WO2001003750A1 - Appareil revetu d'une matrice extracellulaire naturellement secretee par l'homme - Google Patents

Appareil revetu d'une matrice extracellulaire naturellement secretee par l'homme Download PDF

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Publication number
WO2001003750A1
WO2001003750A1 PCT/US2000/018461 US0018461W WO0103750A1 WO 2001003750 A1 WO2001003750 A1 WO 2001003750A1 US 0018461 W US0018461 W US 0018461W WO 0103750 A1 WO0103750 A1 WO 0103750A1
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cells
extracellular matrix
poly
framework
stromal
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PCT/US2000/018461
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English (en)
Inventor
Gail K. Naughton
Joan Zeltinger
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Advanced Tissue Sciences, Inc.
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Priority to AU60722/00A priority Critical patent/AU6072200A/en
Priority to EP00947054A priority patent/EP1196206A1/fr
Publication of WO2001003750A1 publication Critical patent/WO2001003750A1/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
    • A61L27/3683—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • 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
    • A61L27/28—Materials for coating prostheses
    • A61L27/34—Macromolecular materials
    • 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
    • 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

Definitions

  • the present invention is directed to a human naturally secreted extracellular matrix composition, as well as methods for the production and use thereof .
  • the present invention is directed to methods of soft tissue repair by injecting a formulated human naturally secreted extracellular matrix composition into a subject in need thereof.
  • the present invention is also directed to a formulated human naturally secreted extracellular matrix composition-coated prosthetic device for implantation into a subject, preferably a human, and more particularly to a prosthetic device such as a vascular graft or stent coated with a formulated human naturally secreted extracellular matrix composition of the present invention.
  • Collagen is the principal extracellular structural protein of the animal body. At least fourteen types of mammalian collagen have been described. The common 0 characteristic amongst them is a three stranded helix, consisting of three polypeptide chains, called alpha-chains. All alpha-chains have the same configuration, but differ in the composition and sequence of their amino acids. Although this leads to different types of alpha-chains, however, they all have glycine at every third position in the amino acid sequence. The glycine at every third position allows for the helical structure of the alpha-chains.
  • Type I collagen is composed of two alpha- L -chains and one alpha 2 -chain and is the principal extracellular material of skin, tendon and bone. When clinicians mention "collagen", they are usually referring to type I collagen. See Table I, infra, for a detailed listing of collagen types I-V and in which tissues they are found.
  • Collagen has been used as an implant material to replace or augment hard or soft connective tissue, such as skin, tendon, cartilage, bone and interstitium.
  • collagen implants have been used for cosmetic purposes for a number of years since collagen can help cellular ingrowth at the placement site.
  • Early collagen implants were often solid collagen masses which were cross- linked with chemical agents, radiation or other means to improve mechanical properties, decrease immunogenicity and/or increase resistance to resorption.
  • the collagen utilized was in a variety of forms, including cross-linked and non-cross- linked fibrillar collagens, gelatins, and the like and sometimes was combined with various other components, such as lubricants, osteogenic factors and the like, depending on use.
  • a major disadvantage of solid cross-linked collagen implants is the requirement for surgical implantation by means of incision. In addition, lack of deformability and flexibility are other disadvantages of solid collagen implants .
  • U.S. Patent No. 3,949,073 describes the use of atelopeptide solutions of bovine collagen as an injectable implant material for augmenting soft tissue.
  • the bovine collagen is reconstituted before implantation and forms a fibrous mass of tissue when implanted.
  • the patent suggests adding particles of insoluble bovine collagen microfibrils to control the shrinkage of the fibrous mass formed at the augmentation site.
  • the commercial embodiment of the material described in the patent is c composed of reconstituted atelopeptide bovine collagen in saline that contains a small amount of local anesthetic. While effective, the implant shrinks in volume after implantation due primarily to absorption of its fluid component by the body.
  • U.S. Patent No. 4,803,075 describes bovine collagen compositions including a lubricant material to enhance injectability through narrow diameter needles for soft tissue repair.
  • the present invention relates to injectable materials for soft tissue augmentation and methods for use and manufacture of the same, which overcome the shortcomings 5 of bovine injectable collagen and other injectable materials, including silicone, of the prior art.
  • the injectable materials used in accordance with the present invention comprise naturally secreted extracellular matrix preparations as well as preparations derived from naturally secreted 0 extracellular matrix. These preparations are biocompatible, biodegradable and are capable of promoting connective tissue deposition, angiogenesis, reepithelialization and fibroplasia, which is useful in the repair of skin and other tissue defects. These extracellular matrix preparations may be used to repair tissue defects by injection at the site of 5 the defect .
  • the injectable preparations of the present invention have many advantages over conventional injectable collagen preparations used for the repair of skin defects.
  • the extracellular matrix preparations of the present 0 invention contain only human proteins, therefore, there is a reduced risk of an immune response due to foreign, e . g. , xenogeneic, proteins or peptides, especially the type of immune response seen with bovine collagen found in conventional injectable collagen preparations. Additionally, the injected preparations of the present invention should persist longer, and, even if multiple injections are c required, the injections should not be subject to the "no more than three injections per year" rule of bovine collagen- based preparations due to the lack of immunogenicity.
  • Another advantage provided by the present invention is that the preparations of naturally secreted extracellular matrix contain a mixture of extracellular matrix proteins that 0 closely mimics the compositions under physiologically normal conditions; for example, in an extracellular matrix derived from dermal cells, type I and III collagens, hyaluronic acid as well as various glycosaminoglycans and natural growth factors are present. Many of these extracellular matrix proteins and growth factors have been studied extensively and have been shown to be critical for wound healing and tissue restoration.
  • the present invention also relates to a prosthetic device suitable for use or implantation into a subject, preferably a human.
  • the device is coated with a formulated human naturally secreted extracellular matrix composition.
  • the device for example, is a annuloplasty ring, heart sewing ring, stent, artificial joint, artificial heart, etc.
  • the device is a suture material, gauze pad or an adhesive or non-adhesive bandage.
  • the formulated materials used in accordance with the present invention comprise human naturally secreted extracellular matrix preparations as well as preparations derived from human naturally secreted extracellular matrix. These compositions are biocompatible, biodegradable and are capable of promoting connective tissue deposition, angiogenesis, reepithelialization and fibroplasia, which is useful in the promotion of wound healing and tissue regeneration.
  • the present invention also provides new and advantageous processes for generating the extracellular matrix coated devices suitable for implantation.
  • the devices of the present invention have many c advantages over conventional devices used for wound repair or in surgery.
  • the extracellular matrix preparations of the present invention which coat or seal the device contain only human proteins, therefore, there is a reduced risk of an immune response due to foreign, e . g. , xenogeneic, proteins or peptides.
  • Another advantage provided by the present 0 invention is that the preparations of naturally secreted extracellular matrix contain a mixture of extracellular matrix proteins which closely mimics the compositions under physiologically normal conditions, for example, in an extracellular matrix derived from dermal cells, type I and 5 in collagens, hyaluronic acid as well as various glycosaminoglycans and natural growth factors are present .
  • the extracellular matrix is formulated with a drug, e . g. , an antibiotic, angiogenesis factor, other therapeutic agent., such that the implantable device coated with the formulated matrix also acts as a drug delivery system.
  • the composition is an autologous composition prepared according to the methods of the invention using cells or tissues obtained from the subject in which the device is to be implanted.
  • the matrix preparations can be used in highly improved systems of in 0 vitro tissue culture.
  • naturally secreted extracellular matrix coated three-dimensional frameworks can be used to culture cells, which require attachment to a support in order to grow, but do not attach to conventional tissue culture vessels.
  • the extracellular matrix secreted by the cells onto the framework can be collected and used to coat c vessels used in tissue culture.
  • the extracellular matrix, acting as a base substrate, may allow cells normally unable to attach to conventional tissue culture dish base substrates to attach and subsequently grow.
  • Yet another embodiment of the present invention is directed to a novel method for determining the ability for 0 cellular taxis of a particular cell.
  • the method involves inoculating one end of a naturally secreted extracellular matrix coated three-dimensional framework with the cell type in question and over time measure the distance traversed across the framework by the cell. Because the extracellular matrix is secreted naturally by the cells onto the framework, it is an excellent in vi tro substitute of extracellular matrix found in the body.
  • Such an assay may inform whether isolated tumor cells are metastatic or whether certain immune cells can migrate, or even chemotact, across the framework, thus, indicating that the cell has such cellular taxis ability.
  • Adherent Layer cells attached directly to the three-dimensional framework or connected indirectly by attachment to cells that are themselves attached directly to the matrix.
  • Naturally Secreted in context of a naturally secreted three- dimensional extracellular matrix, naturally secreted means that the extracellular matrix is secreted by cells growing in three dimensions as opposed to cells growing in monolayer culture, such that the matrix composition secreted by the cells more closely resembles the matrix as secreted by cells in vivo .
  • Pharmaceutically Acceptable Carrier an aqueous medium at physiological isotonicity and pH and may contain other elements such as local anesthetics and/or fluid lubricants.
  • Stromal Cells fibroblasts with or without other cells and/or elements found in loose connective tissue, including but not limited to, endothelial cells, pericytes, macrophages, monocytes, plasma cells, mast cells, adipocytes, chondrocytes, etc.
  • Three-Dimensional Framework a three dimensional support composed of any material and/or shape that (a) allows cells to attach to it (or can be modified to allow cells to attach to it) ; and (b) allows cells to grow in more than one layer.
  • This support is inoculated with stromal cells to form the living stromal matrix.
  • Living Stromal Tissue a three dimensional framework which has been inoculated with stromal cells. Whether confluent or subconfluent , stromal cells according to the invention continue to grow and divide.
  • the living stromal tissue prepared in vi tro is the source of the extracellular matrix proteins used in the injectable formulations of the invention.
  • FIGURE 1 is a scanning electron micrograph depicting fibroblast attachment to the three-dimensional matrix and extension of cellular processes across the mesh opening. Fibroblasts are actively secreting matrix proteins and are at the appropriate stage of subconfluency which should be obtained prior to inoculation with tissue-specific cells.
  • FIGURE 2A-D are transmission electron micrographs of collagen isolated from extracellular matrix prepared from dermal tissue grown in vi tro ( Figure 2A- B) or from a normal adult human dermal sample ( Figure 2C-D) .
  • One embodiment of the present invention involves the preparation and use of an injectable extracellular matrix composition for the treatment of skin defects.
  • the extracellular matrix proteins are derived from a living stromal tissue prepared in vi tro by growing stromal cells on a three-dimensional framework resulting in a multi-layer cell culture system.
  • cells are grown in a monolayer.
  • Cells grown on a three-dimensional framework support, in accord with the present invention grow in multiple layers, forming a cellular matrix.
  • This matrix system approaches physiologic conditions found in vivo to a greater degree than previously described monolayer tissue culture systems.
  • the three-dimensional cell culture system is applicable to the proliferation of different types of stromal cells and formation of a number of different stromal tissues, including but not limited to, dermis, bone marrow stroma, glial tissue, cartilage, etc.
  • the pre-established living stromal tissue comprises stromal cells grown on a three-dimensional framework or network.
  • the stromal cells can comprise fibroblasts with or without additional cells and/or elements described more fully herein.
  • the fibroblasts and other cells and/or elements that comprise the stroma can be fetal or adult in origin, and can be derived from convenient sources such as skin, liver, pancreas, etc. Such tissues and/or organs can be obtained by appropriate biopsy or upon autopsy. In fact, cadaver organs may be used to provide a generous supply of stromal cells and elements .
  • the stromal cells will proliferate on the framework, and elaborate growth factors, regulatory factors and extracellular matrix proteins that are deposited on the support.
  • the living stromal tissue will sustain active proliferation of the culture for long periods of time. Growth and regulatory factors can be added to the culture, but are not necessary since they are elaborated by the stromal support matrix.
  • the naturally secreted extracellular matrix is collected from the three-dimensional framework and is processed further with a pharmaceutically acceptable carrier and placed in a syringe for precise placement of the biomaterial into tissues, such as the facial dermis, or into the sac of an aneurysm.
  • a pharmaceutically acceptable carrier for precise placement of the biomaterial into tissues, such as the facial dermis, or into the sac of an aneurysm.
  • the injectable compositions are also useful for the repair of spinal and craniofacial defects.
  • the naturally secreted extracellular matrix is collected from the three-dimensional framework and is processed further for coating or sealing a prosthetic device which is suitable, e . g. , for implantation into a subject, preferably a human.
  • a prosthetic device which is suitable, e . g. , for implantation into a subject, preferably a human.
  • examples of such devices include, but are not limited to, a stent, graft, stent/graft, synthetic graft, tissue engineered vascular graft, as well as sewing and annuloplasty rings used in cardiac valve reconstruction and replacement. Further, such devices may be constructed from metal or plastic or biopolymers .
  • the extracellular matrix is used to coat or seal internal or external surgical sutures, as well as for coating Band-Aid ® -type adhesive bandages and other wound healing coverings, e. g. , non- adhesive gauze.
  • the present invention is based, in part, on the discovery that during the growth of human stromal cells on a biodegradable or non-biodegradable three-dimensional support framework, the cells synthesize and deposit on the three-dimensional support framework a human extracellular matrix as produced in normal human tissue.
  • the extracellular matrix is secreted locally by cells and not only binds cells and tissues together but also influences the development and behavior of the cells it contacts.
  • the extracellular matrix contains various fiber- forming proteins interwoven in a hydrated gel composed of a network of glycosaminoglycan c chains .
  • the glycosaminoglycans are a heterogeneous group of long, negatively charged polysaccharide chains, which (except for hyaluronic acid) are covalently linked to protein to form proteoglycan molecules .
  • the fiber-forming proteins are of two functional types: (a) mainly structural (collagens and elastin) , and (b) 0 mainly adhesive (such as fibronectin and laminin) .
  • the fibrillar collagens (types I, II, and III) are rope-like, triple-stranded helical molecules that aggregate into long cable- like fibrils in the extracellular space; these in turn can assemble into a variety of highly ordered arrays .
  • Type 5 iv collagen molecules assemble into a sheetlike meshwork that forms the core of all basal laminae.
  • Elastin molecules form an extensive cross-linked network of fibers and sheets that can stretch and recoil, imparting elasticity to the matrix.
  • Fibronectin and laminin are examples of large adhesive 0 Sflycoproteins in the matrix; fibronectin is widely distributed in connective tissues, whereas laminin is found mainly in basal laminae. By means of their multiple binding domains, such proteins help cells adhere to and become organized by the extracellular matrix.
  • a naturally secreted human dermal extracellular matrix contains type I and type III collagens, fibronectin, tenascin, glycosaminoglycans, acidic and basic
  • FGF, TGF- ⁇ and TGF- ⁇ , KGF, decorin and various other secreted human dermal matrix proteins are produced in the quantities and ratios similar to that existing in vivo .
  • growth of the stromal cells in three dimensions will sustain active proliferation of cells in culture for much longer time periods than will monolayer systems.
  • the three-dimensional system supports the maturation, differentiation, and segregation of cells in culture in vi tro to form components of adult tissues analogous to counterparts found in vivo .
  • the extracellular matrix created by the cells in culture is more analogous to native tissues.
  • the three-dimensional framework provides a greater surface area for protein attachment, and consequently, for the adherence of stromal cells.
  • stromal cells continue to actively grow in contrast to cells in monolayer cultures, which grow to confluence, exhibit contact inhibition, and cease to grow and divide. The elaboration of growth and regulatory factors by replicating stromal cells may be partially responsible for stimulating proliferation and regulating differentiation of cells in culture.
  • the increase in potential volume for cell growth in the three-dimensional system may allow the establishment of localized microenvironments analogous to native counterparts found m vivo .
  • the three-dimensional stromal support, the culture 0 system itself, and its maintenance, as well as various uses of the three-dimensional cultures and of the naturally secreted extracellular matrix are described in greater detail in the subsections below. Solely for ease of explanation, the detailed description of the invention is divided into the 5 three sections, (i) growth of the three-dimensional stromal cell culture, (ii) isolation of the naturally secreted human extracellular matrix, and (iii) formulation of the isolated extracellular matrix into preparations for injection at the site of soft tissue defects and into preparations for coating 0 or sealing a prosthetic device.
  • the three-dimensional support used to culture stromal tissue may be of any material and/or shape that:
  • (b) allows cells to grow in more than one layer.
  • non-biodegradable or biodegradable 0 materials include but are not limited to: nylon (polyamides) , dacron (polyesters) , polystyrene, polypropylene, polyacrylates, polyvinyl compounds ⁇ e . g. , polyvinylchloride) , polycarbonate
  • PVC polytetrafluorethylene
  • PTFE polytetrafluorethylene
  • TPX polymethylmethacrylate
  • polyethylene poly (ethylene terephthalate) , polyalkylene oxalates, polyurethanes, polysiloxanes, poly (dimethyl siloxane) , polycyanoacrylates, polyphosphazenes, poly (amino acids), ethylene glycol I dimethacrylate, poly(methyl methacrylate) , poly(2- hydroxyethyl methacrylate), poly(HEMA), polyhydroxyalkanoates, etc.
  • biodegradable material may also be utilized, including but not limited to: nitrocellulose, cotton, polyglycolic acid (PGA) , cat gut sutures, cellulose, gelatin, dextran, collagen, chitosan, hyaluronic acid, alginate, poly (glycollde-lactide) co- polymer, polylactic acid, poly (e-caprolactone) , poly( ⁇ - hydroxybutyrate) , polydioxanone, poly( ⁇ -ethyl glutamate) , polyiminocarbonates, poly (ortho ester), polyanhydrides, etc. Any of these materials, bio- or non-biodegradable, can be, e . g.
  • the materials can be used to form other types of three-dimensional frameworks, for example, a sponge with interconnected pores of about less than 90 to 700 microns, such as collagen sponges .
  • nylon polystyrene, etc.
  • nylon frameworks can be treated with 0.1 M acetic acid, and incubated in polylysine, FBS, and/or collagen to coat the nylon.
  • Polystyrene can be similarly treated using sulfuric acid.
  • a convenient nylon mesh which can be used in accordance with the invention is Nitex, a nylon filtration mesh having an average pore size of 210 ⁇ m and an average nylon fiber diameter of 90 ⁇ m (#3-210/36, Tetko, Inc., N.Y.).
  • Stromal cells comprising fibroblasts derived from adult or fetal tissue, with or without other cells and elements described below, are inoculated onto the framework. These fibroblasts may be derived from organs, such as skin, liver, pancreas, etc. which can be obtained by biopsy, where appropriate, or upon autopsy. In fact, fibroblasts can be obtained in quantity rather conveniently from any appropriate cadaver organ. In a preferred embodiment, fetal fibroblasts can be obtained in high quantity from foreskin.
  • Fibroblasts may be readily isolated by disaggregating an appropriate organ or tissue which is to serve as the source of the fibroblasts. This can be readily accomplished using techniques known to those skilled in the art.
  • the tissue or organ can be disaggregated mechanically and/or treated with digestive enzymes and/or chelating agents that weaken the connections between neighboring cells making it possible to disperse the tissue into a suspension of individual cells without appreciable cell breakage.
  • Enzymatic dissociation can be accomplished by mincing the tissue and treating the minced tissue with any of a number of digestive enzymes either alone or in combination.
  • Such enzymes include, but are not limited to, trypsin, chymotrypsin, collagenase, elastase, hyaluronidase, DNase, pronase, and/or dispase etc.
  • Mechanical disruption can also be accomplished by a number of methods including, but not limited to the use of grinders, blenders, sieves, homogenizers, pressure cells, or insonators to name but a few.
  • the suspension can be fractionated into subpopulations from which the fibroblasts and/or other c stromal cells and/or elements can be obtained.
  • This also may be accomplished using standard techniques for cell separation including, but not limited to, cloning and selection of specific cell types, selective destruction of unwanted cells (negative selection) , separation based upon differential cell agglutinability in the mixed population, freeze-thaw 0 procedures, differential adherence properties of the cells in the mixed population, filtration, conventional and zonal centrifugation, centrifugal elutriation (counter-streaming centrifugation) , unit gravity separation, countercurrent distribution, electrophoresis and fluorescence-activated cell 5 sorting.
  • fibroblasts for example, can be o carried out as follows: fresh tissue samples are thoroughly washed and minced in Hanks' balanced salt solution (HBSS) in order to remove serum. The minced tissue is incubated from HBSS.
  • HBSS Hanks' balanced salt solution
  • a dissociating enzyme such as trypsin.
  • the dissociated cells are suspended, pelleted by centrifugation 5 and plated onto culture dishes. All fibroblasts will attach before other cells, therefore, appropriate stromal cells can be selectively isolated and grown. The isolated fibroblasts can then be grown to confluency, lifted from the confluent culture and inoculated onto the three-dimensional framework, 0 see Naughton et al . , 1987, J. Med. 18 (3&4) : 219-250.
  • Inoculation of the three-dimensional framework with a high concentration of stromal cells e . g. , approximately 10 6 to 5 x 10 7 cells/ml, will result in the establishment of the three-dimensional stromal support in shorter periods of time.
  • fibroblasts In addition to fibroblasts, other cells can be added to form the three-dimensional stromal cell cultureproducing extracellular matrix.
  • other cells found in loose connective tissue may be inoculated onto the three-dimensional support framework along with fibroblasts.
  • Such cells include, but are not limited to, endothelial cells, pericytes, macrophages, monocytes, plasma cells, mast cells, adipocytes, chondrocytes, etc.
  • endothelial cells include, but are not limited to, endothelial cells, pericytes, macrophages, monocytes, plasma cells, mast cells, adipocytes, chondrocytes, etc.
  • These other cells can be readily derived from appropriate organs such as skin, liver, etc., using methods known, such as those discussed above .
  • stromal cells which are specialized for the particular tissue to be cultured, can be added to the fibroblast stroma for the production of a tissue type specific extracellular matrix.
  • dermal fibroblasts can be used to form the three-dimensional subconfluent stroma for the production of skin-specific extracellular matrix in vi tro .
  • stromal cells of hematopoietic tissue including, but not limited to, fibroblast endothelial cells, macrophages/monocytes, adipocytes and reticular cells, can be used to form the three-dimensional subconfluent stroma for the production of a bone marrow-specific extracellular matrix in vi tro, see infra .
  • Hematopoietic stromal cells can be readily obtained from the "buffy coat" formed in bone marrow suspensions by centrifugation at low forces, e . g. , 3000x g.
  • Stromal cells of liver may include fibroblasts, Kupffer cells, and vascular and bile duct endothelial cells.
  • glial cells can be used as the stroma to support the proliferation of neurological cells and tissues. Glial cells for this purpose can be obtained by trypsinization or collagenase digestion of embryonic or adult brain. Ponten and Westermark, 1980, In Federof, S. Hertz, L. , eds,
  • the stromal cells For certain uses in vivo it is preferable to obtain the stromal cells from the subject's own tissues.
  • the growth of cells in the presence of the three-dimensional stromal support framework can be further enhanced by adding to the framework, or coating the framework support with natural or recombinant molecules, including but not limited to, proteins, such as collagens, elastic fibers, reticular fibers, and glycoproteins; glycosaminoglycans, such as heparin sulfate, chondroitin-4 -sulfate, chondroitin-6-sulfate, dermatan sulfate, keratan sulfate, etc.; a cellular matrix, and/or other materials, such as whole blood, serum, growth factors, fibronectin, Pronectin F, RGD peptide, or cell or tissue extracts.
  • proteins such as collagens, elastic fibers, reticular fibers, and glycoproteins
  • glycosaminoglycans such as heparin
  • the three-dimensional framework is incubated in an appropriate nutrient medium under physiologic conditions favorable for cell growth, i . e . , promoting mitosis (cell division).
  • an appropriate nutrient medium under physiologic conditions favorable for cell growth, i . e . , promoting mitosis (cell division).
  • Many commercially available media such as RPMI 1640, Fisher's,
  • Iscove's, McCoy's, and the like may be suitable for use. It is important that the three-dimensional stromal culture be suspended or floated in the medium during the incubation period in order to maximize proliferative activity. In addition, the culture should be "fed” periodically to remove the spent media, depopulate released cells, and to add fresh media.
  • the stromal cells will grow linearly along and envelop the three-dimensional framework before beginning to grow into the openings of the framework.
  • the cells are grown to an appropriate degree to allow for adequate deposition of extracellular matrix proteins .
  • the openings of the framework should be of an appropriate size to allow the stromal cells to stretch across the openings. Maintaining actively growing stromal cells which stretch across the framework enhances the production of growth factors which are elaborated by the stromal cells, and hence, will support long term cultures. For example, if the openings are too small, the stromal cells may rapidly achieve confluence but be unable to easily exit from the mesh. Trapped cells can exhibit contact inhibition and cease production of the appropriate factors necessary to support proliferation and maintain long term cultures.
  • openings are too large, the stromal cells are unable to stretch across the opening. This will also decrease stromal cell production of the appropriate factors necessary to support proliferation and maintain long term cultures.
  • a mesh type of matrix as exemplified herein, it has been found that openings ranging from about 150 ⁇ m to about 220 ⁇ m will work satisfactorily. However, depending upon the three-dimensional structure and intricacy of the framework, other sizes work equally well. In fact, an Y shape or structure that allows the stromal cells to stretch and continue to replicate and grow for lengthy time periods will work in accordance with the present invention.
  • the secreted matrix contains collagen types III, IV and I in an approximate ratio of 6:3:1.
  • collagen types I and III are deposited in the matrix.
  • the proportions of collagen types deposited can be manipulated or enhanced by selecting fibroblasts which elaborate the appropriate extracellular matrix proteins. This can be accomplished using monoclonal antibodies of an appropriate isotype or subclass which are capable of activating complement, and which define particular collagen types. These antibodies in combination with complement can be used to negatively select the fibroblasts which express the desired collagen type.
  • the stroma used to inoculate the framework can be a mixture of cells which synthesize the appropriate collagen types desired. The distribution and origins of the five types of collagen is shown in Table I .
  • Lens capsule of eye Lens fibers V Fetal membranes ; Fibroblast placenta
  • the appropriate stromal cell(s) can be selected to inoculate the three-dimensional framework.
  • the three-dimensional extracellular matrix producing culture of the present invention affords a vehicle for introducing gene products in vivo .
  • the cells may be genetically engineered to express the gene product, or altered forms of the gene product that are immobilized in the extracellular matrix laid down by the stromal cells.
  • a gene of interest can be placed under the control of an inducible promoter.
  • the recombinant DNA construct containing the gene can be used to transform or transfect a host cell which is cloned and c then clonally expanded in the three-dimensional culture system.
  • the use of the three-dimensional culture in this regard has a number of advantages. First, since the culture comprises eukaryotic cells, the gene product will be properly expressed and processed in culture to form an active product.
  • the number of transfected cells can be substantially 0 enhanced to be of clinical value, relevance, and utility.
  • the three-dimensional cultures of the present invention allow for expansion of the number of transfected cells and amplification (via cell division) of transfected cells.
  • the expression control elements used should allow for the regulated expression of the gene so that the product can be over synthesized in culture.
  • the transcriptional promoter chosen generally, and promoter elements specifically, depends, in part, upon the type of tissue and cells cultured. Cells and tissues which are capable of secreting proteins (e . g. , those characterized by abundant rough endoplasmic reticulum and golgi complex) are preferable .
  • proliferating cells are released from the framework. These released cells can stick to the walls of the culture vessel where they can continue to proliferate and form a confluent monolayer. This should be prevented or minimized, for example, by removal of the released cells during feeding, or by transferring the three-dimensional framework to a new culture vessel .
  • the presence of a confluent monolayer in the vessel will "shut down" the growth of cells in the three-dimensional framework and/or culture. Removal of the confluent monolayer or transfer of the stromal culture to fresh media in a new vessel will restore proliferative activity of the three-dimensional culture system. Such removal or transfers should be done in any culture vessel which has a stromal monolayer exceeding 25% confluency.
  • the culture system can be agitated to prevent the released cells from sticking, or instead of periodically feeding the cultures, the culture system could be set up so that fresh media continuously flows through the system.
  • the flow rate can be adjusted to both maximize proliferation within the three-dimensional culture, and to wash out and 0 remove cells released from the matrix, so that they will not stick to the walls of the vessel and grow to confluence.
  • the released stromal cells can be collected and crypreserved for future use.
  • fibroblasts Once inoculated onto the three-dimensional framework, adherence of the fibroblasts is seen quickly (e . g. , within hours) and the fibroblasts begin to stretch across the framework openings within days. These fibroblasts are metabolically active, secrete extracellular matrix and rapidly form a dermal equivalent consisting of active fibroblasts and collagen.
  • Figure 1 illustrates the ability of the fibroblasts to arrange themselves into parallel layers between the naturally-secreted collagen bundles. These fibroblasts exhibit a rapid rate of cell division and protein secretion.
  • the cells After the cells have been inoculated onto the framework and cultured under conditions favoring cellular growth, such that a desired amount of extracellular matrix is secreted on to the three-dimensional framework, the cells are killed and the naturally secreted extracellular matrix is processed further.
  • This process is carried out in a number of different ways.
  • the cells can be c killed by flash-freezing the living stromal tissue prepared in vi tro in liquid nitrogen without a cryopreservative .
  • Another way to kill the cells is to irrigate the inoculated three-dimensional framework with sterile water, such that the cells burst in response to osmotic pressure.
  • a mild detergent rinse such as EDTA, CHAPS or a zwitterionic detergent
  • a cryoprotectant such as DMSO, propylene glycol, butanediol, raffinose, polyvinyl pyrrolidone, dextran or sucrose and vitrified in liquid nitrogen.
  • the framework can be subjected to enzymatic digestion and/or extracting with reagents that break down the cellular membranes and allow removal of cell contents.
  • reagents include non- ionic detergents (for example, TRITON X-100, octylphenoxy polyethoxyethanol , (Rohm and Haas); BRIJ-35, a polyethoxyethanol lauryl ether (Atlas Chemical Co.), TWEEN
  • Enzymes can be used also and can include nucleases (for example, deoxyribonuclease and ribonuclease) , phospholipases and Upases.
  • the collection of the naturally secreted 0 human extracellular matrix can be accomplished in a variety of ways which depend on whether the three-dimensional framework is composed of material that is biodegradable or non-biodegradable. For example, if the framework is composed of non-biodegradable material, one can remove the extracellular matrix from a non-biodegradable support by subjecting the three-dimensional framework to sonication, or to high pressure water jets, or to mechanical scraping, or to a mild treatment with detergents and/or enzymes to remove the attached extracellular matrix from the framework.
  • the extracellular matrix is deposited on a biodegradable three-dimensional framework, after killing and removing the cells and cellular debris, the extracellular matrix can be recovered, for example, by simply allowing the framework to degrade in solution, i . e . , allow the framework to dissolve, thus freeing the extracellular matrix.
  • the biodegradable support is composed of a material which can be injected, like the extracellular matrix itself, one can process the entire extracellular matrix coated framework into syringes for injection or process the framework for coating or sealing a prosthetic device.
  • the matrix can be removed by the same methods as if the matrix had been deposited on a non- biodegradable support, i . e . , by subjecting the three- dimensional framework to sonication, or to high pressure water jets, or to mechanical scraping, or to a mild treatment with detergents and/or enzymes to remove the attached 5 extracellular matrix from the framework. None of the removal processes are designed to damage and/or denature the naturally secreted human extracellular matrix produced by the cells .
  • extracellular matrix can be homogenized to fine particles, such that it can pass through a surgical needle. Homogenization is well known in the art, for example, by sonication.
  • the extracellular matrix can be native, i.e., not cross-linked, or the extracellular matrix can be cross-
  • the matrix is cross-linked by gamma irradiation without the use of chemical cross-linking agents, such as glutaraldehyde, which are toxic.
  • the gamma irradiation should be a minimum of 20 M rads to sterilize the material since all bacteria, fungi, and viruses are destroyed at 0.2 M
  • the extracellular matrix can be irradiated from 0.25 to 2 M rads to sterilize and cross-link the extracellular matrix.
  • the amounts and/or ratios of the collagens and other proteins may be adjusted by mixing extracellular 30 matrices secreted by other cell types prior to placing the material in a syringe.
  • biologically active substances such as proteins and drugs
  • exemplary biologically active substances can include tissue growth factors, such as TGF- ⁇ , and the c like which promote healing and tissue repair at the site of the injection.
  • tissue growth factors such as TGF- ⁇ , and the c like which promote healing and tissue repair at the site of the injection.
  • genetically engineered cells which express a biologically active substance can be incorporated into the compositions of the present invention.
  • Final formulation of the aqueous suspension of naturally secreted human extracellular matrix will typically 0 involve adjusting the ionic strength of the suspension to isotonicity (i.e., about 0.1 to 0.2) and to physiological pH (i.e., about pH 6.8 to 7.5) and adding a local anesthetic, such as lidocaine, (usually at a concentration of about 0.3% by weight) to reduce local pain upon injection.
  • the final 5 formulation will also typically contain a fluid lubricant, such as maltose, which must be tolerated by the body.
  • Exemplary lubricant components include glycerol, glycogen, maltose and the like.
  • Organic polymer base materials such as polyethylene glycol and hyaluronic acid as well as non- 0 fibrillar collagen, preferably succinylated collagen, can also act as lubricants.
  • Such lubricants are generally used to improve the injectability, intrudability and dispersion of the injected biomaterial at the site of injection and to decrease the amount of spiking by modifying the viscosity of the compositions.
  • This final formulation is by definition the processed extracellular matrix in a pharmaceutically acceptable carrier.
  • “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum oil such as mineral oil, vegetable oil such as peanut oil, soybean oil, and c sesame oil, animal oil, or oil of synthetic origin. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly. for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol 0 monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the therapeutic composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of 5 solutions, suspensions, emulsion, powders, sustained-release formulations and the like.
  • the composition can be formulated with traditional binders and carriers such as triglycerides .
  • compositions contain a therapeutically effective amount of the therapeutic composition, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.
  • suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin.
  • Such o compositions contain a therapeutically effective amount of the therapeutic composition, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.
  • the formulation should suit the mode of administration.
  • the formulated matrix and carrier composition is 5 subsequently placed in a syringe or other injection apparatus for precise placement of the matrix at the site of the tissue defect.
  • injectable means the formulation can be dispensed from syringes having a gauge as low as 25 under normal 0 conditions under normal pressure without substantial spiking.
  • the maximum particle size that can be extruded through such needles will be a complex function of at least the following: particle maximum c dimension, particle aspect ratio (length: width) , particle rigidity, surface roughness of particles and related factors affecting particle :particle adhesion, the viscoelastic properties of the suspending fluid, and the rate of flow through the needle. Rigid spherical beads suspended in a
  • Newtonian fluid represent the simplest case, while fibrous or 0 branched particles in a viscoelastic fluid are likely to be more complex.
  • the above described steps in the process for preparing injectable naturally secreted human extracellular matrix are preferably carried out under sterile conditions 5 using sterile materials.
  • the processed extracellular matrix in a pharmaceutically acceptable carrier can be injected intradermally or subcutaneously to augment soft tissue, to repair or correct congenital anomalies, acquired defects or cosmetic defects.
  • anomalies or defects 0 include but are not limited to congenital anomalies as hemifacial microsomia, malar and zygomatic hypoplasia, unilateral mammary hypoplasia, pectus excavatum, pectoralis agenesis (Poland's anomaly) and velopharyngeal incompetence secondary to cleft palate repair or submucous cleft palate
  • post-traumatic, post-surgical, post-infectious such as depressed scars, subcutaneous atrophy (e . g. , secondary to discoid lupis erythematosus) , keratotic lesions, enophthalmos in the unucleated eye (also superior sulcus syndrome) , acne pitting of the face, linear scleroderma with subcutaneous atrophy, saddle-nose deformity, Romberg's disease and unilateral vocal cord paralysis, post-surgical incisions including amputations; and cosmetic defects such as glabellar frown lines, deep nasolabial creases, circum-oral geographical wrinkles, sunken cheeks and mammary hypoplasia.
  • compositions of the present invention can be used to correct abnormal skin pigmentation, or encourage hair growth and/or retention, or to fill in skin biopsy areas to minimize scar formation.
  • the processed extracellular matrix compositions of the present invention can also be injected into internal tissues, such as the tissues defining body sphincters, e . g. , to augment such tissues.
  • Such internal tissues also include arteries and veins, such that the compositions can be used to fill in the sac of an aneurysm, or cardiac chamber, or to plug a hole in a blood vessel wall made by, for example, the insertion of a stent, catheter or angioplasty device.
  • compositions of the present invention can be used to fill in the remaining sac space to provide further structural integrity.
  • the collected naturally secreted extracellular matrix can also be processed for use in coating or sealing a prosthetic device, for example, an implantable device such as a stent.
  • the extracellular matrix can be homogenized to fine particles, such that it can be processed into a slurry or a suspension. Homogenization is well known in the art, for example, by sonication.
  • the extracellular matrix can be cross-linked by any method known in the art. In a preferred embodiment, the extracellular matrix can be cross- linked by gamma irradiation without the use of chemical cross-linking agents, such as glutaraldehyde, which are toxic. The presence of crosslinks aids in preserving the integrity of the coating and rendering it resistant to indigenous enzymes which would otherwise weaken it .
  • the gamma irradiation should be a minimum of 20 M rads to sterilize the material since all bacteria, fungi, and viruses are destroyed at 0.2 M rads.
  • the extracellular matrix can be irradiated from 0.25 to 2 M rads to sterilize and cross-link the extracellular matrix.
  • the amounts and/or ratios of the collagens and other proteins may be adjusted by mixing extracellular matrices secreted by other cell types prior to coating the material onto a device.
  • Biologically active substances such as proteins and drugs, can also be incorporated in the compositions of the present invention for release or controlled release of such active substances after implantation of the device.
  • Exemplary biologically active substances include tissue growth factors, such as ⁇ -FGF, ⁇ - FGF, NGF, EGF, HGF, TGF- ⁇ , angiogenesis factors, e . g. , VEGF, and the like, which promote healing and tissue repair at the site of placement of the device.
  • the extracellular matrix compositions can also be formulated with live cells, including mesenchymal stem cells or genetically engineered cells, to provide factors that, e . g. , facilitate healing and repair.
  • Final formulation of the slurry or aqueous suspension of naturally secreted human extracellular matrix can involve adjusting the ionic strength of the suspension to isotonicity (i.e., about 0.1 to 0.2) and to physiological pH (i . e . , about pH 6.8 to 7.5) .
  • the final formulation will also typically contain a fluid lubricant, such as maltose, which allows for ease of applying the matrix composition to the device by spraying.
  • Exemplary lubricant components include glycerol, glycogen, maltose and the like.
  • Organic polymer base materials such as polyethylene glycol and hyaluronic acid as well as non-fibrillar collagen, preferably succinylated collagen, can also act as lubricants.
  • Such lubricants are generally used to improve the dispersion of the matrix composition onto the device and to decrease the amount of spiking by modifying the viscosity of the compositions.
  • This final formulation is by definition the processed extracellular matrix in a pharmaceutically acceptable carrier.
  • the composition can be lyophilized and processed as a powder. Based on the teaching of the present invention, the exact formulation may be determined empirically by one of skill in the art .
  • the term "pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum oil such as mineral oil, vegetable oil such as peanut oil, soybean oil, and sesame oil, animal oil, or oil of synthetic origin. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the therapeutic composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, powders, and the like.
  • the composition can be formulated with traditional binders and carriers such as triglycerides . Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions contain an effective amount of the matrix composition, c together with a suitable amount of carrier so as to provide the proper physical form for application to a device.
  • the matrix composition is formulated in accordance with routine procedures as a composition adapted for application to suture.
  • Such exemplary formulations include, but are not limited to, an 0 ointment or salve or other viscous solution.
  • the matrix composition is formulated in accordance with routine procedures as a composition adapted for application to a synthetic vascular graft.
  • the extracellular 5 matrix is formulated with a hydrogel polymer.
  • the matrix is mixed with a hydrogel, or in another aspect, placed on the surface of a hydrogel, before the hydrogel is placed at a desired site within or on a subject, and subsequently polymerized, either in si tu or in 0 vi tro .
  • hydrogel polymers are well known in the art and are described in, for example, International Patent
  • the metal screen is coated with a matrix/hydrogel composition and polymerized.
  • the matrix/hydrogel is polymerized and placed on top of the screen.
  • a matrix/hydrogel composition is used instead of the metal screen.
  • cells including genetically modified cells, and drugs and/or growth factors can be added to the matrix/hydrogel composition before polymerization to facilitate healing.
  • the matrix/hydrogel composition is added directly to the excised area of the skull and polymerized in situ.
  • the matrix/hydrogel composition is added to a mold in the shape of the excised piece of skull, polymerized, and then implanted into the excised site.
  • the matrix/hydrogel compositions are also useful in creating fusions of adjacent vertebrae of the spine.
  • the matrix/hydrogel composition and cells, drugs and/or growth factors are polymerized together in a mold, and the polymerized composition is used in addition to, or as a replacement of, the screen.
  • the above described steps for preparing naturally secreted human extracellular matrix compositions for coating a device are preferably carried out under sterile conditions using sterile materials.
  • the processed extracellular matrix can be applied to at least one surface of a prosthetic device by any method known in the art, such as, e . g. , spraying, dipping, etc.
  • Exemplary devices that can be coated or sealed with a formulated human naturally secreted extracellular matrix of the present invention include, but are not limited to, a metal implant, such as a metal hip joint, cranial plate, orthopedic plate, pin or screw, as well as an orthodontic pin.
  • the device can also be the exterior of an artificial heart.
  • the device can also be an artificial tendon or ligament.
  • the device can also be a vascular plug which is used to plug a hole in a blood vessel wall made by, for example, the insertion of a stent, catheter or angioplasty device.
  • exemplary devices are a stent or stent/graft, or a commercial synthetic vascular graft or a biologic vascular graft. Any stent, stent/graft or tissue engineered vascular graft ("tubes") known in the art can be coated or sealed with the matrix compositions of the present invention.
  • the tubes can be metallic, or made from a biocompatible 10 polymer, as well as a biodegradable polymer, such as, e . g. , dacron polyester, poly (ethylene terephthalate) , polycarbonate, polymethylmethacrylate, polypropylene, polyalkylene oxalates, polyvinylchloride, polyurethanes, polysiloxanes, nylons, poly (dimethyl siloxane) ,
  • a biodegradable polymer such as, e . g. , dacron polyester, poly (ethylene terephthalate) , polycarbonate, polymethylmethacrylate, polypropylene, polyalkylene oxalates, polyvinylchloride, polyurethanes, polysiloxanes, nylons, poly (dimethyl siloxane) ,
  • polycyanoacrylates polyphosphazenes, poly(amino acids), ethylene glycol I dimethacrylate, poly(methyl methacrylate), poly (2-hydroxyethyl methacrylate), poly(HEMA), polyhydroxyalkanoates, polytetrafluorethylene, polycarbonate, poly (glycolide-lactide) co-polymer, polylactic acid, poly(e-
  • caprolactone poly ( ⁇ -hydroxybutyrate) , polydioxanone, poly( ⁇ -ethyl glutamate) , polyiminocarbonates, poly (ortho ester) , polyanhydrides, alginate, dextran, chitin, cotton, polyglycolic acid, polyurethane, or derivatized versions thereof, i.e., polymers which have been modified to include, for example, attachment sites or cross-linking groups in
  • the tubes can also be fabric- coated metal structures.
  • the tubes can also be made from combinations of metal and polymer.
  • the tubes can be 30 configured into any desired shape or conformation, such as, for example, linear, tapered, bifurcated, etc., and may be prepared using fiber technology, such as, e . g. , crimped, woven, knitted, velour, double velour, with or without coils.
  • the tubes can also be prepared by chemical extrusion, casting or molding using, for example, porous materials having linear or random pores that are circular or geometric in shape.
  • the tubes can also be prepared using living three-dimensional stromal tissues, as described in U.S. Patent Nos. 4,963,489;
  • a tissue engineered vascular graft which can be coated with a extracellular matrix composition of the invention, is prepared according to the method described in U.S. Patent
  • FIG. 1 A xenogeneic heart valve obtained from a bovine source is coated with the extracellular matrix of the present invention such that infiltration and colonization of host cells now occurs.
  • a sewing ring can also be coated or sealed with a human naturally secreted extracellular matrix composition in accordance with the present invention such that wound healing is facilitated and cellular migration and infiltration is promoted, which in turn reduces scar tissue formation.
  • Other types of artificial heart valves that can be coated with the matrix composition of the present invention are described in International Patent Publication WO 96/08213, published March 21, 1996.
  • the matrix compositions of the present invention can be applied to a gauze pad or a gauze pad on an adhesive bandage to promote wound healing.
  • the matrix compositions can also be applied to suture material (thread) for internal and external stitches, as well as being added to cosmetics, cuticle cream for nails, shampoo or conditioner for hair, body lotion, lip balm, antibiotic gels, ointments, and added to powders .
  • the matrix can be applied to the device by spraying at least one surface of the device with the matrix in suspension, and allowing the applied surface to dry.
  • the device can be dipped into such a suspension, or by casting a suspension of the matrix over the device, or by layering a device the a suspension of the matrix over the device, or by impregnating a device with a suspension of the extracellular matrix.
  • a formulated extracellular matrix is applied to the outside surface of a tube.
  • the growth factors and proteins present in the extracellular matrix promote host cell integration, wound healing and/or angiogenesis .
  • a formulated extracellular matrix is applied to the inside surface of a tube.
  • the growth factors and proteins present in the c matrix promote reendothelialization of the lumen wall, promote wound healing and/or prevent one or more cardiovascular disease states, such as stenosis, restenosis or intimal and neointimal hyperplasias .
  • the extracellular matrix is applied to both the outside and the inside surfaces of the device.
  • a space is provided between the coating and the original prosthetic device.
  • This can be accomplished by using a mold which has been coated with the extracellular matrix suspension, which mold is slightly larger than the prosthetic device to be coated.
  • a model prosthesis is enhanced in size by coating of, for example, paraffin.
  • the enlarged prosthesis is then used as a model for a two- piece mold of casting resin which can be split away into the two halves after solidifying around the enlarged prosthesis.
  • the mold is then washed and each half supplied with a sufficient amount of the matrix suspension so that when a new prosthesis is inserted and the mold resealed around it, the matrix suspension spreads over the entire surface to form a uniform coating. After sealing, the assembly is incubated to solidify partially the matrix layer. After removing the mold, any uncoated areas of the device are covered with additional extracellular matrix.
  • the surface of the device before the surface of the device is coated or sealed with an extracellular matrix composition of the present invention, the surface of the device can be derivatized with an attachment moiety.
  • an attachment moiety for example, an antibody or a small molecule capable of binding to a component of the matrix composition can be attached to a surface of a device prior to the application of the matrix composition.
  • the thickness of extracellular matrix applied to a device is in the range of about 1 to 100 microns. In a preferred embodiment, the thickness is in the range of about 1-50 5 microns. In a more preferred embodiment, the thickness is in the range of about 10 to 30 microns.
  • the thickness is in the range of about 20-25 microns.
  • more than one coat of the extracellular matrix, either untreated or crosslinked, can be 0 applied to a device. It is highly desirable to inspect the device once coated to insure that there are no gaps or breaks present in the coating.
  • a synthetic vascular graft which is coated on the exterior surface with the naturally secreted human extracellular matrix of the present invention, and which provides a reservoir for the slow release of a drug after implantation, is provided.
  • the extracellular matrix proteins in the graft are complexed with a drug, such as an antibiotic agent or an antiviral agent, or mixtures thereof, in order to insure against graft rejection.
  • the extracellular matrix proteins are complexed with a drug, such as an angiogenesis factor, to insure that the implant or graft helps to promote tissue regeneration and angiogenesis .
  • the methods used for implanting the devices coated with the human naturally secreted extracellular matrix are analogous to those used for the implantation of such devices 5 without the extracellular matrix coating, and, of course, depend on the nature of the condition to be modified or corrected.
  • the surgery can be performed under either local or systemic anesthesia and, generally, involves an incision, spacing to accommodate the implant, insertion, and suture.
  • the three-dimensional culture system of the invention is described based upon the type of tissue and cells used in various systems. These descriptions specifically include but are not limited to bone 5 marrow, skin, smooth muscle cells, epithelial cells, and cartilage but it is expressly understood that the three-dimensional culture system can be used with other types of cells and tissues.
  • the invention is also illustrated by way of examples, which demonstrate characteristic data o generated for each system described.
  • Skin fibroblasts were isolated by mincing dermal tissue, trypsinization for 2 hours, and separation of cells into a suspension by physical means. Fibroblasts were grown to confluency in 25 cm 2 Falcon tissue culture dishes and fed with RPMI 1640 (Sigma, MO) supplemented with 10% fetal bovine serum (FBS) , fungizone, gentamicin, and penicillin/streptomycin. Fibroblasts were lifted by mild trypsinization and cells were plated onto nylon filtration mesh, the fibers of which are approximately 90 ⁇ m in diameter and are assembled into a square weave with a mesh opening of 210 ⁇ m (Tetko, Inc., NY) . The mesh was pretreated with a mild acid wash and incubated in polylysine and FBS.
  • FBS fetal bovine serum
  • FIG. 1 is a scanning electron micrograph depicting fibroblast attachment and extension of cellular processes across the mesh opening.
  • Bone marrow was aspirated from multiple sites on the posterior iliac crest of hematologically normal adult volunteers after informed consent was obtained. Specimens were collected into heparinized tubes and suspended in 8 ml of RPMI 1640 medium which was conditioned with 10% FBS and
  • the three-dimensional stromal culture was generated using oral fibroblasts and 8 mm x 45 mm pieces of nylon filtration screen (#3-210/36, Tetko Inc., NY). The mesh was soaked in 0.1 M acetic acid for 30 minutes and treated with 10 mM polylysine suspension for 1 hour. The meshes were placed in a sterile petri dish and inoculated with 1 X 10 s oral fibroblasts collected as described above in 5 DMEM complete medium. After 1-2 hours of incubation at 5% C0 2 the meshes were placed in a Corning 25 cm 2 tissue culture flask, floated with an additional 5 ml of medium, and allowed to reach subconfluence, being fed at 3 day intervals. Cultures were maintained in DMEM complete medium at 37°C and
  • Small vessel endothelial cells isolated from the 15 brain according to the method of Larson et al . , 1987, Microvasc. Res. 3 . 4:184 were cultured in vi tro using T-75 tissue culture flasks. The cells were maintained in Dulbecco ' s Modified Eagle Medium/Hams-F-12 medium combination
  • FCS calcium sulfate
  • glutamine glutamine
  • antibiotics The cells were seeded at a concentration of 1 x 10 6 cells per flask, and reached a confluent state within one week. The cells were passaged once a week, and, in addition, were fed once a week with
  • Nylon filtration screen mesh (#3-210/36, Tetko, Inc., NY) was prepared essentially as described above. The mesh was soaked in an acetic acid solution (1 ml glacial acetic acid plus 99 ml distilled H 2 0) for thirty minutes, was rinsed with copious amounts of distilled water and then autoclaved. Meshes were coated with 6 ml fetal bovine serum per 8 x 8 cm mesh and incubated overnight . The meshes were then stacked, three high, and 3 x 10 7 small vessel endothelial cells (cultured as described) were seeded onto the stack, and incubated for three hours at 37°C under 5% C0 2 in a humidified atmosphere. The inoculated meshes were fed with 10 ml of DMEM/Hams-F-12 medium every 3-4 days until complete confluence was reached (in approximately two weeks) .
  • Cartilage was harvested from articular surfaces of human joints.
  • the cartilage pieces were digested with collagenase (0.2% w/v) in complete medium (DMEM with 10% fetal bovine serum, glutamine, non-essential amino acids, sodium pyruvate, 50 ⁇ g/ml ascorbate and 35 ⁇ g/ml gentamicin) for 20 hours at 37°C.
  • DMEM complete medium
  • Liberated chondrocytes were spun, resuspended in complete medium, counted and plated at 1 X 10 6 cells per T-150 flask. Cells were routinely passed at confluence (every 5-7 days) .
  • Polyglycolic acid mesh (1 mm diameter x 2 mm thick) was sterilized by ethylene oxide or electron beam treatment and presoaked overnight in complete medium. The mesh was seeded in 6 well plates with 3-4 X 10 6 cells per mesh in a total volume of 10 ⁇ l and incubated for 3-4 hours at 37°C in a tissue culture incubator. At this time, 1.5 ml of media was added. The seeded mesh was incubated overnight. 5 ml of media was added the next day. Media was changed three times per week until confluence is reached.
  • the extracellular matrix has been characterized by a number of analytic methods to determine its content of matrix proteins, each value is the average of at least two independent determinations.
  • the matrix contained type I and type III collagens, fibronectin, tenascin, sulfated glycosaminoglycans, decorin and various other secreted human 10 extracellular matrix proteins. Additionally, the secreted matrix proteins were found throughout the three-dimensional support framework.
  • the extracellular matrix contained a total protein amount of 292 mg/cm 2 ⁇ 0.06; fibronectin was present at 3.4 mg/cm 2 ⁇ 1.2; and tenascin at 1.7 mg/cm 2 ⁇ 0.6.
  • Collagen content of the extracellular matrix was 20 3 determined using the Sirius Red assay.
  • the binding of Sirius Red F3BA in saturated picric acid solution has been used widely to estimate fibrotic collagen deposition. Bedossa et al., 1989, Digestion 44(1):7-13; Finkelstein et al . , 1990,
  • Sirius Red binding to collagen is based largely on its use as a histological stain.
  • collagen content measured by Sirius Red binding shows strong correlation with hydroxyproline content.
  • histological staining with Sirius Red is birefringent , indicating directional binding related to the orientation of the collagen strands.
  • Sirius Red is known to bind to proteins other than the classical collagens that contain collagen-like triple helices, such as the complement component Cl .
  • collagens I and III showed the expected molecular weight distributions on immunoblots.
  • Tris buffer pH 8.0. After mixing for two hours on 20 a wrist shaker, the Tris buffer was removed and the specimen placed in a homogenization cylinder along with 30 ml fresh 0.05 M Tris buffer. The sample was homogenized for 30 seconds in buffer alone and then for two 30 second bursts following the addition of a dispersing agent as described in
  • Glycosaminoglycans have been shown to play a variety of structural and functional roles in the body and their presence in the secreted extracellular matrix is important. Table II lists a number of examples of glycosaminoglycans which have been determined to be found in the extracellular matrix as well as their functional importance in normal dermis.
  • the extracel lular matrix was found to contain a total of 2 . 8 mg/cm 2 ⁇ 0 . 1 sulfated glycosaminoglycans .
  • the cells producing and depositing the extracellular matrix also expressed a number of different growth factors .
  • Growth factors are important in the extracellular matrix for two reasons. During the growth of and deposition of the extracellular matrix, naturally seeded growth factors help to control cell proliferation and activity. Further, growth factors remain attached to the extracellular matrix. A variety of growth factors have been determined to be expressed during the deposition of the matrix.
  • acidic and basic FGF, TGF- ⁇ and TGF- ⁇ , and KGF mRNA transcripts were present as were several others as shown in Table III, including PDGF, amphiregulin, HBEGF, IGF, SPARC and VEGF .
  • PDGF and TGF- ⁇ 3 are thought to be involved in regulation of cell proliferation and matrix deposition in culture, while TGF- ⁇ l, HBEGF, KGF, SPARC, VEGF and decorin are deposited in the matrix.
  • Amphiregulin, IGF-1, IGF-2 and IL-1 were not expressed at the sensitivity used in these experiments.
  • a small amount of PDGF B chain is seen in some preparations .
  • the following experiment shows that more extracellular matrix is secreted by fibroblast cells when culture on a three-dimensional framework than when cultured in traditional monolayer tissue culture dishes.
  • Fibroblasts were cultured in monolayer 6 well plates with DMEM tissue culture medium containing 10% serum.
  • samples of the culture medium were taken and subjected to analysis for detecting the soluble C-propeptide of collagen, which is an indirect measurement of collagen synthesis.
  • the assay used was a commercial ELISA kit distributed by PanVera, Madison, WI .
  • the amount of collagen synthesis on a per cell 5 basis was calculated using the following formula: C- propeptide released (ng/ml) * 1 ml * 50 (dilution factor) / cell number / days in culture. In monolayer culture, the amount of collagen synthesized per cell is in the range of 6.5 to 16 ng/10 6 cells/day.
  • Fibroblasts were also cultured on a 11 x 11 square 0 three-dimensional framework with DMEM tissue culture medium containing 10% serum in accordance with the disclosure of the present invention.
  • 5 and 8 samples of the culture medium were taken and subjected to analysis for detecting the soluble C-propeptide of collagen.
  • the assay used was a 5 commercial ELISA kit distributed by PanVera, Madison, WI .
  • the amount of collagen synthesis on a per cell basis was calculated using the following formula: C-propeptide released (ng/ml) * 1 ml * 50 (dilution factor) / cell number / days in culture.
  • the amount Q of collagen synthesized per cell is in the range of 26 to 116 ng/10 6 cells/day based on an estimate of 1.2 x 10 6 cells per

Abstract

La présente invention concerne des compositions comprenant une matrice extracellulaire naturellement sécrétée par l'homme, et leurs méthodes d'utilisation. L'invention concerne, en particulier des compositions et des méthodes permettant de réparer les défauts de la peau par injection d'une matrice extracellulaire humaine naturelle. La présente invention concerne également des appareils prothétiques revêtus d'une composition renfermant une matrice extracellulaire sécrétée naturellement ou scellés par ladite composition, ainsi que leurs méthodes d'utilisation.
PCT/US2000/018461 1999-07-09 2000-07-06 Appareil revetu d'une matrice extracellulaire naturellement secretee par l'homme WO2001003750A1 (fr)

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AU60722/00A AU6072200A (en) 1999-07-09 2000-07-06 Human naturally secreted extracellular matrix-coated device
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WO2005009498A2 (fr) * 2003-07-17 2005-02-03 Boston Scientific Limited Matrice extracellulaire de moelle osseuse decellularisee
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WO2008092440A2 (fr) * 2007-02-01 2008-08-07 Stiftung Caesar Composition thérapeutique et utilisation d'une substance acellulaire
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US8137965B2 (en) 2002-09-06 2012-03-20 Dfb Technology Holdings, Llc Methods and compositions for tissue regeneration
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WO2020163329A1 (fr) * 2019-02-05 2020-08-13 Corning Incorporated Substrats de culture cellulaire tissés
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US8137965B2 (en) 2002-09-06 2012-03-20 Dfb Technology Holdings, Llc Methods and compositions for tissue regeneration
US9173906B2 (en) 2002-09-06 2015-11-03 Smith & Nephew, Inc. Methods and compositions for tissue regeneration
US8323638B2 (en) 2002-09-06 2012-12-04 Dfb Technology Holdings, Llc Methods and compositions for tissue regeneration
AU2009240818B2 (en) * 2002-09-06 2012-08-16 Smith & Nephew Orthopaedics Ag Methods and Compositions for Tissue Regeneration
US8679475B2 (en) 2002-09-06 2014-03-25 Smith & Nephew, Inc. Methods and compositions for tissue regeneration
US7326571B2 (en) 2003-07-17 2008-02-05 Boston Scientific Scimed, Inc. Decellularized bone marrow extracellular matrix
WO2005009498A3 (fr) * 2003-07-17 2005-03-24 Scimed Life Systems Inc Matrice extracellulaire de moelle osseuse decellularisee
WO2005009498A2 (fr) * 2003-07-17 2005-02-03 Boston Scientific Limited Matrice extracellulaire de moelle osseuse decellularisee
US8790920B2 (en) 2003-07-17 2014-07-29 Boston Scientific Scimed, Inc. Decellularized bone marrow extracellular matrix
US8864844B2 (en) 2004-05-11 2014-10-21 Synthasome, Inc. Tissue scaffold
US8874204B2 (en) 2004-12-20 2014-10-28 Cardiac Pacemakers, Inc. Implantable medical devices comprising isolated extracellular matrix
WO2006102063A3 (fr) * 2005-03-19 2007-03-08 Cook Biotech Inc Implants prothetiques comprenant un materiau composite a matrice extracellulaire
WO2006102063A2 (fr) * 2005-03-19 2006-09-28 Cook Biotech Incorporated Implants prothetiques comprenant un materiau composite a matrice extracellulaire
US8454678B2 (en) 2005-03-19 2013-06-04 Cook Biotech Incorporated Prosthetic implants including ECM composite material
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EP1962920A1 (fr) * 2005-12-01 2008-09-03 Agency for Science, Technology and Research Matrices extracellulaires reconstituees en trois dimensions servant d'echafaudage pour le genie tissulaire
WO2008092440A3 (fr) * 2007-02-01 2009-01-08 Stiftung Caesar Composition thérapeutique et utilisation d'une substance acellulaire
WO2008092440A2 (fr) * 2007-02-01 2008-08-07 Stiftung Caesar Composition thérapeutique et utilisation d'une substance acellulaire
US20130344161A1 (en) * 2008-01-30 2013-12-26 Histogen, Inc. Conditioned Medium and Extracellular Matrix Compositions from Cells Cultured under Hypoxic Conditions
US9512403B2 (en) * 2008-01-30 2016-12-06 Histogen, Inc. Conditioned medium and extracellular matrix compositions from cells cultured under hypoxic conditions for methods of treating tissue injury
US10538736B2 (en) 2008-01-30 2020-01-21 Histogen, Inc. Conditioned medium and extracellular matrix compositions from cells cultured under hypoxic conditions
US11274276B2 (en) 2008-01-30 2022-03-15 Histogen, Inc. Conditioned medium and extracellular matrix compositions from cells cultured under hypoxic conditions
US20120226218A1 (en) * 2009-09-04 2012-09-06 Mary Phillips Extracellular matrix material created using non-thermal irreversible electroporation
US8835166B2 (en) * 2009-09-04 2014-09-16 The Regents Of The University Of California Extracellular matrix material created using non-thermal irreversible electroporation
US9186377B2 (en) 2011-06-03 2015-11-17 Maguire Abbey, Llc Method, composition, and articles for improving joint lubrication
WO2015101711A1 (fr) 2013-12-30 2015-07-09 Upm-Kymmene Corporation Membrane structurée
WO2015101712A1 (fr) 2013-12-30 2015-07-09 Upm-Kymmene Corporation Dispositif biomédical
US11111470B2 (en) 2019-02-05 2021-09-07 Corning Incorporated Packed-bed bioreactor systems and methods of using the same
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WO2020163329A1 (fr) * 2019-02-05 2020-08-13 Corning Incorporated Substrats de culture cellulaire tissés
JP2022519651A (ja) * 2019-02-05 2022-03-24 コーニング インコーポレイテッド 織布細胞培養基材
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