in the treatment of full thickness skin wounds: specifically, thin (less than 0.010 inch) STSGs can be harvested and used with the intestinal submucosa carrier to treat these types of wounds instead of needing to harvest a thick (greater than 0.010 inch) STSG which leaves a deep wound at the graft harvest site with significant morbidity and subsequent scarring.
  • This type of study shows the utility of the composite intestinal submucosa plus primary epithelial cells and its superiority over the use of either alone.
  • articular cartilage As articular cartilage has a limited ability for repair, once it has been damaged by trauma or disease, it may deteriorate, resulting in an osteoarthritic joint
  • joint replacement with a prosthesis is the treatment of choice, these have a limited lifespan, and replacement of a failed implant is a difficult procedure. Consequently, biological resurfacing has been developed as a way to treat localized, or early cartilage damage in order to delay or preferably prevent the onset of osteoarthritis.
  • Porcine small intestinal submucosa is a resorbable biomaterial that upon implantation induces tissue remodeling. This material was investigated for use as a suitable substratum for the formation of cartilage in vitro, which could then be used to resurface damaged joints.
  • the submucosa—cartilaginous tissue composites were implanted into full thickness articular cartilage defects. After 4 weeks, the composites had survived and retained their hyaline-like appearance, although fibrovascular and fibrocartilaginous tissue was also present. Defects receiving intestinal submucosa alone contained predominantly fibrovascular tissue, whereas ungrafted defects were filled with fibrocartilage.
  • submucosa-cartilaginous tissue-resurfaced defects scored significantly better than submucosa-filled defects, but were no different from the unfilled defects. However, the repair tissue in the composite-filled defects scored significantly higher than that in the unfilled defects. This pilot study suggests that submucosa-cartilaginous tissue grafts may be useful for joint resurfacing.
  • chondrocytes are cultured in vitro to forma cell layer on the surface of intestinal submucosa and then implanted to support bone ingrowth while the submucosa is bioabsorbed. Such a construct promotes graft fixation without jeopardizing the integrity of the overlying cartilage.
  • Intestinal submucosa was prepared as a two-layer woven sheet and was stored at -20 °C. One week prior to use, the intestinal submucosa was soaked in three changes of antibiotics (10,000 U/ml penicillin G, 10 mg/ml streptomycin sulphate,
  • GIBCO BRL 25 ⁇ g/ml fungizone; GIBCO BRL, Burlington, Canada
  • Ham's F12 GIBCO BRL
  • a biopsy punch Premier Medical Products, Norristown, PA
  • chondrocytes the stifle joints of New Zealand White (NZW) rabbits (male, 2 kg) were opened, and the superficial portion of the articular cartilage was dissected off and discarded.
  • NZW New Zealand White
  • the remaining cartilage (lower third) was harvested and the cells isolated by sequential digestion with 0.25% proteinase (Sigma Chemical Co., St Louis, MO) for 1 h, followed by 0.1% collagenase (Collagenase A, Boehringer Mannheim, Laval, Canada) for 4 h.
  • the chondrocytes were resuspended in Ham's F12 supplemented with 5% normal rabbit serum (NRS) and plated on intestinal submucosa (2 X 10 5 cells/disc of woven sheet Intestinal submucosa).
  • the rabbit serum was obtained by intracardiac puncture of NZW rabbits (University of Toronto, Toronto, Canada), heat inactivated (60 °C for 30 min), and filter sterilized prior to being stored at -20 °C.
  • DMEM Dulbecco's modified Eagles medium
  • NRS N-reactive serum
  • AA ascorbic acid
  • the DNA content in the digests was measured using Hoescht 33258 dye (Polysciences Inc., Warrington, PA) and fluorometry using techniques known to those skilled in the art. Calf thymus DNA (Pharmacia, Montreal, Canada) in PBS was used to generate the standard curve.
  • the amount of proteoglycans was estimated by measuring the glycosaminoglycan (GAG) content of the papain digests using the dimethylmethylene blue dye (Polysciences Inc.) binding assay using techniques known to those skilled in the art. The assay was performed in 96-well plates, and the absorbance at 525 nm was measured by an automatic plate reader (Titrek Multiscan, Inter Sciences Inc., Finland). Chondroitin sulfate (Sigma) was used to generate the standard curve. All values were normalized to dry weight. The GAG and DNA content of the cartilaginous tissue formed on the intestinal submucosa was estimated by correcting for the intestinal submucosa contribution (submucosa cultured for 8 weeks in the absence of cells).
  • proteoglycans were extracted from proteoglycans (PG).
  • type I or type II collagen was determined by Western blot using antibodies reactive with type I collagen (polyclonal; Southern Biotechnology Associates Inc., Birmingham, AL) or type II collagen (monoclonal antibody CIICI; Developmental Studies Hybridoma Bank, Baltimore, MD). Reactivity was detected using affinity purified, secondary antibodies conjugated with alkaline phosphatase (BioRad, Mississauga, Canada). Nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate were added for substrate and colour reaction (GIBCO BRL).
  • NZW rabbits females, 4.5 kg were divided into two groups: seven experimental and six control.
  • the rabbits were anesthetized with Acepromazine (1 mg/kg s.c.) and Somnotol (25 mg/kg i.v.).
  • the stifle joints were exposed using a lateral longitudinal parapatellar incision, and the patella was dislocated.
  • Bilateral incisions were made in the experimental group and a unilateral incision in the control group.
  • a 4-mm diameter defect was made in the articular cartilage of the trochlear groove of the distal femur using a disposable biopsy punch in order to minimize damage to the surrounding cartilage.
  • the defect was then extended into the subchondral bone using an electric drill (Variable speed rotary drill, Sears model no. 924375).
  • the patella was reduced, and the capsule and skin were sutured closed.
  • the features assessed were (a) the tissue filling the defect (type of tissue, staining characteristics and appearance of the surface of the repair tissue) (b) fusion with the adjacent cartilage and bone, (c) filling of the defect, and (d) whether the adjacent tissue showed degenerative changes.
  • Differentiation between hyaline-like and fibrocartilaginous tissue was based on histological features and whether collagen fibers could be visualized by polarized light microscopy, which is a feature of fibrocartilage.
  • Data were analyzed statistically using the Student's t test and significance was assigned at ap value of ⁇ 0.05.
  • the intestinal submucosa-cartilaginous tissue composites were examined histologically after 3, 6, and 8 weeks in culture.
  • the chondrocytes attached to the woven intestinal submucosa, accumulated extracellular matrix, and formed cartilaginous tissue.
  • the chondrocytes were round and in lacunae.
  • the extracellular matrix stained metachromatically with toluidine blue, indicating the presence of sulfated proteoglycans. Some cells had infiltrated into the intestinal submucosa, and these were surrounded by small amounts of matrix.
  • the "as received" intestinal submucosa samples had a DNA content of 2.90 ⁇ 0.2 ⁇ g/mg dry weight (mean ⁇ SE) and a GAG content 25.35 ⁇ 5.0 ⁇ g/mg dry weight (mean ⁇ SE), whereas the intestinal submucosa analyzed after 8 weeks of culture ("post culture”) had DNA and GAG values of 2.04 ⁇ 0.1 and 8.66 ⁇ 0.6 ⁇ g/mg dry weight (mean ⁇ SE), respectively.
  • proteoglycans and type II collagen are the major macromolecules present in the matrix of cartilage
  • the proteoglycan and collagen present in the cartilaginous tissue formed in vitro was analyzed to determine whether the chondrocyte phenotype was retained under these culture conditions.
  • Agarose gel electrophoresis showed that the 8-week intestinal submucosa-cartilage cultures synthesized a population of large proteoglycans that were similar in size to those extracted from rabbit deep articular cartilage, although some of the proteoglycans were larger than those extracted from the in vivo tissue.
  • Proteoglycans extracted from intestinal submucosa were much smaller and ran as two broad bands near the bottom of the gel.
  • Pepsin extracts of 8-week intestinal submucosa-cartilaginous tissue composites were analyzed by SDS-PAGE and autoradiography. A band similar in size to the a (I) chain of type II collagen was seen. No band, corresponding to ⁇ chain of type I collagen, was present. A higher molecular weight band, likely representing dimers of the (H) chains, was also present. Western blot analysis of these extracts confirmed the presence of type II collagen. Type I collagen was detected in both the pepsin extracts of intestinal submucosa-cartilaginous tissue composites and from intestinal submucosa alone. No type II collagen was detected in the intestinal submucosa extracts by Western blot analysis.
  • Tissue (mg) (mg/mg dry weight) (mg/mg dry weight) (mg/mg dry weight)
  • a NA not applicable.
  • the dry weight, and the amount of DNA and glycosaminoglycan (GAG) were determined as described in Materials and Methods.
  • the data for the cartilaginous tissue formed in vitro were estimated by subtracting the mean dry weight or mean total DNA and GAG for intestinal submucosa after 8 weeks in culture ("post culture"), from the dry weight and total DNA and GAG respectively of each Intestinal submucosa-cartilaginous tissue sample.
  • post culture the dry weight and total DNA and GAG respectively of each Intestinal submucosa-cartilaginous tissue sample.
  • the analysis was performed on at least two different samples from each of three different experiments.
  • the data are expressed as mean ⁇ SE.
  • the defects that received intestinal submucosa grafts were filled with fibrovascular tissue. There was no or minimal amounts of fibrocartilage. If fibrocartilage was present, it was present at the edge of the defect. Residual intestinal submucosa was only seen in one defect. Although the articular cartilage surrounding the defect merged with the fibrovascular tissue filling the defect, it was possible to distinguish the margins of the articular cartilage from the reparative tissue. In contrast, the joint defects that had not received a transplant were entirely filled with fibrocartilage, which showed variable amounts of bonding to the adjacent cartilage and bone.
  • the fibrocartilage could be easily differentiated from the adjacent articular cartilage as it was more cellular, the matrix was fibrous in appearance, and collagen fibers could be visualized under polarized light.
  • the surface of the fibrocartilage showed degenerative changes with loss of proteoglycans.
  • Intestinal submucosa 1.0 ⁇ 0.7 3.8 ⁇ 0.8° None 2.6 ⁇ 0.6 d 6.2 ⁇ 0.8 a Values out of 6 points, expressed as mean ⁇ SD.
  • chondrocytes attached to Intestinal submucosa, accumulated extracellular matrix, and formed cartilaginous tissue in vitro.
  • the chondrocytes under these conditions maintain their phenotype and synthesize large proteoglycans and type II collagen, macromolecules characteristic of hyaline cartilage.
  • cartilaginous tissue formed by chondrocytes obtained from the deep zone of bovine articular cartilage that consistently showed a continuous layer of mineralization when cultured on filter inserts, the tissue on the intestinal submucosa more commonly had only focal areas of mineralization.
  • Type I collagen was detected by Western blot, but was not seen on autoradiographs suggesting that the type I Collagen was derived from the Intestinal submucosa. However, it is possible that small amounts of newly synthesized collagen were not detected by autoradiography.
  • the thickness of the cartilaginous tissue that formed on the intestinal submucosa in vitro appeared to be influenced by the method of intestinal submucosa processing (data not shown), intestinal submucosa woven into a sheet supported cartilaginous tissue formation most consistently, which is why it was used in these experiments.
  • intestinal submucosa woven into a sheet supported cartilaginous tissue formation most consistently, which is why it was used in these experiments.
  • chondrocytes As the seeding density of chondrocytes is critical for maintenance of chondrocyte phenotype and matrix synthesis, and as the nonwoven intestinal submucosa curled or provided an irregular surface, it is likely that the initial distribution of cells over the surface of the intestinal submucosa was uneven, which may explain the variability.
  • the in vitro population also contained larger proteoglycans.
  • intestinal submucosa contains fibroblast growth factor (FGF-2) and transforming growth factor ⁇ (TGF- ⁇ ) related protein, both of which have been shown to stimulate chondrocytes to synthesize larger proteoglycans. It is therefore possible that the intestinal submucosa influenced the chondrocytes to synthesize larger proteoglycans in vitro.
  • FGF-2 fibroblast growth factor
  • TGF- ⁇ transforming growth factor ⁇
  • intestinal submucosa supports the formation of cartilaginous tissue in vitro, intestinal submucosa-cartilaginous tissue grafts survive upon transplantation into a joint and are able to fuse with the adjacent cartilage.

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Abstract

Produit de construction amélioré de greffe de tissu comprenant une sous-muqueuse d'un vertébré à sang chaud et un groupe de cellules eucaryotes présélectionné. Selon la présente invention, les produits de construction améliorés de greffe de tissu peuvent être utilisés afin d'accroître la réparation in vivo de tissus endommagés ou malades.
PCT/US1999/020996 1998-09-11 1999-09-10 Produits de construction ameliores de greffe de tissu sous-muqueux WO2000015765A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CA002342589A CA2342589A1 (fr) 1998-09-11 1999-09-10 Produits de construction ameliores de greffe de tissu sous-muqueux
EP99945660A EP1109888A4 (fr) 1998-09-11 1999-09-10 Produits de construction ameliores de greffe de tissu sous-muqueux
JP2000570292A JP2002525043A (ja) 1998-09-11 1999-09-10 改良粘膜下組織グラフト構成物
AU58224/99A AU766719B2 (en) 1998-09-11 1999-09-10 Enhanced submucosal tissue graft constructs

Applications Claiming Priority (2)

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US15179098A 1998-09-11 1998-09-11
US09/151,790 1998-09-11

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WO2000015765A1 true WO2000015765A1 (fr) 2000-03-23
WO2000015765A9 WO2000015765A9 (fr) 2000-08-17

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PCT/US1999/020996 WO2000015765A1 (fr) 1998-09-11 1999-09-10 Produits de construction ameliores de greffe de tissu sous-muqueux

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EP (1) EP1109888A4 (fr)
JP (1) JP2002525043A (fr)
AU (1) AU766719B2 (fr)
CA (1) CA2342589A1 (fr)
WO (1) WO2000015765A1 (fr)

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003045277A1 (fr) 2001-11-20 2003-06-05 Artiss Gmbh Implant de trachee bioartificiel et procede de realisation associe
WO2003092381A1 (fr) 2002-05-02 2003-11-13 Purdue Research Foundation Constructions de greffe ameliorees de vascularisation
JP2003534101A (ja) * 2000-05-29 2003-11-18 アウグスチナス バデル， 生物学的人工移植片の作製方法
US6783776B2 (en) 1999-12-22 2004-08-31 Acell, Inc. Tissue regenerative composition, method of making, and method of use thereof
EP1503652A2 (fr) * 2002-05-02 2005-02-09 Purdue Research Foundation Greffons hybrides a vascularisation amelioree
EP1585426A2 (fr) * 2002-05-02 2005-10-19 Cook Biotech Incorporated Greffons de matrice extracellulaire ensemencee de cellules
US7615083B2 (en) 2002-04-09 2009-11-10 Orthowaz, Ltd. Biologically reabsorbable acetabular constraining components and materials for use with a hip replacement prosthesis and bioreabsorbable materials to augment hip replacement stability and function
US7771717B2 (en) 1995-02-10 2010-08-10 Purdue Research Foundation Enhanced submucosal tissue graft constructs
US7795022B2 (en) 2002-05-02 2010-09-14 Purdue Research Foundation Vascularization enhanced graft constructs
US8298586B2 (en) 2009-07-22 2012-10-30 Acell Inc Variable density tissue graft composition
US8409625B2 (en) 2003-06-25 2013-04-02 Acell, Inc. Conditioned decellularized native tissues for tissue restoration
US8652500B2 (en) 2009-07-22 2014-02-18 Acell, Inc. Particulate tissue graft with components of differing density and methods of making and using the same
US11478574B2 (en) 2013-10-16 2022-10-25 Purdue Research Foundation Collagen compositions and methods of use
US11739291B2 (en) 2017-04-25 2023-08-29 Purdue Research Foundation 3-dimensional (3D) tissue-engineered muscle for tissue restoration
US11919941B2 (en) 2015-04-21 2024-03-05 Purdue Research Foundation Cell-collagen-silica composites and methods of making and using the same

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997017038A1 (fr) * 1995-11-09 1997-05-15 University Of Massachusetts Regeneration de surfaces tissulaires a l'aide de compositions hydrogel-cellules
US5753267A (en) * 1995-02-10 1998-05-19 Purdue Research Foundation Method for enhancing functional properties of submucosal tissue graft constructs

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5753267A (en) * 1995-02-10 1998-05-19 Purdue Research Foundation Method for enhancing functional properties of submucosal tissue graft constructs
WO1997017038A1 (fr) * 1995-11-09 1997-05-15 University Of Massachusetts Regeneration de surfaces tissulaires a l'aide de compositions hydrogel-cellules

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DE LUCA ET AL.: "Evidence that Human Oral Epithelium Reconstituted in vitro and Transplanted onto Patients with Defects in Oral Mucosa Retains Properties of the Original Donor Site", vol. 50, no. 3, September 1990 (1990-09-01), pages 454 - 459, XP002925137 *
FREED ET AL.: "Joint resurfacing using allograft chondrocytes and synthetic biodegradable polymer scaffolds", J. BIOMEDICAL MATERIALS RESEARCH, vol. 28, August 1994 (1994-08-01), pages 891 - 899, XP002925138 *
See also references of EP1109888A4 *

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US7771717B2 (en) 1995-02-10 2010-08-10 Purdue Research Foundation Enhanced submucosal tissue graft constructs
US6861074B2 (en) 1999-12-22 2005-03-01 Acell, Inc. Tissue regenerative composition, method of making, and method of use thereof
US9265860B2 (en) 1999-12-22 2016-02-23 Acell, Inc. Tissue regenerative composition, method of making, and method of use thereof
US6783776B2 (en) 1999-12-22 2004-08-31 Acell, Inc. Tissue regenerative composition, method of making, and method of use thereof
US6849273B2 (en) 1999-12-22 2005-02-01 Acell, Inc. Tissue regenerative composition, method of making, and method of use thereof
US6869619B2 (en) 1999-12-22 2005-03-22 Acell, Inc. Tissue regenerative composition, method of making, and method of use thereof
US9433701B2 (en) 1999-12-22 2016-09-06 Acell, Inc. Extracellular matrix for the treatment of intestinal disease and methods thereof
US10092678B2 (en) 1999-12-22 2018-10-09 Acell, Inc. Extracellular matrix for the treatment of intestinal disease and methods thereof
US6893666B2 (en) 1999-12-22 2005-05-17 Acell, Inc. Tissue regenerative composition, method of making, and method of use thereof
US6852339B2 (en) 1999-12-22 2005-02-08 Acell, Inc. Tissue regenerative composition, method of making, and method of use thereof
US6887495B2 (en) 1999-12-22 2005-05-03 Acell, Inc. Tissue regenerative composition, method of making, and method of use thereof
US6890562B2 (en) 1999-12-22 2005-05-10 Acell, Inc. Tissue regenerative composition, method of making, and method of use thereof
US6890563B2 (en) 1999-12-22 2005-05-10 Acell, Inc. Tissue regenerative composition, method of making, and method of use thereof
US6890564B2 (en) 1999-12-22 2005-05-10 Acell, Inc. Tissue regenerative composition, method of making, and method of use thereof
JP2003534101A (ja) * 2000-05-29 2003-11-18 アウグスチナス バデル， 生物学的人工移植片の作製方法
WO2003045277A1 (fr) 2001-11-20 2003-06-05 Artiss Gmbh Implant de trachee bioartificiel et procede de realisation associe
US7615083B2 (en) 2002-04-09 2009-11-10 Orthowaz, Ltd. Biologically reabsorbable acetabular constraining components and materials for use with a hip replacement prosthesis and bioreabsorbable materials to augment hip replacement stability and function
AU2003231209B2 (en) * 2002-05-02 2009-08-06 Cook Biotech Incorporated Cell-seeded extracellular matrix grafts
WO2003092381A1 (fr) 2002-05-02 2003-11-13 Purdue Research Foundation Constructions de greffe ameliorees de vascularisation
EP1503652A4 (fr) * 2002-05-02 2006-02-08 Purdue Research Foundation Greffons hybrides a vascularisation amelioree
EP1585426A4 (fr) * 2002-05-02 2006-09-20 Cook Biotech Inc Greffons de matrice extracellulaire ensemencee de cellules
AU2003231248B2 (en) * 2002-05-02 2009-03-05 Purdue Research Foundation Vascularization enhanced graft constructs
EP1585426A2 (fr) * 2002-05-02 2005-10-19 Cook Biotech Incorporated Greffons de matrice extracellulaire ensemencee de cellules
EP2103684A1 (fr) * 2002-05-02 2009-09-23 Purdue Research Foundation Constructions de greffe à vascularisation améliorée
JP2005524699A (ja) * 2002-05-02 2005-08-18 パーデュー・リサーチ・ファウンデーション 血管新生が促進された移植片構成物
JP2005524426A (ja) * 2002-05-02 2005-08-18 パーデュー・リサーチ・ファウンデーション 血管新生が促進された移植片構成物
US7776596B2 (en) 2002-05-02 2010-08-17 Purdue Research Foundation Vascularization enhanced graft constructs
US7795022B2 (en) 2002-05-02 2010-09-14 Purdue Research Foundation Vascularization enhanced graft constructs
US7815686B2 (en) 2002-05-02 2010-10-19 Purdue Research Foundation Vascularization enhanced graft constructs
US8084048B2 (en) 2002-05-02 2011-12-27 Purdue Research Foundation Vascularization enhanced graft constructs
EP1503625A4 (fr) * 2002-05-02 2006-01-11 Purdue Research Foundation Constructions de greffe ameliorees de vascularisation
US9492267B2 (en) 2002-05-02 2016-11-15 Cook Biotech Incorporated Cell-seeded extracellular matrix grafts
EP1503625A1 (fr) * 2002-05-02 2005-02-09 Purdue Research Foundation Constructions de greffe ameliorees de vascularisation
EP1503652A2 (fr) * 2002-05-02 2005-02-09 Purdue Research Foundation Greffons hybrides a vascularisation amelioree
US8409625B2 (en) 2003-06-25 2013-04-02 Acell, Inc. Conditioned decellularized native tissues for tissue restoration
US9056078B2 (en) 2009-07-22 2015-06-16 Acell, Inc. Particulate tissue graft with components of differing density and methods of making and using the same
US8968761B2 (en) 2009-07-22 2015-03-03 Acell, Inc. Particulate tissue graft with components of differing density and methods of making and using the same
US8652500B2 (en) 2009-07-22 2014-02-18 Acell, Inc. Particulate tissue graft with components of differing density and methods of making and using the same
US9561307B2 (en) 2009-07-22 2017-02-07 Acell, Inc. Particulate tissue graft with components of differing density and methods of making and using the same
US8298586B2 (en) 2009-07-22 2012-10-30 Acell Inc Variable density tissue graft composition
US10898610B2 (en) 2009-07-22 2021-01-26 Acell, Inc. Particulate tissue graft with components of differing density and methods of making and using the same
US11000628B2 (en) 2009-07-22 2021-05-11 Acell, Inc. Particulate tissue graft with components of differing density and methods of making and using the same
US11013829B2 (en) 2009-07-22 2021-05-25 Acell, Inc. Particulate tissue graft with components of differing density and methods of making and using the same
US11478574B2 (en) 2013-10-16 2022-10-25 Purdue Research Foundation Collagen compositions and methods of use
US11919941B2 (en) 2015-04-21 2024-03-05 Purdue Research Foundation Cell-collagen-silica composites and methods of making and using the same
US11739291B2 (en) 2017-04-25 2023-08-29 Purdue Research Foundation 3-dimensional (3D) tissue-engineered muscle for tissue restoration

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CA2342589A1 (fr) 2000-03-23
EP1109888A1 (fr) 2001-06-27
WO2000015765A9 (fr) 2000-08-17
AU5822499A (en) 2000-04-03
EP1109888A4 (fr) 2005-02-09
AU766719B2 (en) 2003-10-23
JP2002525043A (ja) 2002-08-13

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