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WO1999043270A1 - Augmentation des tissus dermiques, sous-cutanes et des cordes vocales et reparation de leurs defauts - Google Patents

Augmentation des tissus dermiques, sous-cutanes et des cordes vocales et reparation de leurs defauts Download PDF

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Publication number
WO1999043270A1
WO1999043270A1 PCT/US1998/003538 US9803538W WO9943270A1 WO 1999043270 A1 WO1999043270 A1 WO 1999043270A1 US 9803538 W US9803538 W US 9803538W WO 9943270 A1 WO9943270 A1 WO 9943270A1
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tissue
cells
fibroblasts
subject
defect
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PCT/US1998/003538
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English (en)
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Gregory S. Keller
Don A. Kleinsek
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Gerigene Medical Corporation
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Priority to PCT/US1998/003538 priority Critical patent/WO1999043270A1/fr
Priority to AU66649/98A priority patent/AU6664998A/en
Publication of WO1999043270A1 publication Critical patent/WO1999043270A1/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
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • 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
    • A61L27/3683—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
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • 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
    • A61L27/3895—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/60—Materials for use in artificial skin
    • 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
    • C12N5/0653—Adipocytes; Adipose tissue
    • 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
    • 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
    • C12N5/066—Tenocytes; Tendons, Ligaments
    • 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
    • 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
    • A61F2310/00365—Proteins; Polypeptides; Degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells

Definitions

  • the field of the present invention is the long-term augmentation and/or repair of defects in dermal, subcutaneous, or vocal cord tissue.
  • these aforementioned three-dimensional substrates are inoculated with the cells to be cultured, which subsequently penetrate the substrate and establish a "tissue-like" histology similar to that found in vivo.
  • Several attempts to regenerate "tissue-like" histology from dispe rsed monolayers of cells utilizing three-dimensional substrates have been reported.
  • three-dimensional collagen substrates have been utilized to culture a variety of cells including breast epithelium (Yang, Cancer Res. 47:1021 (1981 ) ), vascular epithelium (Folkman et al, Nature 288 :551 (1980) ), and hepatocytes (Sirica et al, Cancer Res. 76:3259 (1980) ).
  • Non-biological injectable materials e g., paraffin
  • injectable silicone became prevalent in the 1960's for the correction of minor defects, although various inherent complications also limited the use of this substance.
  • Complications associated with the utilization of injectable liquid silicone include local and systemic inflammatory reactions, formation of scar tissue around the silicone droplets, rampant and frequently distant, unpredictable migration throughout the body, and localized tissue breakdown.
  • Injectable atelocollagen solution also was shown to be absorbed from the injection site, without replacement by host material, within a period of weeks to months.
  • Clinical protocols calling for repeated injections of atelocollagen are, in practice, primarily limited by the development of immunogenic reactions to the bovine collagen.
  • bovine atelocollagen was further processed by cross-linking with 0.25% glutaraldehyde, followed by filtration and mechanical shearing through fine mesh. The methodologies involved in the preparation and clinical utilization of this material are disclosed in U.S. Patent Nos. 4,582,640 and 4,642,1 17.
  • the modified atelocollagen was marketed as ZYPLAST ® brand cross-linked bovine atelocollagen.
  • cross-linking of the bovine atelocollagen was found to decrease the number of host cells which infiltrated the injected collagen site.
  • bovine atelocollagen cross-linked with glutaraldehyde may retain this agent as a high molecular weight polymer which is continuously hydrolyzed, thus facilitating the release of monomeric glutaraldehyde.
  • the monomeric form of glutaraldehyde is detectable in body tissues for up to 6 weeks after the initial injection of the cross-linked atelocollagen.
  • cytotoxic effect of glutaraldehyde on in vitro fibroblast cultures is indicative of this substance's not being an ideal cross-linking agent for a dermal equivalent which is eventually infiltrated host cells and in which the bovine atelocollagen matrix is rapidly degraded, thus resulting in the release of monomeric glutaraldehyde into the bodily tissues and fluids.
  • chondroitin-6-sulfate which weakly binds to collagen at neutral pH, has also been utilized to chemically modify bovine protein for tissue graft implantation. See Hansborough and Boyce, JAMA 136:2125 ( 1989). However, like glutaraldehyde, GAG may be released into the tissue causing unforeseen long-term effects on human subjects.
  • GAG has been reported to increase scar tissue formation in wounds, which is to be avoided in grafts. Additionally, a reduction of collagen blood clotting capacity may also be deleterious in the application in bleeding wounds, as fibrin clot contributes to an adhesion of the graft to the surrounding tissue.
  • Human collagen for injection derived from a sample of the patient's own tissue, is currently available and is marketed as AUTOLOGEN ® . It should be noted, however, that there is no quantitative evidence which demonstrates that human collagen injection results in lower levels of implant degradation than that which is found with bovine collagen preparations. Furthermore, the utilization of autologous collagen preparation and injection is limited to those individuals who have previously undergone surgery, due to the fact that the initial culture from which the collagen is produced is derived from the tissue removed during the surgical procedure. Therefore, it is evident that, although human collagen circumvents the potential for immunogenicity exhibited by bovine collagen, it fails to provide long-term therapeutic benefits and is limited to those patients who have undergone prior surgical procedures.
  • FIBREL a mixture of gelatin powder, e-aminocaproic acid, and the patient's plasma marketed as FIBREL ® .
  • the action of the FIBREL product appears to be dependent upon the initial induction of a sclerogenic inflammatory response to the augmentation of the soft tissue via the subcutaneous injection of the material. See e.g., Gold, J. Dermatologic Surg. Oncology, 20:586 (1994).
  • the present invention utilizes the surgical engraftment of autologous adipocytes which have been cultured on a solid support typically derived from, but not limited to, collagen or isolated extracellular matrix. The culture may be established from a simple skin biopsy specimen and the amount of adipose tissue which can be subsequently cultured in vitro is not limited by the amount of adipose tissue initially excised from the patient.
  • split thickness autographs have been used with a varying degree of success.
  • these forms of treatment have all exhibited numerous disadvantages.
  • split thickness autographs generally show limited tissue expansion, require repeated surgical operations, and give rise to unfavorable aesthetic results.
  • E pidermal autographs require long periods of time to be cultured, have a low success ('"take") rate of approximately 30-48%, frequently form spontaneous blisters, exhibit contraction to 60-70% of their original size, are vulnerable during the first 15 days of engraftment, and are of no use in situations where there is both epidermal and dermal tissue involvement.
  • epidermal allografts cultured allogenic keratinocytes
  • Additional methodologies have been examined which involve the utilization of irradiated cadaver dermis. However, this too has met with limited success due to, for example, graft rejection and unfavorable aesthetic results.
  • Living skin equivalents comprising a dermal layer of rodent fibroblast cells cast in soluble collagen and an epidermal layer of cultured rodent keratinocytes have been successfully grafted as allografts onto Sprague Dawley rats by Bell et al., J. Investigative Dermatology 81 :2 (1983). Histological examination of the engrafted tissue revealed that the epidermal layer had fully differentiated to form desmonosomes, tonofilaments, keratohyalin, and a basement lamella. However, subsequent attempts to reproduce the 1 iving skin equivalent using human fibroblasts and keratinocytes has met with only limited success. In general, the keratinocytes failed to fully differentiate to form a basement lamella and the dermo-epidermal junction was a straight line.
  • the present invention includes the following methodologies for the repair and/or augmentation of various skin defects: (1) the injection of autologously cultured dermal or fascial fibroblasts into various layers of the skin or injection directly into a "pocket” created in the region to be repaired or augmented, or (2) the surgical engraftment of "strands" derived from autologous dermal and fascial fibroblasts which are cultured in such a manner as to form a three-dimensional "tissue-like" structure similar to that which is found in vivo.
  • the present invention also differs on a two-dimensional level in that "true" autologous culture and preparation of the cells is performed by utilization of the patient's own cells and serum for in vi tro culture.
  • VOCAL CORD TISSUE AUGMENTATION AND/OR REPAIR Phonation is accomplished in humans by the passage of air past a pair of vocal cords located within the larynx. Striated muscle fibers within the larynx, comprising the constrictor muscles, function so as to vary the degree of tension in the vocal cords, thus regulating both their overall rigidity and proximity to one another to produce speech. However, when one (or both) of the vocal cords becomes totally or partially immobile, there is a diminution in the voice quality due to inability to regulate and maintain the requisite tension and proximity of the damaged cord in relation to that of the operable cord. Vocal cord paralysis may be caused by cancer, surgical or mechanical trauma, or similar afflictions which render the vocal cord incapable of being properly tensioned by the constrictor muscles.
  • SILASTIC ® siliconized rubber
  • the surgeon hand carves SILASTIC block during the procedure in order to maximize the ability of the patient to phonate.
  • the patient is kept under local anesthesia so that he or she can produce sounds to test the positioning of the implant.
  • the implanted blocks are formed into the shape of a wedge which is totally implanted within the thyroid cartilage or a flanged plug which can be moved back-and-forth within the opening in the thyroid cartilage to fine-tune the voice of the patient.
  • SILASTIC implants have proved to be superior over TEFLON injections, there are several areas of dissatisfaction with the procedure including difficulty in the carving and insertion of the block, the large amount of time required for the procedure, and a lack of an efficient methodology for locking the block in place within the thyroid cartilage.
  • vocal-cord edema due to the prolonged nature of the procedure and repeated voice testing during the operation, may also prove problematic in obtaining optimal voice quality.
  • the present invention discloses a methodology for the longterm augmentation and/or repair of dermal, suboutaneous, or vocal cord tissue by the injection or direct surgical placement/implantation of: (1) autologous cultured fibroblasts derived from connective tissue, dermis, or fascia; (2) lamina intestinal tissue; (3) fibroblasts derived from the lamina propria or (4) adipocytes.
  • the fibroblast cultures utilized for the augmentation and/or repair of skin defects are derived from either connective tissue, dermal, and/or fascial fibroblasts.
  • Typical defects of the skin which can be corrected with the injection or direct surgical placement of autologous fibroblasts or adipocytes include rhytids, stretch marks, depressed scars, cutaneous depressions of traumatic or non-traumatic origin, hypoplasia of the lip, and/or scarring from acne vulgaris.
  • Typical defects of the vocal cord which can be corrected by the injection or direct surgical placement of lamina intestinal or autologous cultured fibroblasts from lamina intestinal include scarred, paralyzed, surgically or traumatically injured, or congenitally underdeveloped vocal cord(s).
  • fibroblasts derived from the dermis, fascia, connective tissue, or lamina propria mitigates the possibility of an immunogenic reaction due to a lack of tissue histocompatibility. This provides vastly superior post-surgical results.
  • fibroblasts of connective tissue, dermal, or fascial origin as well as adipocytes are derived from full biopsies of the skin.
  • lamina propria tissue or fibroblasts obtained from the lamina intestinal are obtained from vocal cord biopsies.
  • the present invention further provides a methodology of rendering the cultured cells substantially free of potentially immunogenic serum-derived proteins by late-stage passage of the cultured fibroblasts, lamina intestinal tissue, or adipocytes in serum-free medium or in the patient's own serum.
  • immunogenic proteins may be markedly reduced or eliminated by repeated washing in phosphate-buffered saline (PBS) or similar physiologically-compatible buffers.
  • PBS phosphate-buffered saline
  • the skin is composed of two distinct layers: the epidern a specialized epithelium derived from the ectoderm, and beneath this, the dermis, a vascular dense connective tissue, a derivative of mesoderm. These two layers are firmly adherent to one another and form a region which varies in overall thickness from approximately 0.5 to 4 mm in different areas of the body. Beneath the dermis is a layer of loose connective tissue which varies from areolar to adipose in character. This is the superficial fascia of gross anatomy, and is sometimes referred as the hypodermis, but is not considered to be part of the skin. The dermis is connected to the hypodermis by connective tissue fibers which pass from one layer to the other.
  • EPIDERMIS The epidermis, a stratified squamous epithelium, is composed of cells of two separate and distinct origins. The majority of the epithelium, of ectodermal origin, undergoes a process of keratinization resulting in the formation of the dead superficial layers of skin. The second component comprises the melanocytes which are involved in the synthesis of pigmentation via melanin. The latter cells do not undergo the process of keratinization. The superficial keratanized cells are continuously lost from the surface and must be replaced by cells that arise from the mitotic activity of cells of the basal layers of the epidermis.
  • the average thickness of the dermis varies from 0.5 to 3 mm and is further subdivided into two strata - the papillary layer superficially and the reticular layer beneath.
  • the papillary layer is composed of thin collagenous, reticular, and elastic fibers arranged in an extensive network. Just beneath the epidermis, reticular fibers of the dermis form a close network into which the basal processes of the cells of the stratum germinativum are anchored. This region is referred to as the basal lamina.
  • the reticular layer is the main fibrous bed of the dermis.
  • the papillary layer contains more cells and smaller and finer connective tissue fibers than the reticular layer. It consists of coarse, dense, and interlacing collagenous fibers, in which are intermingled a small number of reticular fibers and a large number of elastic fibers. The predominant arrangement of these fibers is parallel to the surface of the skin.
  • the predominant cellular constituent of the dermis are fibroblasts and macrophages.
  • adipose cells may be present either singly or, more frequently, in clusters. Owing to the direction of the fibers, lines of skin tension, Langer's lines, are formed.
  • Smooth muscle fibers may also be found in the dermis. These fibers are arranged in small bundles in connection with hair follicles (arrectores pilorum muscles) and are scattered throughout the dermis in considerable numbers in the skin of the nipple, penis, scrotum, and parts of the perineum. Contraction of the muscle fibers gives the skin of these regions a wrinkled appearance. In the face and neck, fibers of some skeletal muscles terminate in delicate elastic fiber networks of the dermis.
  • ADIPOSE TISSUE/ADIPOCYTES Fat cells, or adipocytes, are scattered in areolar connective tissue.
  • adipocytes When adipocytes form large aggregates, and are the principle cell type, the tissue is designated adipose tissue. Adipocytes are fully differentiated cells and are thus incapable of undergoing mitotic division. New adipocytes therefore, which may develop at any time within the connective tissue, arise as a result of differentiation of more primitive cells. Although adipocytes, prior to the storage of lipid, resemble fibroblasts, it is likely that they arise directly from undifferentiated mesenchymal tissue.
  • Each adipocyte is surrounded by a web of fine reticular fibers; in the spaces between are found fibroblasts, lymphoid cells, eosinophils, and some mast cells.
  • the closely spaced adipocytes form lobules, separated by fibrous septa.
  • adipose tissue is not static There is a dynamic balance between lipid deposit and withdrawal.
  • the lipid contained within adipocytes may be derived from three sources.
  • Adipocytes under the influence of the hormone insulin, can synthesize fat from carbohydrate. They can also produce fat from various fatty acids which are derived from the initial breakdown of dietary fat. Fatty acids may also be synthesized from glucose in the liver and transported to adipocytes as serum lipoproteins. Fats derived from different sources also differ chemically. Dietary fats may be saturated or unsaturated, depending upon the individual diet. Fat which is synthesized from carbohydrate is generally saturated. Withdrawals of fat result from enzymatic hydrolysis of stored fat to release fatty acids into the blood stream.
  • the larynx is that part of the respiratory system which connects the pharynx and trachea. In addition to its function as part of the respiratory system, it plays an important role in phonation (speech).
  • the wall of the larynx is composed of a "skeleton" of hyaline and elastic cartilages, collagenous connective tissue, striated muscle, and mucous glands.
  • the major cartilages of the larynx (the thyroid, cricoid, and arytenoids) are hyaline, whereas the smaller cartilages (the corniculates, cuneiforms, and the tips of the arytenoids) are elastic, as is the cartilage of the epiglottis.
  • the aforementioned cartilages, together with the hyoid bone, are connected by three large, flat membranes: the thyrohyoid, the quadrates, and the cricovocal. These are composed of dense fibroconnective tissue in which many elastic fibers are present, particularly in the cricovocal membrane.
  • the true and false vocal cords are, respectively, the free upper boarders of the cricovocal (cricothyroid) and the free lower boarders of the quadrate (aryspiglottic) membranes. Extending laterally on each side between the true and false cords are the sinus and saccule of the larynx, a small slit-like diverticulum.
  • the posterior wall of the pharynx is formed by the striated muscle of the pharyngeal constrictor muscles.
  • the epithelium of the mucous membrane of the larynx varies with location. For example, over the vocal folds, the lamina intestinal tract of the vocal ligament is extremely dense and firmly bound to the underlying connective tissue of the vocal ligament. While there is no true submucosa in the larynx, the lamina intestinal of the mucous membrane is thick and contains large numbers of elastic fibers.
  • tissue culture techniques which are suitable for the propagation of non-differentiated mesenchymal cells may be used to expand the aforementioned cells/tissue and practice present invention as further discussed below. See e.g., Culture of Animal Cells: A Manual of Basic Techniques, Freshney, R. I., ed., (Alan R.
  • Autologous engraftment is a preferred therapeutic methodology due to the potential for graft rejection associated with the use of allograft-based engraftment.
  • Autologous grafts i.e., those derived directly from the patient ensure histocompatibility by initially obtaining a tissue sample via biopsy directly from the patient who will be undergoing the corrective surgical procedure and then subsequently culturing fibroblasts derived from the dermal, connective tissue, fascial, or lamina intestinall regions contained therein.
  • An autologous fibroblast culture is preferably initiated by the following methodology.
  • a full-thickness biopsy of the skin (-3x6 mm) is initially obtained through, for example, a punch biopsy procedure. The specimen is repeatedly washed with antibiotic and anti-fungal agents prior to culture.
  • the keratinized tissue-containing epidermis and subcutaneous adipocyte-containing tissue is removed, thus ensuring that the resultant culture is substantially free of non-fibroblast cells (e.g., adipocytes and keratinocytes).
  • the isolated adipocytes-containing tissue may then be utilized to establish adipocyte cultures. Alternately, whole tissue may be cultured and fibroblast-specific growth medium may be utilized to "select" for these cells.
  • Two methodologies are generally utilized for the autologous culture of fibroblasts in the practice of the present invention - mechanical and enzymatic.
  • the fascia, dermis, or connective tissue is intially dissected out and finely divided with scalpal or scissors.
  • the finely minced pieces of the tissue are initially placed in 1-2 ml of medium in either a 5 mm petri dish (Costar), a 24 multi-well culture plate (Corning), or other appropriate tissue culture vessel.
  • the tissue is incubated with 200-1000 U/ml of collagenase type II for a time period ranging from 30 minutes to 24 hours, as collagenase type II was found to be highly efficacious in providing a high yield of viable fibroblasts.
  • the cells are collected by centrifugation and resuspended into fresh medium in culture flasks.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • CCS cosmic calf serum
  • v/v concentrations of fetal bovine serum
  • CCS cosmic calf serum
  • substantial reductions in the concentration of serum results in a loss of cell viability in culture.
  • the complete culture medium typically contains Lglutamine, sodium bicarbonate, pyridoxine hydrochloride, lg/liter glucose, and gentamycin sulfate.
  • Cells are preferably removed for freezing and long-term storage during the early passage stages of culture, rather thane the later stages due to the fact that human fibroblasts are capable of undergoing a finite numbers of passages.
  • Culture medium containing 70% DMEM growth medium, 10% (v/v) serum, and 20% (v/v) tissue culture grade dimethyleulfoxide (DM SO, Gibco/BRL) may be effectively utilized for freezing of fibroblast cultures.
  • Frozen cells can subsequently be used to inoculate secondary cultures to obtain additional fibroblasts for use inthe original patient, thus doing away with the requirement to obtain a second biopsy specimen.
  • the removal of the various antigenic constituent proteins contained within the serum may be facilitated by collection of the fibroblasts by centrifugation, washing the cells repeatedly in phosphate-buffered saline (PBS) and then either re-suspending or culturing the washed fibroblas for a period of 2-24 hours in serum-free medium containing requisite growth factors which are well known in the field.
  • Culture media include, but are not limited to, Fibroblast Basal Medium (FBM). Alternately, the fibroblasts may be cultured utilizing the patient's own serum in the appropriate growth medium.
  • Fibroblasts utilized for injection consist of cells suspended in a collagen gel matrix or extracellular matrix.
  • the collagen gel matrix is preferably comprised of a mixture of 2 ml of a collagen solution containing 0.5 to 1.5 mg/ml collagen in 0. 05% acetic acid, 1 ml of DMEM medium, 270 ⁇ l of 7.5% sodium bicarbonate, 48 microliters of 100 micrograms/ml solution of gentamycin sulfate, and up to 5x10 6 fibroblast cell/ml of collagen gel.
  • the collagen may be derived from human or bovine sources, or from the patient and may be enzymatically- or chemically-modified (e.g., atelocollagen).
  • Three-dimensional "tissue" is formed by initially suspending the fibroblasts in the collagen gel matrix as described above.
  • full-length collagen is utilized, rather than truncated or
  • microporous membrane typically
  • system may be fabricated in any desired shape or size (e.g.,
  • Adipocytes require a "feeder-layer" or other type of solid
  • the solid support may be provided by
  • adipocytes are "seeded" onto the surface
  • adipocytes are removed by gentle
  • the extracellular matrix may be isolated in either a cellular or acellular form. Constituent materials which form the
  • ECM include, but are not limited to, collagen, elastin, fibrin,
  • fibrinogen fibrinogen, proteases, fibronectin, laminin, fibrellins, and
  • ECM is typically isolated by the initial
  • the ECM may be obtained by
  • the isolated ECM may then be utilized as a "filler" material in the various augmentation or repair procedures disclosed in the present application.
  • the ECM may possess certain cell growth- or metabolism-promoting characteristics.
  • fibroblasts are injected initially into the lower dermis, next in
  • fibroblast suspension is injected via a syringe with a needle
  • the gauge are used with general and local anesthesia, respectively.
  • the needle ' is placed at approximately a 45° angle to the skin
  • Subcutaneous injection is accomplished by initial placement of the needle into the subcutaneous tissue and
  • the needle is preferably inserted into the skin from various directions such
  • the injections may pass into deeper
  • philtrum may also be injected.
  • the suspension is subsequently injected into the deeper tissues of the lip, including the
  • passer needle is selected which is larger in diameter
  • the passer needle is then placed into the skin and threaded down
  • position may be adjusted by pulling on the distal point guide suture or, alternately, the guide suture closest to the passer needle entry point. While the dermal or fascial strand is held
  • the fascial or dermal graft following the final cutting of the remaining suture.
  • the fascial or dermal graft is placed into the subcutaneous layer of the skin. However, in some situations, it
  • a subcutaneous "pocket" may be created with a
  • lamina propria tissue finely minced if required
  • fibroblasts derived from lamina limbal tissue
  • preferable methodology consists of injection directly into the space containing the lamina intestinal, specifically into Reinke ' s
  • the material is injected via a
  • needles ranging from 22 to 18 gauge and 30 to 27
  • the materials are preferably injected
  • the fibroblasts may be injected into scar, Reinke 's
  • muscle Tne procedure may be performed under general, local,
  • a "pocket" may be created by needle dissection. Alternately, laryngeal
  • the desired material is then placed into the pocket with

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Abstract

Cette invention concerne un procédé permettant d'effectuer une opération chirurgicale et de correction de défauts qui peuvent être rectifiés par l'augmentation des tissus sous-jacents. Ce procédé consiste à prélever des cellules viables chez un sujet, un nouveau-né ou un foetus humain. Les cellules sont ensuite cultivées in vitro puis placées dans les tissus du sujet qui se trouvent en une position sous-jacente par rapport au défaut à rectifier. Dans une autre variante, les cellules peuvent être cultivées dans une matrice au collagène ou mises en suspension dans une matrice au collagène avant d'être placées en une position sous-jacente par rapport au défaut à rectifier. Dans un autre mode de réalisation, les cellules sont cultivées et la matrice extracellulaire produite in vitro est recueillie puis placée en une position sous-jacente par rapport au défaut à rectifier. Cette invention concerne également un procédé permettant de corriger les défauts des cordes vocales.
PCT/US1998/003538 1998-02-24 1998-02-24 Augmentation des tissus dermiques, sous-cutanes et des cordes vocales et reparation de leurs defauts WO1999043270A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/US1998/003538 WO1999043270A1 (fr) 1998-02-24 1998-02-24 Augmentation des tissus dermiques, sous-cutanes et des cordes vocales et reparation de leurs defauts
AU66649/98A AU6664998A (en) 1998-02-24 1998-02-24 Augmentation and repair of dermal, subcutaneous, and vocal cord tissue defects

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1998/003538 WO1999043270A1 (fr) 1998-02-24 1998-02-24 Augmentation des tissus dermiques, sous-cutanes et des cordes vocales et reparation de leurs defauts

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WO2003068287A1 (fr) * 2002-02-11 2003-08-21 Neocutis Sa Compositions comprenant des cellules embryonnaires indifferenciees destinees au traitement d'affections de la peau
WO2004029230A2 (fr) * 2002-09-27 2004-04-08 Verigen Ag Cellules autologues semees sur une matrice de support pour effectuer des reparations tissulaires
EP1653807A2 (fr) * 2003-05-01 2006-05-10 Medgenics, Inc. Micro-organes dermiques, leurs procedes et appareils de production et d'utilisation
US7326571B2 (en) 2003-07-17 2008-02-05 Boston Scientific Scimed, Inc. Decellularized bone marrow extracellular matrix
US7767452B2 (en) 1997-02-20 2010-08-03 Kleinsek Don A Tissue treatments with adipocyte cells
US20110081323A1 (en) * 2005-09-21 2011-04-07 Dask Technologies, Llc Methods and compositions for organ and tissue functionality
US8088568B2 (en) 2001-11-05 2012-01-03 Medgentics, Inc. Dermal micro-organs, methods and apparatuses for producing and using the same
US8142990B2 (en) 2001-11-05 2012-03-27 Medgenics Inc. Dermal micro-organs, methods and apparatuses for producing and using the same
US8449879B2 (en) 2004-09-15 2013-05-28 Neogyn, Inc. Fetal skin cell protein compositions for the treatment of skin conditions, disorders or diseases and methods of making and using the same
US8501396B2 (en) 2001-11-05 2013-08-06 Medgenics Medical Israel Ltd. Dermal micro-organs, methods and apparatuses for producing and using the same
US8877175B2 (en) 2006-09-14 2014-11-04 Medgenics Medical Israel Ltd. Long lasting drug formulations
US9127084B2 (en) 2006-09-14 2015-09-08 Medgenics Medical Israel Ltd. Long lasting drug formulations
US9155749B2 (en) 2006-09-14 2015-10-13 Medgenics Medical Israel Ltd. Long lasting drug formulations

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FR3083246B1 (fr) * 2018-06-29 2023-12-08 Oreal Modeles cellulaires et tissulaires comprenant des fibroblastes de la jonction dermo-hypodermique et ses applications

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US7767452B2 (en) 1997-02-20 2010-08-03 Kleinsek Don A Tissue treatments with adipocyte cells
US8142990B2 (en) 2001-11-05 2012-03-27 Medgenics Inc. Dermal micro-organs, methods and apparatuses for producing and using the same
US8088568B2 (en) 2001-11-05 2012-01-03 Medgentics, Inc. Dermal micro-organs, methods and apparatuses for producing and using the same
US9468667B2 (en) 2001-11-05 2016-10-18 Medgenics Medical Israel Ltd. Dermal micro-organs, methods and apparatuses for producing and using the same
US9107896B2 (en) 2001-11-05 2015-08-18 Medgenics Medical Israel Ltd. Dermal micro-organs, methods and apparatuses for producing and using the same
US8293463B2 (en) 2001-11-05 2012-10-23 Medgenics Inc. Dermal micro-organs, methods and apparatuses for producing and using the same
US8530149B2 (en) 2001-11-05 2013-09-10 Medgenics Medical Israel Ltd Dermal micro-organs, methods and apparatuses for producing and using the same
US8501396B2 (en) 2001-11-05 2013-08-06 Medgenics Medical Israel Ltd. Dermal micro-organs, methods and apparatuses for producing and using the same
WO2003068287A1 (fr) * 2002-02-11 2003-08-21 Neocutis Sa Compositions comprenant des cellules embryonnaires indifferenciees destinees au traitement d'affections de la peau
US9078903B2 (en) 2002-02-11 2015-07-14 Merz Gmbh & Co. Kgaa Compositions for the treatment of skin conditions, disorders or diseases and methods of making and using the same
US8394371B2 (en) 2002-02-11 2013-03-12 Neocutis Sa Compositions for the treatment of skin conditions, disorders or diseases and methods of making and using the same
WO2004029230A3 (fr) * 2002-09-27 2004-10-14 Verigen Ag Cellules autologues semees sur une matrice de support pour effectuer des reparations tissulaires
WO2004029230A2 (fr) * 2002-09-27 2004-04-08 Verigen Ag Cellules autologues semees sur une matrice de support pour effectuer des reparations tissulaires
US8685635B2 (en) 2002-11-05 2014-04-01 Medgenics Medical Israel Ltd. Dermal micro-organs, methods and apparatuses for producing and using the same
US8771291B2 (en) 2002-11-05 2014-07-08 Medgenics Medical Israel Ltd. Dermal micro-organs, methods and apparatuses for producing and using the same
US9101595B2 (en) 2002-11-05 2015-08-11 Medgenics Medical Israel Ltd. Dermal micro-organs, methods and apparatuses for producing and using the same
EP2377401A1 (fr) * 2003-05-01 2011-10-19 Medgenics, Inc. Micro-organe dermique génétiquement modifié exprimant l'érythropoïétine
EP2936984A1 (fr) * 2003-05-01 2015-10-28 Medgenics, Inc. Appareil et procédé pour récolter un micro-organe dermique
EP1653807B1 (fr) * 2003-05-01 2012-05-23 Medgenics, Inc. Utilisation de micro-organes dermiques
EP2377403A1 (fr) * 2003-05-01 2011-10-19 Medgenics, Inc. Micro-organe dermique génétiquement modifié exprimant le facteur VIII
EP2377404A1 (fr) * 2003-05-01 2011-10-19 Medgenics, Inc. Micro-organe dermique qui est un explant de tissus vivants
US9572593B2 (en) 2003-05-01 2017-02-21 Medgenics Medical Israel Ltd. Dermal micro-organs, methods and apparatuses for producing and using the same
EP1653807A2 (fr) * 2003-05-01 2006-05-10 Medgenics, Inc. Micro-organes dermiques, leurs procedes et appareils de production et d'utilisation
EP2377402A1 (fr) * 2003-05-01 2011-10-19 Medgenics, Inc. Micro-organe dermique génétiquement modifié exprimant l'interféron
US7326571B2 (en) 2003-07-17 2008-02-05 Boston Scientific Scimed, Inc. Decellularized bone marrow extracellular matrix
US8790920B2 (en) 2003-07-17 2014-07-29 Boston Scientific Scimed, Inc. Decellularized bone marrow extracellular matrix
US8449879B2 (en) 2004-09-15 2013-05-28 Neogyn, Inc. Fetal skin cell protein compositions for the treatment of skin conditions, disorders or diseases and methods of making and using the same
US9278122B2 (en) 2004-09-15 2016-03-08 Merz Gmbh & Co. Kgaa Fetal skin cell protein compositions for the treatment of skin conditions, disorders or diseases and methods of making and using the same
US20110081323A1 (en) * 2005-09-21 2011-04-07 Dask Technologies, Llc Methods and compositions for organ and tissue functionality
US9127084B2 (en) 2006-09-14 2015-09-08 Medgenics Medical Israel Ltd. Long lasting drug formulations
US9155749B2 (en) 2006-09-14 2015-10-13 Medgenics Medical Israel Ltd. Long lasting drug formulations
US8877175B2 (en) 2006-09-14 2014-11-04 Medgenics Medical Israel Ltd. Long lasting drug formulations
US9687564B2 (en) 2006-09-14 2017-06-27 Medgenics Medical Israel Ltd. Long lasting drug formulations

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