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WO1999060951A1 - Compositions pour la regeneration de tissu degenere et procedes d'utilisation desdites compositions - Google Patents

Compositions pour la regeneration de tissu degenere et procedes d'utilisation desdites compositions Download PDF

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Publication number
WO1999060951A1
WO1999060951A1 PCT/US1999/011283 US9911283W WO9960951A1 WO 1999060951 A1 WO1999060951 A1 WO 1999060951A1 US 9911283 W US9911283 W US 9911283W WO 9960951 A1 WO9960951 A1 WO 9960951A1
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WIPO (PCT)
Prior art keywords
subject
composition
tissue
fibroblasts
biodegradable
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PCT/US1999/011283
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English (en)
Inventor
William K. Boss
Olga Marko
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Isolagen Technologies, Inc.
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Publication date
Application filed by Isolagen Technologies, Inc. filed Critical Isolagen Technologies, Inc.
Priority to AU40933/99A priority Critical patent/AU4093399A/en
Publication of WO1999060951A1 publication Critical patent/WO1999060951A1/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
    • 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
    • A61L27/02—Inorganic materials
    • A61L27/12—Phosphorus-containing materials, e.g. apatite
    • A—HUMAN NECESSITIES
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    • 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
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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    • 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
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    • 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
    • A61L27/3895—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 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
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    • A61L31/026—Ceramic or ceramic-like structures, e.g. glasses
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    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
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    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
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    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/04—Macromolecular materials
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    • A61L31/045—Gelatin
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    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L31/148—Materials at least partially resorbable by the body
    • 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
    • C12N5/0656—Adult fibroblasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F13/00—Bandages or dressings; Absorbent pads
    • A61F2013/00361—Plasters
    • A61F2013/00365—Plasters use
    • A61F2013/0037—Plasters use for cosmesis
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    • A61F2310/00—Prostheses classified in A61F2/28 or A61F2/30 - A61F2/44 being constructed from or coated with a particular material
    • A61F2310/00005—The prosthesis being constructed from a particular material
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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Definitions

  • the present invention concerns the regeneration of tissues in a subject that have degenerated as a result of a disease or disorder in the subject. More particularly, the present invention concerns novel compositions for use in nonsurgical techniques that promote regeneration of tissue whose mass has been diminished due to a disease or disorder in a subject, correct defects in the skin of subjects, or augment tissue in subjects. Also disclosed is the use of a novel composition in conjunction with a biodegradable acellular matrix for ameliorating defects in the tissues, and methods for using the novel composition.
  • Periodontal disease is the term commonly used to describe inflammatory disease of the periodontium, i.e., the tissue surrounding and securing teeth to the jawbone. The condition is characterized by inflammatory and degenerative processes that develop at the gingival margin (gingivitis) and lead to a progressive breakdown and resorption of the periodontal ligament and bone (periodontitis) , oftentimes resulting in severe diminution of the periodontium. Periodontal disease is the leading cause of tooth loss in adults after middle age. [Anderson's Pathology, p. 2000, John M. Kissane ed. , 9th ed. (1992) ] .
  • Periodontal disease results from the accumulation of bacterial plaque in the gap between the gingiva and the tooth. While anaerobic bacteria are the primary etiologic agents, the destructive process is believed to be mediated in large part by immunologic reactions of the host. As the disease progresses, a periodontal pocket is established below the gingival margin, thus prolonging and promoting the inflammatory process. Successive inflammatory reactions result in the progressive erosion of the tooth-supporting tissues, i.e., the collagenous fibers making up the periodontal ligament and the bone pocket in which the tooth sits. [Reviewed in Anderson's Pathology, pp. 1999-2000, John M. Kissane ed. , 9th ed. (1992); Shafer et al . , A Textbook of Oral Pathology, 4th ed. (1983) ] .
  • Periodontal disease can be diagnosed by checking the gingiva for inflammation, probing the depths of periodontal pockets, checking clinical attachment level, and assessing bone loss by means of autoradiography . [Jeffcoat, M.K., et al . , J. Am. Dent. Assoc, 128:713-724 (1997) ] .
  • Surgical techniques comprise reflecting the gingival tissues to provide access to root surfaces and bone defects, in order that mechanical debridement may be accomplished directly. Following debridement, the gingival tissue is sutured back in position.
  • Currently available surgical approaches entail substantial patient discomfort and fail to consistently provide satisfactory outcome.
  • Tetracyclines in particular have shown promise as inhibitors of extracellular collagenases, but cause the same side effects associated with antibiotics in general. Modified forms of tetracycline have been developed which are non-antimicrobial and retain their ability to inhibit collagenases, but these chemically modified tetracyclines are not commercially available. [Ciancio, G.C. et al . , J. Am. Dent. Assoc. 123:34-43 (1992)].
  • tissue growth factors for example insulin-like growth factor, platelet-derived growth factor, and transforming growth factor to promote periodontal regeneration.
  • Bone grafting techniques involve the use of natural bone or synthetic bone materials. Natural bone grafts are typically either autografts (grafts transferred from one position in the body of a patient to another position in the body of the same patient) or allografts (grafts transferred from one person to another) . Clinicians using natural bone grafts have had limited success in inducing new bone growth.
  • Freeze-dried, demineralized bone has been used as an allograft and shown to promote bone formation.
  • the predictability and the amount of bone fill achieved varies. [Jeffcoat, M.K. et al . , J. Am. Dent. Assoc ' ., 128:713-724 (1997)]. Since allografts are transferred from one person to another, the potential exists that viruses or other pathogens might be transferred to the patient.
  • Synthetic bone materials which have been investigated include plaster, calcium carbonates, and ceramics such as hydroxyapatite .
  • Clinical trials have demonstrated that the use of synthetic grafts has resulted in improvements in probing depth and attachment level. Histologic findings, however, indicate that, in general, synthetic grafts act primarily as space fillers, with little if any regeneration. [Jeffcoat, M.K. et al . , J. Am. Dent. Assoc, 128:713-724 (1997)].
  • Guided tissue regeneration is a surgical approach based on placing a membrane barrier under a soft tissue flap above the area of bone loss to enhance wound healing potential. [Ciancio, G.C. et al . , J. Am. Dent.
  • compositions and methods to correct defects in skin, such as scars and wrinkles, or to augment the tissue of a subject in order to improve the appearance of the skin, particularly facial skin.
  • the principal method employed to correct such defects involves injecting a filler composition into the dermal layer of the skin proximate to the defect or desired tissue augmentation.
  • non-biological filler compositions used in these roles include mineral oil, paraffin, silicone fluid, autologous fat, gelatin powder mixes, polymethylmethacrylate microspheres, cross-linked polydimethylsiloxane, ""TEFLON”” paste, reconstituted bovine collage, and autologous human collagen.
  • compositions comprises inherent limitations.
  • the use of mineral oil, paraffin and similar oils and waxes has resulted in complications such as local chronic edema, lymphadenopathy, scarring and ulcerations (Devore et al . , Effectiveness of injectable filler materials for smoothing wrinkle lines and depressed scars . Medical Progress Through Technology 20:243-250 (1994 which is hereby incorporated by reference in its entirety) .
  • reconstituted bovine collagen to correct defects or augment tissue also possesses inherent limitations. For example, it has been reported that reconstituted bovine collage is only moderately effective, and is associated with infrequent, but controversial, adverse reactions. In addition, it is rapidly broken down and resorbed in vivo, providing only a temporary correction of a skin defect or augmentation. More importantly, reconstituted bovine collagen may elicit an immune response in the subject. Id.
  • gelatin matrix implant such as that sold under the mark "FRIBEL"
  • FPIBEL is a composite material of porcine gelatin powder and o-aminocaproic acid which are dispersed in 0.9% (by volume) sodium chloride solution and an aliquot of the recipient's plasma mixed in a 1:1 ratio
  • this material also possess inherent limitations.
  • gelatin matrix does not appear to have applications in the treatment of wrinkle lines.
  • a large bore needle 27 gauge or greater
  • treatment with gelatin results in greater discomfort and pain to the subject as opposed to the injection of other fillers. Id.
  • the use of autologous fat injections to correct a skin defect or augment tissue in a subject also possesses disadvantages. More specifically, prior to its injection, fat must be processed by skilled clinicians in aseptic conditions to maintain sterility. Also, a very large bore needle (as large as 22 gauge) is needed to inject the fat into a subject, resulting in great pain, moderate bruising, and formation of visible puncture holes. Moreover, fat injections are subject to rapid resorption, and must be repeated in order to maintain skin augmentation or defect correction.
  • the use of autologous, injectable dermal collagen to correct defects or augment tissue has also met with limited success. For example, if large concentrations of collagen are injected, a 27 gauge needle or larger is used, resulting in the infliction of pain on the subject. Furthermore, serial injections are required in order to compensate for the gradual resorption of autologous collagen.
  • compositions and methods for promoting regeneration of tissue that does not elicit an immune response in the subject at the site of desired tissue regeneration.
  • compositions that can be used in non-surgical methods to correct defects in skin, such as scars or wrinkles, and augment tissue in a subject, particularly facial tissue, which is not rapidly resorbed by the body so that additional injections are required.
  • the present invention provides a composition for promoting regeneration of tissue in a subject that has degenerated as a result of a disease or disorder, and a method of using the composition that does not suffer from the shortcomings of other methods described above .
  • the present invention is based on the inventors' discovery of the successful use of autologous fibroblasts to regenerate tissue in a subject, correct skin defects in the subject, or augment tissue in the subject.
  • Fibroblasts are connective-tissue cells involved in tissue repair. When a tissue is injured, nearby fibroblasts migrate into the wound, proliferate, and produce large amounts of collagenous -matrix, which helps to is.olate and repair the damaged tissue. [Alberts et al . , Molecular Biology of the Cell , p. 987, 2nd ed. , (1992) ] .
  • the present invention extends to a method for regenerating a subject's tissue that has degenerated as a result of a disease or disorder, comprising the steps of providing a pharmaceutical composition comprising autologous, passaged fibroblasts, identifying a site of tissue degeneration, and injecting an effective amount of the composition into tissue at the site of tissue degeneration so that the tissue is augmented and regeneration of tissue is promoted.
  • Injection of a pharmaceutical composition of the present invention can be into tissues of the subject comprising the periodontal pocket and/or the periodontal tissue adjacent to the area of degeneration or into tissue subadj acent to a defect in the oral mucosa, or into the tissue of the palate of a human subject, in order promote regeneration of tissue in the oral mucosa, the gingiva, or the palate.
  • Typical defects in the oral mucosa or palate that can be corrected with this embodiment of the present invention include those caused by trauma, dermatoses, recurrent aphthous stomatitis, and infections, or a disease or disorder.
  • the present invention can be used to correct defects in the skin, such as scars or wrinkles.
  • a disease or disorder which results in tissue degeneration in a subject that can be treated with the present invention includes, but is not limited to, defects of the oral mucosa, periodontal disease, trauma to the oral mucosa (e.g., extraction of a tooth), diabetes, cutaneous ulcers, or venous stasis.
  • periodontal disease can include periodontal degeneration, gingivitis, or a non-healing wound of the palatal mucosa or the gingival mucosa.
  • the present invention further extends to a method of forming a composition comprising autologous, passaged fibroblasts which are substantially free of immunogenic proteins, and are histocompatible with a subject.
  • This method comprises the steps of collecting a biopsy of dermis from a subject, isolating the autologous fibroblasts contained in the biopsy from extracellular matrix and other cells contained in the biopsy, culturing the autologous fibroblasts in a culture medium that permits expansion of the autologous fibroblasts, incubating the autologous fibroblasts in a protein free medium for at least about 2 hours between about 30°C and about 37.5°C, and exposing the incubated autologous fibroblasts to a proteolytic enzyme so as to suspend the fibroblasts.
  • An example of a culture medium that permits expansion of autologous fibroblasts comprises between
  • the biopsy from dermis can comprise tissue form the gums, palate or skin of the subject.
  • autologous fibroblasts from the gums, plate or skin have applications in the present invention.
  • the passaged autologous fibroblasts can be added to a pharmaceutically acceptable carrier to form a pharmaceutical composition.
  • antigenic proteins are removed from the autologous fibroblasts, thereby avoiding an immunological reaction in a subject when such cells are reintroduced to the subject proximate to the site of -tissue degeneration or defect .
  • the present invention extends to a device delivering a pharmaceutical composition of the present invention to a site proximate to the site of tissue degeneration or defect in a subject, wherein the device comprises a hypodermic syringe having a syringe chamber, a piston disposed therein, an orifice communicating with the chamber, a pharmaceutical composition comprising autologous passaged fibroblasts and a pharmaceutically acceptable carrier, such that the pharmaceutical composition is disposed in the chamber, and a hypodermic needle is fixed to the orifice.
  • Tissues which have suffered degeneration or have a defect that can be treated with a device of the present invention include the oral mucosa, the gingival mucosa, and the palatal mucosa.
  • diseases or disorders which can be treated with this device include periodontal degeneration, gingivitis, or a non-healing wound of the palatal mucosa or the gingival mucosa.
  • skin defects, such as scars and wrinkles can be treated with the present invention.
  • the present invention further extends to a composition for promoting the regeneration of tissue that has degenerated in a subject, wherein the composition comprises a biodegradable acellular matrix, and autologous passaged fibroblasts substantially free of immunogenic proteins, wherein the autologous fibroblasts are integrated into the biocompatible biodegradable acellular matrix.
  • the biocompatible biodegradable acellular matrix comprises exogenous proteins, such as any type of collagen.
  • the biodegradable acellular matrix can be comprised of any type of collagen and glycosaminoglycans (GAG) cross-linked with, for example, glutaraldehyde, or any type of collagen.
  • the biodegradable acellular matrix comprises one or more of gelatin, polyglycolic acid, cat gut, demineralized bone, or hydroxyapatite.
  • Other appropriate matrices consist of bone from which substantially all (e.g., at least 80%, at least 90%, at least 95%, at least 99%, or even 100% by weight) organic material has been removed (referred to herein as "anorganic bone”); such matrices can, optionally, include exogenous collagen in various amounts (e.g., about 1%, about 2%, about 5%, about 10%, or about 20% by dry weight) .
  • diseases, disorders or defects resulting in degeneration of tissue in a subject which can be treated with the present invention, comprise defects of the oral mucosa, trauma to the oral mucosa (e.g., extraction of a tooth) , periodontal disease, diabetes, cutaneous ulcers, or venous stasis.
  • periodontal disease which result in tissue degeneration include, but are not limited to, periodontal degeneration, gingivitis, or non-healing wounds of the palatal mucosa or gingival mucosa .
  • defects in skin such as scars or wrinkles, can be treated with the present invention.
  • the present invention further extends to a method of using a composition for promoting regeneration of tissue, wherein the method comprises passaged autologous fibroblasts substantially free of immunogenic proteins, providing a biodegradable acellular matrix, incubating the biodegradable acellular matrix with the passaged autologous fibroblasts so that the fibroblasts integrate into the biodegradable acellular matrix, forming a composition for promoting regeneration of tissue, identifying a site of tissue degeneration or defect in the subject, and applying the composition to the site of tissue degeneration or defect so that tissue regeneration at the site is promoted.
  • Autologous passaged fibroblasts used herein can comprise fibroblasts from the gums, palate or skin of the subject.
  • Diseases or disorders which can be treated with this method include, but are not limited to, defects of the oral mucosa, trauma to the oral mucosa (e.g., extraction of a tooth) , periodontal disease, diabetes, cutaneous ulcers, or venous stasis.
  • periodontal disease that can be treated with the present invention comprise periodontal degenexation, gingivitis, or a non-healing wound of the palatal mucosa or the gingival mucosa.
  • defects in skin such as scars or wrinkles can be treated with the composition of the present invention. In a preferred embodiment, such defects are treated with a composition comprising fibroblasts from the palate.
  • biodegradable acellular matrices having applications in the present invention may comprise exogenous proteins.
  • examples of such matrices include matrices comprising any type of collagen, or any type of collagen and glycosaminoglycans (GAG) cross-linked with, for example, glutaraldehyde.
  • GAG glycosaminoglycans
  • Other examples of biodegradable acellular matrices having applications in the present invention include one or more of gelatin, polyglycolic acid, cat gut, demineralized bone, or hydroxyapatite.
  • matrices consist of bone from which substantially all (e.g., at least 80%, at least 90%, at least 95%, at least 99%, or even 100% by weight) organic material has been removed (referred to herein as "anorganic bone”); such matrices can, optionally, include exogenous collagen, in various amounts (e.g., about 1%, about 2%, about 5%, about 10%, or about 20% by dry weight) .
  • the present invention further extends to an injectable composition for correcting a defect in skin of a subject, or augmenting tissue of a subject, said injectable composition comprising passaged, autologous fibroblasts substantially free of immunogenic proteins, and a biodegradable, acellular injectable filler material.
  • Passaged autologous fibroblasts having applications in an injectable composition of the present invention are from gums, palate or skin of the subject.
  • the present invention extends to an injectable composition as described above, wherein the biodegradable, acellular injectable filler material comprises endogenous proteins.
  • the acellular injectable filler material of an injectable composition of the present invention comprises an injectable dispersion of autologous collagen fibers having a concentration in the composition of at least 24 mg/ml ' of composition.
  • the present invention extends to an injectable composition as described above, wherein the biodegradable acellular injectable filler material comprises exogenous proteins, such as any type of collagen.
  • exogenous proteins such as any type of collagen.
  • An example of an exogenous collagen having applications in an injectable composition of the present invention is reconstituted bovine collagen fibers cross- linked with glutaraldehyde.
  • the filler material can comprise any type of solubilized gelatin either alone, or in combination with other materials.
  • the filler material comprises porcine gelatin powder and o-aminocaproic acid dispersed in sodium chloride solution and an aliquot of plasma from the subject to be injected with the composition.
  • the ratio of sodium chloride to serum is 1:1 by volume.
  • Other examples of materials having applications in the present invention as biodegradable, acellular injectable filler material include, but are not limited to polyglycolic acid or cat gut.
  • the present invention further extends to a method for correcting a defect in skin of a subject, or augmenting tissue of a subject, wherein the method comprises injecting an effective amount of an injectable composition comprising autologous passaged fibroblasts substantially free of immunogenic proteins and a biodegradable, acellular injectable filler material, into the skin of the subject at the site of the skin defect or desired tissue augmentation, so that regeneration of tissue at the site is promoted at the site.
  • the present invention extends to a method for correcting a defect in skin of a subject, or augmenting tissue of a subject, the method comprising the steps of injecting autologous fibroblasts substantially free of immunogenic proteins into the subject at a site of a skin defect or desired tissue augmentation, and subsequently injecting a biodegradable, acellular injectable filler material into the s-ite .
  • the duration between injecting the autologous fibroblasts into the subject and injecting the biodegradable acellular injectable filler into the subject is about two weeks.
  • Autologous fibroblasts having applications in methods of the present invention for correcting a defect in skin of a subject, or augmenting tissue of a subject can be obtained from the gums, palate of skin of the subject .
  • the present invention further extends to a method for correcting a defect in skin of a subject, or augmenting tissue of the subject, as described above, wherein the biodegradable, acellular injectable filler material comprises endogenous proteins.
  • the biodegradable acellular injectable filler material can comprise an injectable dispersion of autologous collagen fibers, preferably at a concentration of at least 24 mg of autologous fibers per ml of composition.
  • the present invention extends to a method for correcting a defect in skin of a subject, or augmenting tissue of the subject, as described above, wherein the biodegradable, acellular filler material of the composition comprises exogenous proteins such as, for example, any type of collagen.
  • An example of collagen having applications in a method of the present invention comprises reconstituted bovine collagen fibers cross- linked with glutaraldehyde.
  • Other examples of biodegradable, acellular injectable filler material for use in a method for correcting a defect in skin or a subject, or augmenting tissue of the subject include, but are not limited to solubilized gelatin, polyglycolic acid, or cat gut sutures.
  • an example of acellular injectable filler material having applications in the present invention comprises porcine gelatin powder and aminocaproic acid dispersed in sodium chloride solution, and an aliquot of plasma from the subject.
  • the ratio of sodium chloride solution to the aliquot of serum is 1:1 by volume.
  • the sodium chloride solution comprises 0.9% sodium chloride by volume.
  • the present invention extends to a method for correcting a defect in skin of a subject, or augmenting tissue of a subject, as described above, wherein the ratio of autologous fibroblasts substantially free of immunogenic proteins to biodegradable, acellular injectable filler material is approximately 1:1 by volume . Accordingly, it is an object of the present invention to provide a composition for augmenting tissue, or promoting regeneration of tissue such as the oral mucosa, the gingival mucosa, or the palatal mucosa or skin, which has degenerated as a result of a disease or disorder. Examples of such disorders include periodontal disease, trauma, dermatoses, recurrent aphthous stomatitis, infections, scars, or wrinkles.
  • the present invention is based, in part, on the recognition that living cells normally present in tissue that has degenerated, particularly fibroblasts, are the ideal material to augment the volume of tissue in order to promote regeneration of tissue. Hence the present invention ameliorates and reverses the degenerative effects of a disease or disorder which results in tissue degeneration .
  • the present invention is based on the recognition that an ideal composition with which to augment the oral mucosa subadj acent to a defect, to treat defects in the palate or gingiva, or to promote regeneration of tissue that has degenerated as a result of periodontal disease, would comprise living cells normally present in such tissues, particularly fibroblasts .
  • the present invention is based on the recognition that an abundant supply of autologous cells of the desired type can be obtained by culturing a biopsy specimen taken from the skin, palate or gums of a subject several weeks prior to treating the tissue degenerating disease, disorder, or defect.
  • the invention is further based on the recognition that, after such a tissue culture expansion, the autologous cells will contain a significant quantity of antigenic proteins, but that the antigenic proteins can be removed, prior to treatment of the subject.
  • biodegradable denotes a composition that is not biologically harmful and can be chemically degraded or decomposed by natural effectors (e.g., weather, soil bacteria, plants, animals) .
  • autologous refers to cells removed from a donor and administered to a recipient, wherein the donor and recipient are the same individual.
  • an effective amount refers to the injection of an amount of pharmaceutical composition of the present invention to promote tissue regeneration in of tissue that has degenerated in a subject.
  • EXAMPLE I Administration of a Suspension of Autologous Fibroblasts to Promote Tissue Regeneration and Correct Defects in Tissues
  • one embodiment of the present invention comprises a method for regenerating tissue that has been damaged in a subject as a result of a disease or disorder in the subject, wherein the method comprises providing a pharmaceutical composition comprising of autologous, passaged fibroblasts substantially free of immunogenic proteins, identifying a site of tissue degeneration or a defect in tissue, injecting an effective amount of the pharmaceutical composition into the tissue at the site of the tissue degeneration or defect so that the tissue is augmented, and the growth of tissue is promoted at the site of degenerated tissue.
  • a disease or disorder which promotes tissue degeneration in a subject, and can be treated with this aspect of the present invention includes, but is not limited to, defects of the oral mucosa, trauma to the oral mucosa, periodontal disease, diabetes, cutaneous ulcers, or venous stasis.
  • examples of periodontal disease which can be treated with this aspect of the present invention include periodontal degeneration, gingivitis, or a non-healing wound of the palatal mucosa or the gingival mucosa.
  • defects in skin, such as scars or wrinkles can be treated with the present invention.
  • the invention can be practiced by injecting any undifferentiated mesenchymal cell that can be expanded in culture.
  • dermal fibroblasts are injected because they can be readily obtained and expanded, and because they are a cell type normally present beneath the gingival mucosa or palatal mucosa. Fibroblasts taken from a biopsy of the gums, palate, or skin of the subject can be used in the present invention.
  • a dermal fibroblast culture is initiated from a 1 to 5 mm full thickness biopsy specimen of the gums, palate or skin of a subject suffering from tissue degeneration. Because of the phenomenon of allograft rejection, which is well known to transplantation surgeons and immunologists, it is essential that the cultured fibroblasts be histocompatible with the host. Histocompatibility can be ensured by obtaining a biopsy of the subject to be treated and culturing the fibroblasts from this specimen.
  • the biopsy Before the initiation of the culture, the biopsy is washed repeatedly with antibiotic and antifungal agents. The specimen of dermis is then separated into small pieces. The pieces of the specimen are individually placed onto the dry surface of a tissue culture flask and allowed to attach for between about 5 and about 10 minutes, before a small -amount of medium is slowl-y added, taking care not to displace the attached tissue fragments. After about 48 hours of incubation, the flask is fed with additional medium. When a T-25 flask is used to start the culture, the initial amount of medium is about 1.5-2.0 ml. The establishment of a cell line from the biopsy specimen ordinarily takes between about 2 and 3 weeks, at which time the cells can be removed from the initial culture vessel for expansion.
  • the fibroblasts can be stimulated to grow by a brief exposure to EDTA-trypsin, according to techniques well known to those skilled in the art. The exposure to trypsin is too brief to release the f-ibroblasts from their attachment to the culture vessel wall.
  • samples of the fibroblasts can be processed for frozen storage, such as in liquid nitrogen. Presently, numerous methods for successfully freezing cells for later use are known in the art, and are included in the present invention.
  • the frozen storage of early rather than late passage fibroblasts is preferred because the number of passages in cell culture of normal human fibroblasts is limited.
  • the fibroblasts can be frozen in any freezing medium suitable for preserving fibroblasts.
  • a medium consisting of about 70% (v/v) growth medium, about 20% (v/v) fetal bovine serum and about 10% (v/v) dimethylsulfoxide (DMSO) can be used -with good effect.
  • DMSO can also be substituted with, for example, glycerol .
  • Thawed cells can be used to initiate secondary cultures to obtain suspensions for use in the same subject without the inconvenience of obtaining a second specimen.
  • tissue culture technique that is suitable for the propagation of dermal fibroblasts from biopsy specimens may be used to expand the cells to practice the invention.
  • Techniques well known to those skilled in the art can be found in R.I. Freshney, Ed.., ANIMAL CELL CULTURE: A PRACTICAL APPROACH (IRL Press, Oxford, England, 1986) and R.I. Freshney, Ed., CULTURE OF ANIMAL CELLS: A MANUAL OF BASIC TECHNIQUES, Alan R. Liss & Co., New York, 1987) , which are hereby incorporated by reference .
  • the medium can be any medium suited for the growth of primary fibroblast cultures.
  • the medium can be supplemented with human or non-human serum in an amount of between about 0.0% and about 20% (v/v) to promote growth of the fibroblasts. Higher concentrations of serum promote faster growth of the fibroblasts .
  • An example of medium having application herein comprises glucose DMEM supplemented with about 2 mM glutamine, about 10 mg/L sodium pyruvate, about 10% (v/v) fetal bovine serum and antibiotics ("complete medium”), wherein the concentration of glucose ranges from about 1,000 milligrams per liter of medium to about 4,500 milligrams per liter of medium.
  • Fibroblasts can also be expanded in a serum-free medium.
  • Autologous fibroblasts can be passaged into new flasks by trypsinization. For expansion, individual flasks are split 1:3. Triple bottom, T-150 flasks, having a total culture area of 450 cm 2 are suitable for the practice of the invention.
  • a triple bottom T-150 flask can be seeded with about 1X10 6 to about 3X10 6 cells and has a capacity to yield about 8X10 6 to about 1.3X10 7 cells.
  • the capacity of the flask is reached, which typically requires about 5-7 days of culture, the growth medium is replaced by serum- free medium; thereafter the cells are incubated, i.e., held at between bout 30°C and about 37.5°C, for at least 2 hours.
  • serum-free medium comprises glucose DMEM supplemented with about 2 mM glutamine, and about 110 mg/L sodium pyruvate, wherein the concentration of glucose can range from approximately 1,000 mg/L of medium to about 4,500 mg/L of medium. In a preferred embodiment, the concentration of glucose is approximately 4,500 mg/L of medium.
  • the cells are removed from the tissue culture flask by trypsin-EDTA; washed extensively by centrifugation and resuspension; and suspended for injection in an equal volume of injectable isotonic solution with an appropriate physiological osmolarity, which is substantially pyrogen and foreign protein free.
  • injectable isotonic solution is isotonic saline.
  • a pharmaceutically acceptable carrier can then be added to the passaged autologous fibroblasts forming a pharmaceutical composition.
  • compositions that are not deleterious to the cells, are physiologically tolerable, and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • Such compositions include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength.
  • the cells can be transported at 4°C so long as they are injected within 24-48 hours of the time that the pharmaceutical composition is made.
  • the cells can be suspended in an appropriate physiological solution with appropriate osmolarity and tested for pyrogen and endotoxin levels, except for the absence of phenol red pH indicator, and the replacement of the fetal bovine serum by the subject's serum for such transportation (transport medium) .
  • the cells can be suspended in Krebs-Ringer solution comprising 5% dextrose or any other physiological solution. The cells can be aspirated and injected in the transport medium.
  • the volume of saline or transport medium in which the cells are suspended is related to such factors as the number of fibroblasts the practitioner desires to inject, the extent of the damage due to tissue degeneration or defect, or the size, the number of the defects that are to be treated, and the urgency of the subject's desire to obtain the results of treatment. Moreover, the practitioner can suspend the cells in a larger volume of medium and inject correspondingly fewer cells at each injection site.
  • the present invention extends to a device delivering a pharmaceutical composition of autologous passaged cells described above, to a point proximate to the site of tissue degeneration, or defects of the palate, oral mucosa or skin.
  • a device comprises a hypodermic syringe having a syringe chamber, a piston disposed therein, an orifice communicating with the chamber, a pharmaceutical composition comprising autologous, passaged fibroblasts, and a pharmaceutically acceptable carrier thereof, such that the pharmaceutical composition is disposed in the chamber, and a hypodermic needle is fixed to the orifice.
  • a pharmaceutical composition of the invention can be used to treat tissue degeneration in a subject as a result of a disorder or disease, such as periodontal disease, or defects of the oral mucosa or skin, such as scars or wrinkles, by use of the following techniques. Initially, the tissue to be injected is prepped with alcohol and stretched to give a taut surface. If the tissue degeneration is the result of periodontal disease, the tissue to be injected is periodontal tissue, including periodontal pockets. If the tissue to be injected contains defects in the palate or gums of the subject, the tissue to be injected with a pharmaceutical composition of the present invention is subadj acent to the defect.
  • a disorder or disease such as periodontal disease
  • defects of the oral mucosa or skin such as scars or wrinkles
  • the tissue to be injected is the skin, in order to treat defects such as scars or wrinkles, it is also injected subadj acent to the defect.
  • a syringe is filled with a pharmaceutical composition of the present invention and fitted with a 30 gauge needle. The needle is inserted into the tissue as superficially as possible, and the orientation of the bevel is not critical to the success of this method of the present invention.
  • the injection of the pharmaceutical composition is made by gentle pressure until a slight blanch is seen in the injected tissue. Multiple serial injections are made.
  • Example II A Composition for Promoting the
  • Regeneration of Tissue that Has Degenerated in a Subject Also disclosed in the present invention is a composition for promoting the regeneration of tissue that degenerated in a subject.
  • Such degeneration can occur as a result of periodontal disease, trauma, dermatoses, recurrent aphthous stomatitis, or infections to name only a few.
  • examples of periodontal disease which can cause tissue degeneration include periodontal degeneration, gingivitis, or non-healing wounds of the palatal mucosa or gingival mucosa.
  • composition of the present invention can also be used to correct defects in tissue of a subject, such as, for example, defects in the palatal mucosa, gingival mucosa, or defects in skin, such as scars or wrinkles. Such compositions can be used, for example, for healing extraction sockets after extraction of a tooth.
  • a composition of the present invention comprises a biodegradable acellular matrix, and autologous passaged fibroblasts substantially free of immunogenic proteins, wherein the autologous fibroblasts are integrated into the biodegradable acellular matrix.
  • a method of making a composition of the present invention to promote regeneration of tissue in a subject comprises providing a suspension of passaged autologous fibroblasts substantially free of immunogenic proteins, providing a biodegradable acellular matrix, incubating the biodegradable acellular matrix with the suspension of passaged autologous fibroblasts such that the autologous fibroblasts integrate within the biodegradable acellular matrix forming a composition for promoting regeneration of tissue.
  • a method of using a composition of the present invention comprising identifying a site of tissue degeneration, and applying the composition for promoting regeneration of tissue to the site of tissue degeneration.
  • autologous passaged fibroblasts substantially free of antigenic proteins must be made available to avoid the phenomenon of allograft rejection, which is well known to transplantation surgeons and immunologists .
  • the cultured fibroblasts be histocompatible with the host. Histocompatibility can be ensured by obtaining a biopsy of the gums, palate or skin of the subject to be treated, and culturing the fibroblasts from this specimen.
  • a biopsy of 1-5 mm is taken ' from the gums, palate or skin of the subject, and washed repeatedly with antibiotic and antifungal agents.
  • the specimen of dermis is then separated into small pieces.
  • the pieces of the specimen are individually placed onto a dry surface of a tissue culture flask and allowed to attach for between 5 and 10 minutes before a small amount of medium is slowly added, taking care not to displace the attached tissue fragments. After 48 hours of incubation, the flask is fed with additional medium.
  • the initial amount of medium is 1.5- 2.0 ml.
  • the establishment of a cell line from the biopsy specimen ordinarily takes between 2 and 3 weeks, at which time the cells can be removed from the initial culture vessel for expansion.
  • the autologous fibroblasts can be stimulated to grow by a brief exposure to trypsin-EDTA, according to techniques known to those skilled in the art. The exposure to trypsin is too brief to release the fibroblasts form their attachment to the culture vessel wall.
  • samples of the autologous fibroblasts can be processed for frozen storage in, for example, liquid nitrogen.
  • the frozen storage of early rather than late passage fibroblasts is preferred because the number of passages in cell culture of normal human fibroblasts is limited.
  • the autologous fibroblasts can be frozen in any freezing medium suitable for preserving cells.
  • a medium consisting of about 70% growth medium, about 20% (v/v) fetal bovine serum and about 10% (v/v) dimethylsulfoxide (DMSO) can be used with good effect.
  • DMSO can also be substituted with, for example, glycerol .
  • Thawed cells can be used to initiate secondary cultures to obtain suspensions for use in the same subject without the inconvenience of obtaining a second specimen.
  • Any tissue culture technique that is suitable for the propagation of dermal fibroblasts from biopsy specimens may be used to expand the cells to practice the invention. Techniques for propagation known to those skilled in the art can be found in R.I. Freshney, Ed., ANIMAL CELL CULTURE: A PRACTICAL APPROACH (IRL Press,
  • the medium can be any medium suited for the growth of primary autologous fibroblast cultures. In most instances, the medium is supplemented with human or non- human serum in the amount of between about 0.0% and about 20% (v/v) to promote growth of the autologous fibroblasts. Higher concentrations of serum promote faster growth of the fibroblasts. In a preferred embodiment the serum is fetal bovine serum, which is added to a final concentration of about 10% of medium.
  • the medium can be glucose DMEM supplemented with about 2mM glutamine, about 110 mg/L sodium pyruvate, about 10% (v/v) fetal bovine serum and antibiotics ("complete medium"), wherein the concentration of glucose ranges from approximately 1,000 mg/L of medium to approximately 4,500 mg/L of medium. "Preferably, the concentration of glucose in the medium is approximately 4,500 mg/L of medium.
  • Autologous fibroblasts can be passaged into new flasks by trypsinization. For expansion, individual flasks are split 1:3. Triple bottom, T-150 flasks, having a total culture area of 450 cm 2 are suitable for the practice of the invention.
  • a triple bottom T-150 flask can be seeded with about 1X10 6 to about 3X10 S to about 3X10 6 cells and has a capacity to yield about 8X10 6 to about 1.3X10 7 cells.
  • the capacity of the flask is reached, which typically requires 5-7 days of culture, the growth medium is replaced by serum- free medium; thereafter the cells are incubated, i.e., held at between about 30°C and about 37.5°C, for at least 2 hours.
  • the incubation of the cells in serum free" medium substantially removes from the cells proteins that are derived from the fetal bovine serum which, if present, would elicit an immune subject.
  • the serum-free medium comprises glucose and DMEM supplemented with about 2 mM glutamine, about 110 mg/L sodium pyruvate, wherein the concentration of glucose ranges from approximately 1,000 mg/L of medium to approximately 4,500 mg/L of medium, and preferably is 4,500 mg/L of medium.
  • a biodegradable acellular matrix is then provided.
  • matrices which can be used in the present invention include, but are not limited to, acellular matrices comprising exogenous proteins, or matrices comprising biodegradable polymers.
  • biodegradable acellular matrices comprising exogenous proteins are presently available, and have ready applications in the present invention.
  • An embodiment of such biodegradable acellular matrices are matrices comprising any type of collagen, or any type of collagen with glycosaminoglycans (GAG) cross-linked with, for example, glutaraldehyde.
  • GAG glycosaminoglycans
  • Examples of collagen matrices having application in the present invention are absorbable collagen sponges made by the Calcitek Company of Carlsbad, CA.
  • collagen sponge dressings sold under the names COLLATAPE, " "COLLACOTE, “ and “COLLAPLUG” are made from cross-linked collagen extracted from bovine deep flexor (Achilles) tendon, and glycosaminoglycans (GAGS) . These products are soft, pliable, nonfriable, and non-pyrogenic In addition, more than 90% of this product consists of open pores.
  • An alternative biodegradable acellular matrix can consist of collagen
  • Bio- Gide ® has a bilayer structure with one surface that is porous allowing the ingrowth of cells and a second surface that is dense and will prevent the ingrowth of fibrous tissue.
  • Another biodegradable acellular matrix can be made from bone spongiosa formed into granules or blocks.
  • This material consists of animal (e.g., human, non-human primate, bovine, sheep, pig, or goat) bone from which substantially all organic material (e.g., proteins, lipids, nucleic acids, carbohydrates, and small organic molecules such as vitamins and non-protein hormones) have been removed.
  • This type of matrix is referred to herein as an anorganic matrix.
  • One such matrix which is marketed as either Bio-Oss ® spongiosa granules or Bio-Oss ® blocks, is ' manufactured by ED. GEISTLICH SOHNE AG.
  • Bio-Oss ® collagen also consisting of the anorganic bone, but containing in addition approximately 10% by weight of collagen fibers.
  • Other biodegradable acellular matrices having applications in the present invention can contain one or more of gelatin, polyglycolic acid, or cat gut sutures, demineralized bone, hydroxyapatite, or mixtures of these substances. Demineralized bone can be combined, for example, with collagen to produce a matrix in the form of a sponge, block, or membrane.
  • Synthetic polymers made from 1 or more monomers can also be used to make the biodegradable acellular matrices of the invention.
  • the matrices can be made from one or more of such synthetic polymers.
  • the synthetic polymers can also be combined with any of the above-mentioned substances to form matrices.
  • Different polymers forming a single matrix can be in separate compartments or layers.
  • Gore-Tex, Inc. manufactures a porous biodegradable acellular matrix (GORE RESOLUT XT Regenerative Material) that is composed of a synthetic bioabsorbable glycolide and trimethylene carbonate copolymer fiber (into which cells can migrate) attached to an occlusive membrane that does not permit ingrowth of cells and composed of a synthetic bioabsorbable glycolide and lactide copolymer.
  • a concentrated suspension of autologous passaged fibroblasts is evenly distributed on the surface of the matrix.
  • a concentrated suspension is necessary to avoid going beyond the capacity of the matrix to absorb the suspension.
  • a typical distribution of cell suspension using the GORE RESOLUT XT as the matrix comprises applying about 94 ⁇ l to about 125 ⁇ l of cell suspension having about 2.0X10 6 cells to about 4.0X10 6 cells, per square centimeter of matrix. Cells are allowed to attach to the matrix without further addition of media.
  • - incubation of the cells- with the matrix occurs at about 37°C for about 1-2 hours.
  • the cells readily attach and grow into the matrix, and are supplemented with additional growth medium.
  • Cells are then cultured in the matrix for about 3 to 5 days .
  • the matrix with the cells is then washed at least three times, for 10 minutes per wash, with, e.g., phosphate buffered solution (PBS) to substantially remove immunogenic proteins which may elicit an immune response in the subject.
  • PBS phosphate buffered solution
  • Fresh PBS is used for each washing.
  • the matrix is then incubated for at least one hour in fresh PBS prior to use. After incubation, the matrix comprising autologous fibroblasts is placed on the area of tissue degeneration, or defect, such as, for example, the periodontal pockets, and secured "so that it can not be moved from the site.
  • sponge matrix e.g. Collacote ®
  • approximately 1.5 -2.0 x 10 7 fibroblasts in approximately 1.5ml of growth medium are seeded onto 2cm x 4cm thin (approximately 2.5mm to 3.0mm thick) sponges.
  • the sponge is then incubated at 37°C for about 1-2 hours without further addition of medium to allow substantially all the fibroblasts to adhere to the matrix material .
  • additional growth medium is added to the composition of matrix and fibroblasts which is then incubated at 37 °C for 3-4 days (with daily change of medium) to allow for fibroblast cell division and, consequently, an increase in the number of fibroblasts in the matrix.
  • composition is then removed from growth medium containing FBS and washed repeatedly (at least 3 times) with FBS- free PBS. After each addition of PBS, the matrix is incubated for 10-20 minutes prior to discarding of the PBS. After the final wash, the composition is either applied directly to the area of the subject requiring tissue regeneration or is transferred to a shipping vial containing a physiological solution (e.g., Kreb's Ringer solution) and shipped (preferably overnight) to .a practitioner (e.g., a dentist or physician) .
  • a physiological solution e.g., Kreb's Ringer solution
  • a membranous matrix e.g. BiomendTM
  • approximately 3-8 x 10 6 fibroblasts in approximately lOO ⁇ l of growth medium are seeded onto the 15mm x20mm thin (approximately 0.5 to 1.0mm thick) membranes.
  • the membrane is then incubated at 37°C for about 30-60 minutes without further addition of medium to allow substantially all the fibroblasts to adhere to the matrix material.
  • additional growth medium is added to the composition of matrix and fibroblasts which is then incubated at 37°C for 2-3 days (with daily change of medium) to allow for fibroblast cell division and, consequently, an increase in the number of fibroblasts bound to the matrix. Washing of the composition and either immediate use or shipping are as described above for the sponge matrices .
  • a block matrix such as the above described anorganic matrix (e.g., Bio-Oss ® blocks)
  • approximately 5-10 x 10 6 fibroblasts in approximately lOO ⁇ l to 150 ⁇ l of growth medium are seeded into 1cm x 1cm x 2cm cubic blocks of matrix material.
  • Cells are slowly seeded onto one face of the block face. Once the medium and cells have been absorbed into the block, another face of the block is seeded in a similar fashion. The procedure is repeated until all faces of the block have been seeded and the block is fully saturated with medium. Care 'is taken to avoid adding excess medium and thereby causing leaking out of medium and cells from the block.
  • composition is then incubated at 37°C for about 30-60 minutes without further addition of medium to allow substantially all the fibroblasts to adhere to the matrix material. After cell adherence, additional growth medium is added to the composition of matrix and fibroblasts which is then incubated at 37°C for 2-3 days (with daily change of medium) to allow for fibroblast cell division and, consequently, an increase in the number of fibroblasts bound to the matrix. Washing of the composition and either immediate use or shipping are as described above for the sponge matrices.
  • compositions using Bio-Oss collagen, RESOLUT, and COLLACOTE as the matrix material have " been used to heal extra'ction sockets of 18 patients after extraction of a tooth.
  • untreated extraction sockets the subject's fibroblasts migrate into the sockets approximately 10-14 days after extraction of the tooth and thus the healing process only begins at that time.
  • the compositions containing the patient's fibroblasts into the sockets immediately after extraction of the tooth (e.g., within 1-3 hours of the extraction), the healing process is initiated immediately.
  • shrinkage of the socket due to collapse of the socket walls) , which is generally approximately 30% in untreated sockets, is minimized.
  • socket shrinkage be minimized in cases in which it is proposed to implant a dental prosthesis (e.g., a false tooth) at the site of the extraction at a later date.
  • a dental prosthesis e.g., a false tooth
  • fibroblasts obtained from 1 mm punch biopsies of the patients' gum tissues were used and the compositions were shipped to the dentists performing the procedures.
  • the compositions were shaped by the dentist to fit into the extraction sockets.
  • the compositions after shaping had an approximately cylindrical shape with a length of approximately 10 mm and a diameter of approximately 4 mm.
  • fragments of the compositions were prepared for histological analysis. In all cases, fibroblast colonization of and proliferation within the matrices was seen by light microscopy.
  • compositions using Bio-Oss ® collagen (2 patients) as the matrix had the advantage of persistence of the matrix material in the sockets longer than those compositions in which the matrix material consisted of collagen only and thus provided physical support for the fibroblasts before full replacement of the matrix material with components endogenous to the patient.
  • Example III An Injectable Composition for correcting skin defects and augmenting tissue in a Subject
  • the present invention extends to an injectable composition for correcting a defect in skin of a subject, or augmenting tissue of a subject, wherein the injectable composition comprises passaged autologous fibroblasts substantially free of immunogenic proteins, and a biodegradable, acellular injectable filler material.
  • skin defects that can be treated with the present invention include scars, particularly facial scars resulting from trauma or acne, or wrinkles.
  • the present invention can be used to augment tissue in a subject.
  • the injectable composition of the present invention can be injected into the lips of a subject in order to make the lips larger and fuller.
  • Fibroblasts are used in the present invention because they can be readily obtained and expanded, and are a cell type normally present beneath the dermis. Fibroblasts taken from a biopsy of the gums, palate, or skin of the subject can be used in the present invention, and can readily obtained using procedures set forth above .
  • a dermal fibroblast culture is initiated from a 1 to 5 mm full thickness biopsy specimen of the gums, palate or skin of the subject. Because of the phenomenon of allograft rejection, which is well known to transplantation surgeons and immunologists , it is essential that the cultured fibroblasts be histocompatible with the host. Histocompatibility can be ensured by obtaining a biopsy of the subject to be treated and culturing the fibroblasts from this specimen.
  • the biopsy Before the initiation of the culture, the biopsy is washed repeatedly with antibiotic and antifungal agents. The specimen of dermis is then separated into small pieces. The pieces of the specimen are individually placed onto the dry surface of a tissue culture flask and allowed to attach for between about 5 and about 10 minutes, before a small amount of medium is slowly added, taking care not to displace the attached tissue fragments. After about 48 hours of incubation, the flask is fed with additional medium. When a T-25 flask is used to start the culture, the initial amount of medium is about 1.5-2.0 ml. The establishment of a cell line from the biopsy specimen ordinarily takes between about 2 and 3 weeks, at which time the cells can be removed from the initial culture vessel for expansion.
  • the tissue fragments remain attached to the culture vessel bottom; fragments that detach should be reimplanted into new vessels.
  • the fibroblasts can be stimulated to grow by a brief exposure to EDTA-trypsin, according to techniques well known to those skilled in the art. The exposure to trypsin is too brief to release the fibroblasts from their attachment to the culture vessel wall.
  • samples of the fibroblasts can be processed for frozen storage, such as in liquid nitrogen.
  • frozen storage such as in liquid nitrogen.
  • numerous methods for successfully freezing cells for later * use are known in the art and are included in the present invention.
  • the frozen storage of early rather than late passage fibroblasts is preferred because the number of passages in cell culture of normal human fibroblasts is limited.
  • the fibroblasts can be frozen in any freezing medium suitable for preserving fibroblasts.
  • a medium consisting of about 70 % (v/v) growth medium, about 20 % (v/v) fetal bovine serum and about 10 % (v/v) dimethylsulfoxide (DMSO) can be used with good effect.
  • DMSO can also be substituted with, for example, glycerol .
  • Thawed cells can be used to initiate secondary cultures to obtain suspensions for use in the same subject without the inconvenience of obtaining a second specimen.
  • Any tissue culture technique that is suitable for the propagation of dermal fibroblasts- from biopsy specimens may be used to expand the cells to practice the invention. Techniques well known to those skilled in the art can be found in R.I. Freshney, Ed., ANIMAL CELL CULTURE: A PRACTICAL APPROACH (IRL Press, Oxford, England, 1986) and R.I. Freshney, Ed., CULTURE OF ANIMAL CELLS: A MANUAL OF BASIC TECHNIQUES, Alan R.
  • the medium can be any medium suited for the growth of primary fibroblast cultures.
  • the medium can be supplemented with human or non-human serum in an amount of between about 0.0 % and about 20 % (v/v) to promote growth of the fibroblasts. Higher concentrations of serum promote faster growth of the fibroblasts.
  • An example of medium having application herein comprises glucose DMEM supplemented with about 2 mM glutamine, about 110 mg/L sodium pyruvate, about 10 % (v/v) fetal bovine serum and antibiotics ("complete medium"), wherein the concentration of glucose ranges from approximately 1,000 mg/L of medium to 4,500 mg/L of medium, and preferably is 4,500 mg/L.
  • Fibroblasts can also be expanded in a serum-free medium.
  • Autologous fibroblasts can be passaged into new flasks by trypsinization. For expansion, individual flasks are split 1:3. Triple bottom, T-150 flasks, having a total culture area of 450 cm 2 are suitable for the practice of the invention.
  • a triple bottom T-150 flask can be seeded with about 1X10 6 to about 3X10 6 cells and has a capacity to yield about 8X10 6 to about 1.3X10 7 cells.
  • the capacity of the flask is reached, which typically requires about 5-7 days of culture, the growth medium is replaced by serum-free medium; thereafter the cells are incubated, i.e., held at between about 30° C and about 37.5° C, for at least 2 hours.
  • serum-free medium comprises glucose DMEM supplemented with about 2 mM glutamine, and about 110 mg/L sodium pyruvate, wherein the concentration of glucose is approximately 1,000 mg/L of medium to approximately 4,500 mg/L of medium, and preferably 4,500 mg/L of medium.
  • the cells are removed from the tissue culture flask by trypsin-EDTA; washed extensively by centrifugation and resuspension; and suspended for subsequent use in an injectable composition of the present invention, or for injection into the subject.
  • the cells can be transported at 4° C so long as they are injected within 24-48 hours of their suspension.
  • the cells can be suspended in an appropriate physiological solution with appropriate osmolarity and tested for pyrogens and endotoxin levels, except for the absence of phenol red pH indicator, and the replacement of the fetal bovine serum by the subject's serum for such transportation (transport medium) .
  • the cells can be suspended in Krebs-Ringer solution comprising 5 % dextrose or any other physiological solution.
  • the ' cells can be aspirated and injected into the transport medium.
  • the volume of saline or transport medium in which the cells are suspended is related to such factors as the number of fibroblasts the practitioner desires to inject, the extent of the defects to the subject's skin that are to be corrected, the size or number of the defects that are to be corrected, and the urgency of the subject's desire to obtain the results of the treatment. Moreover, the practitioner can suspend the cells in a larger volume of medium and inject correspondingly fewer cells at each injection site.
  • the passaged autologous fibroblasts of the present invention are mixed with a biodegradable, acellular injectable filler material in a ratio of approximately 1:1 by volume .
  • an injectable composition of the present invention also comprises a biodegradable, acellular injectable filler material.
  • a biodegradable, acellular injectable filler material can be comprised endogenous proteins, such as any type of collagen from the subject.
  • An example of such a filler is "AUTOLOGEN” produced by Collagenes-is , Inc. "AUTOLOGEN” is a dispersion of autologous dermal collagen fibers from the subject, and should not elicit an immune response.
  • AUTOLOGEN In order to obtain "AUTOLOGEN” for the subject, a specimen of tissue is obtained from the subject and forwarded to Collagenesis, Inc., where it is turned into “AUTOLOGEN". Approximately a one and a half square inch of tissue yields one cubic centimeter (cc) of "AUTOLOGEN” . After “AUTOLOGEN” has been prepared, its concentration can be adjusted depending upon the amount needed to correct defects in the subject's skin, or augment tissue in the subject. In particular, the concentration of "AUTOLOGEN" in the dispersion can be at least about 25 mg/L.
  • filler material comprises exogenous proteins, such as any type -of collagen.
  • exogenous proteins such as any type -of collagen.
  • numerous collagen products are commercially available and have applications in the present invention.
  • examples of such products are reconstituted bovine collagen products commercially available including, but not limited to, "ZYDERM I", “ZYDERM II” and “ZYPLAST”, which comprise reconstituted bovine collagen fibers cross-linked with glutaraldehyde.
  • FDA U.S. Food and Drug Administration
  • filler materials having applications in the present invention include, but are not limited to, solubilized gelatin, polyglycolic acid, or cat gut sutures.
  • a gelatin matrix implant sold under the mark "FIBRIL”, which comprises porcine gelatin powder plus o-aminocaproic acid, which are dispersed in a 0.9 % (by volume) sodium chloride solution and an aliquot of plasma from the subject, mixed in a 1 : 1 ratio by volume.
  • the present invention extends to methods for correcting a defect in skin of a subject, such as scars or wrinkles, or augmenting tissue in the subject, particularly facial tissue, e.g., augmenting the tissue of lips to make the lips appear fuller.
  • One such method of the present invention comprises injecting an effective amount of an injectable composition comprising autologous fibroblasts substantially free of immunogenic proteins and a biodegradable, acellular injectable filler material, into the subject at the site of the skin defect or desired tissue augmentation so that regeneration of tissue at the site is promoted.
  • Another method encompassed by the present invention comprises the steps of injecting passaged autologous fibroblasts substantially free of immunogenic proteins into the subject at a site of a skin defect or desired tissue augmentation, and injecting a biodegradable, acellular injectable filler material into the site.
  • the duration between injecting the autologous fibroblasts into the subject and injecting the biodegradable acellular injectable filler into the subject is about two weeks.
  • Passaged, autologous fibroblasts substantially free of immunogenic proteins can be readily obtained using procedures set forth above.
  • the various types of biodegradable, acellular filler materials which have been described in detail above, have applications in the methods described herein.
  • Injections set forth in the present invention are typically carried out with a hypodermic syringe having a syringe chamber, a piston disposed therein, an orifice communicating with the chamber, and a hypodermic needle is fixed to the orifice.
  • the size of the needle used in a method for correcting a defect in skin of a subject or augmenting tissue in the subject ranges from approximately 30 gauge to approximately 27 gauge.
  • the tissue to be injected is prepped with 'alcohol and stretched to give a taut surface.
  • a syringe is filled with an injectable composition of the present invention, if the first method described above is to be used, or with passaged, autologous fibroblasts substantially free of immunogenic proteins if the second method for correcting skin defects or augmenting tissue is used.
  • the needle is inserted into the tissue as superficially as possible, and the orientation of the bevel is not critical to the success of this method of present invention.
  • the actual injection is made by gentle pressure until a slight blanch is seen in the injected tissue. Multiple serial injections can be made.
  • an equal' volume of biodegradable, acellular filler is injected using the same procedure as described above into the same location where the passaged autologous fibroblasts were previously injected.

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Abstract

L'invention concerne une composition pour favoriser la régénération de tissu ayant dégénéré chez un sujet, consécutivement à une maladie ou à un trouble, et un procédé d'utilisation de ladite composition. Ladite composition comprend une matrice acellulaire biodégradable, et des fibroblastes autologues à passage, sensiblement exempts de protéines immunogènes intégrées à la matrice. Le procédé d'utilisation de la composition permettant de favoriser la régénération du tissu, consiste à placer la composition sur un site de tissu dégénéré chez un sujet, de manière que la composition favorise la régénération du tissu au niveau dudit site. La composition et son procédé d'utilisation peuvent être utilisés pour favoriser la régénération de tissu ayant dégénéré consécutivement à diverses maladies ou troubles, dont, entre autres, des défauts de la muqueuse buccale (ex. extraction d'une dent), une parodontopathie, le diabète, les ulcérations cutanées, la stase veineuse, les cicatrices, ou les rides. L'invention se rapporte encore à une composition injectable comprenant n'importe quel type de collagène et des fibroblastes autologues à passage, sensiblement exempts de protéines immunogènes, pour la correction de défauts de la peau, tels que les rides ou les cicatrices, et pour l'augmentation tissulaire chez le sujet, notamment du tissu facial.
PCT/US1999/011283 1998-05-22 1999-05-21 Compositions pour la regeneration de tissu degenere et procedes d'utilisation desdites compositions WO1999060951A1 (fr)

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000073418A3 (fr) * 1999-05-28 2001-06-28 Pacgen Technologies Llc Procede d'utilisation de fibroblastes autologues dans le but de favoriser la guerison de blessures et de fistules
WO2000074741A3 (fr) * 1999-06-08 2002-07-18 Universitaetsklinikum Freiburg Produit de synthese biologique pour articulation
US6713085B2 (en) * 2001-04-27 2004-03-30 Ed. Geistlich Soehne Ag Fuer Chemische Industrie Method and membrane for mucosa regeneration
EP1664275A2 (fr) * 2003-09-19 2006-06-07 The Board Of Trustees Of The University Of Illinois Synthese in vivo de tissus conjonctifs
EP1745759A2 (fr) * 2000-05-29 2007-01-24 CelGen AG Dispositifs et membranes médicales pour la régénération osseuse
WO2010052464A2 (fr) 2008-11-07 2010-05-14 Sportcell Compositions cellulaires et leurs utilisations
WO2011011808A1 (fr) * 2009-07-30 2011-02-03 Roman Buga Composition cosmétique comprenant du chlorure de sodium en combinaison avec et/ou au moins une protéine, un collagène, une gélatine, un acide aminé
WO2013166045A1 (fr) * 2012-04-30 2013-11-07 The Johns Hopkins University Procédés pour utiliser des fibroblastes autologues pour modifier l'identité cutanée
US20150165001A1 (en) * 2009-09-04 2015-06-18 Fujifilm Corporation Bone regeneration agent including gelatin
RU2693000C1 (ru) * 2018-05-24 2019-06-28 Общество с ограниченной ответственностью "Кардиоплант" Биологическая резорбируемая мембрана, армированная титановым каркасом, для костной регенерации
CN110193094A (zh) * 2018-02-24 2019-09-03 上海优先生物医学工程有限公司 一种软组织填充修复材料及其制备方法和用途

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5166187A (en) * 1987-06-15 1992-11-24 Centre National De La Recherche Biomaterials with a base of mixtures of collagen, chitosan and glycosaminoglycans, process for preparing them and their application in human medicine
US5591444A (en) * 1995-07-28 1997-01-07 Isolagen Technologies, Inc. Use of autologous dermal fibroblasts for the repair of skin and soft tissue defects
US5711957A (en) * 1993-05-13 1998-01-27 Inoteb Use of a porous calcium carbonate based material as support of a growth factor in the preparation of a bioabsorbable implant

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5166187A (en) * 1987-06-15 1992-11-24 Centre National De La Recherche Biomaterials with a base of mixtures of collagen, chitosan and glycosaminoglycans, process for preparing them and their application in human medicine
US5711957A (en) * 1993-05-13 1998-01-27 Inoteb Use of a porous calcium carbonate based material as support of a growth factor in the preparation of a bioabsorbable implant
US5591444A (en) * 1995-07-28 1997-01-07 Isolagen Technologies, Inc. Use of autologous dermal fibroblasts for the repair of skin and soft tissue defects
US5660850A (en) * 1995-07-28 1997-08-26 Isolagen Technologies, Inc. Use of autologous dermal fibroblasts for the repair of skin and soft tissue defects
US5665372A (en) * 1995-07-28 1997-09-09 Isolagen Technologies, Inc. Autologous dermal fibroblasts for the repair of skin and soft tissue defects

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LAMME E N, ET AL.: "LIVING SKIN SUBSTITUTES: SURVIVAL AND FUNCTION OF FIBROBLASTS SEEDED IN A DERMAL SUBSTITUTE IN EXPERIMENTAL WOUNDS", JOURNAL OF INVESTIGATIVE DERMATOLOGY, NATURE PUBLISHING GROUP, US, vol. 111, no. 06, 1 December 1998 (1998-12-01), US, pages 989 - 995, XP002919324, ISSN: 0022-202X, DOI: 10.1046/j.1523-1747.1998.00459.x *

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7846465B1 (en) 1999-05-28 2010-12-07 Fibrocell Science, Inc. Method of using autologous fibroblasts to promote healing of wounds and fistulas
WO2000073418A3 (fr) * 1999-05-28 2001-06-28 Pacgen Technologies Llc Procede d'utilisation de fibroblastes autologues dans le but de favoriser la guerison de blessures et de fistules
WO2000074741A3 (fr) * 1999-06-08 2002-07-18 Universitaetsklinikum Freiburg Produit de synthese biologique pour articulation
EP1745759A2 (fr) * 2000-05-29 2007-01-24 CelGen AG Dispositifs et membranes médicales pour la régénération osseuse
EP1745759A3 (fr) * 2000-05-29 2007-05-09 CelGen AG Dispositifs et membranes médicales pour la régénération osseuse
EP1709981A1 (fr) * 2001-04-27 2006-10-11 Ed. Geistlich Söhne Ag Für Chemische Industrie Membranes contenant du collagène pour la régénération de muqueuses
EP1252903B1 (fr) * 2001-04-27 2006-11-29 Ed Geistlich Söhne AG Für Chemische Industrie Membranes contenant du collagène pour la régénération de muqueuses
US6713085B2 (en) * 2001-04-27 2004-03-30 Ed. Geistlich Soehne Ag Fuer Chemische Industrie Method and membrane for mucosa regeneration
CZ302044B6 (cs) * 2001-04-27 2010-09-15 Ed. Geistlich Soehne Ag Fuer Chemische Industri Prípravek podporující regeneraci poškozené sliznice a jeho použití
US7709442B2 (en) 2003-09-19 2010-05-04 The Trustees Of Columbia University In The City Of New York In vivo synthesis of connective tissues
EP1664275A4 (fr) * 2003-09-19 2008-11-05 Univ Illinois Synthese in vivo de tissus conjonctifs
EP1664275A2 (fr) * 2003-09-19 2006-06-07 The Board Of Trustees Of The University Of Illinois Synthese in vivo de tissus conjonctifs
WO2010052464A2 (fr) 2008-11-07 2010-05-14 Sportcell Compositions cellulaires et leurs utilisations
WO2011011808A1 (fr) * 2009-07-30 2011-02-03 Roman Buga Composition cosmétique comprenant du chlorure de sodium en combinaison avec et/ou au moins une protéine, un collagène, une gélatine, un acide aminé
US20150165001A1 (en) * 2009-09-04 2015-06-18 Fujifilm Corporation Bone regeneration agent including gelatin
US9968661B2 (en) * 2009-09-04 2018-05-15 Fujifilm Corporation Bone regeneration agent including gelatin
WO2013166045A1 (fr) * 2012-04-30 2013-11-07 The Johns Hopkins University Procédés pour utiliser des fibroblastes autologues pour modifier l'identité cutanée
CN110193094A (zh) * 2018-02-24 2019-09-03 上海优先生物医学工程有限公司 一种软组织填充修复材料及其制备方法和用途
RU2693000C1 (ru) * 2018-05-24 2019-06-28 Общество с ограниченной ответственностью "Кардиоплант" Биологическая резорбируемая мембрана, армированная титановым каркасом, для костной регенерации

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