WO1995030742A1 - Procedes et compositions de reparation de defauts de cartilage articulaire chez les mammiferes - Google Patents

Procedes et compositions de reparation de defauts de cartilage articulaire chez les mammiferes Download PDF

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Publication number
WO1995030742A1
WO1995030742A1 PCT/US1995/005609 US9505609W WO9530742A1 WO 1995030742 A1 WO1995030742 A1 WO 1995030742A1 US 9505609 W US9505609 W US 9505609W WO 9530742 A1 WO9530742 A1 WO 9530742A1
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cartilage
cells
synthetic
cell
well
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PCT/US1995/005609
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English (en)
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Ross A. Tubo
Leesa M. Barone
Courtney A. Wrenn
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Genzyme Corporation
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Priority to AU24354/95A priority Critical patent/AU2435495A/en
Publication of WO1995030742A1 publication Critical patent/WO1995030742A1/fr

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    • 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
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3817—Cartilage-forming cells, e.g. pre-chondrocytes
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    • 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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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Definitions

  • This invention relates to methods and compositions for the repair of articular cartilage defects in a mammal.
  • the methods and synthetic cartilage compositions of the invention are particularly useful in treatment of partial-thickness and full-thickness articular cartilage defects.
  • Cartilage is a hyperhydrated structure with water comprising 70% to 80% of its weight. The remaining 20% to 30% comprises type II collagen and proteoglycan.
  • the collagen usually accounts for 70% of the dry weight of cartilage (in "Pathology” (1988) Eds. Rubin & Farber, J.B. Lippincott Company, PA. pp. 1369-1371).
  • Proteoglycans are composed of a central protein core from which long chains of polysaccharides extend. These polysaccharides, called glycosaminoglycans, include: chondroitin-4-sulfate; chondroitin-6-sulfate; and keratan sulfate.
  • Cartilage has a characteristic structural organization consisting of chondrogenic cells dispersed within an endogenously produced and secreted extracellular matrix.
  • the cavities in the matrix which contain the chondrocytes are called cartilage lacunae.
  • cartilage is neither innervated nor penetrated by either the vascular or lymphatic systems (Clemente (1984) in "Gray's Anatomy, 30 th Edit,” Lea & Febiger).
  • Hyaline cartilage consists of a gristly mass having a firm, elastic consistency, is translucent and is pearly blue in color. Hyaline cartilage is predominantly found on the articulating surfaces of articulating joints. It is found also in epiphyseal plates, costal cartilage, tracheal cartilage, bronchial cartilage and nasal cartilage. Fibrocartilage is essentially the same as hyaline cartilage except that it contains fibrils of type I collagen that add tensile strength to the cartilage.
  • the collagenous fibers are arranged in bundles, with the cartilage cells located between the bundles.
  • Fibrocartilage is found commonly in the anulus fibrosus of the invertebral disc, tendinous and ligamentous insertions, menisci, the symphysis pubis, and insertions of joint capsules.
  • Elastic cartilage also is similar to hyaline cartilage except that it contains fibers of elastin. It is more opaque than hyaline cartilage and is more flexible and pliant. These characteristics are defined in part by the elastic fibers embedded in the cartilage matrix. Typically, elastic cartilage is present in the pinna of the ears, the epiglottis, and the larynx.
  • articular cartilage prevents direct contact of the opposing bone surfaces and permits the near frictionless movement of the articulating bones relative to one another (Clemente, supra) .
  • Two types of articular cartilage defects are commonly observed in mammals and include full-thickness and partial-thickness defects.
  • the two-types of defects differ not only in the extent of physical damage but also in the nature of repair response each type of lesion elicits.
  • Full-thickness articular cartilage defects include damage to the articular cartilage, the underlying subchondral bone tissue, and the calcified layer of cartilage located between the articular cartilage and the subchondral bone.
  • Full-thickness defects typically arise during severe trauma of the joint or during the late stages of degenerative joint diseases, for example, during osteoarthritis. Since the subchondral bone tissue is both innervated and vascularized damage to this tissue is often painful. The repair reaction induced by damage to the subchondral bone usually results in the formation of fibrocartilage at the site of the full-thickness defect. Fibrocartilage, however, lacks the biomechanical properties of articular cartilage and fails to persist in the joint on a long term basis.
  • Partial-thickness articular cartilage defects are restricted to the cartilage tissue itself. These defects usually include fissures or clefts in the articulating surface of the cartilage. Partial- thickness defects are caused by mechanical derangement's of the joint which in turn induce wearing of the cartilage tissue within the joint. In the absence of innervation and vasculature, partial- thickness defects do not elicit repair responses and therefore tend not to heal. Although painless, partial-thickness defects often degenerate into full- thickness defects.
  • the chondrocyte progenitor cells differentiate subsequently into chondrocytes that in turn secrete components of the extracellular matrix thereby to form articular cartilage at the site of the defect _in situ.
  • synthetic cartilage tissue may be grown .in vitro and implanted subsequently into the cartilage defect.
  • the synthetic matrices or biological resorbable immobilization vehicles may be impregnated with allogeneic chondrocytes.
  • synthetic carrier matrices include: three-dimensional collagen gels (U.S. Pat.
  • the defect may be filled with a biocompatible, biodegradable matrix containing growth factors to stimulate the influx of chondrocyte progenitor cells into the matrix _in situ.
  • the matrices optimally contain pores of sufficient dimensions to permit the influx into, and proliferation of the chondrocyte progenitor within the matrix.
  • the matrix also may contain differentiating growth factors to stimulate the differentiation of chondrocyte progenitor cells into chondrocytes. The resulting chondrocytes hopefully secrete extracellular matrix components thereby to form cartilage at the site of the defect _in situ. See for example, U.S. Pat. No. 5,206,023; U.S. Pat. No. 5,270,300; and EP 05 30 804 Al.
  • This approach may have problems similar to those associated with the first approach, hereinabove.
  • chondrocytes may be cultured in vitro thereby to form synthetic cartilage-like material.
  • the resulting cartilage may be implanted subsequently into the cartilage defect.
  • This type of approach has the advantage over the previous methods in that the development of the synthetic cartilage material may be monitored prior to implantation.
  • the resulting cartilage may be characterized biochemically and morphologically prior to implantation.
  • Two general procedures have been developed for growing synthetic cartilage i ⁇ vitro. These include growing chondrogenic cells in either an anchorage-dependent or an anchorage-independent manner.
  • the chondrogenic cells may be cultured as colonies within an agarose gel. See for example: Benya et a ⁇ . (1982) Cell 3_0-.215-224; Aydlotte et al. (1990) in Methods and Cartilage Research Chapter 23:pp. 90-92; Aulthouse et al. (1989) JEn Vitro Cellular and Developmental Biology 25:659-668; Delbruck et al. (1986) Connective Tissue Res. 15:1550-172; and Bohme et al_. (1992) J. Cell Biol. 116:1035-1042.
  • Benya et a ⁇ . (1982) Cell 3_0-.215-224 Aydlotte et al. (1990) in Methods and Cartilage Research Chapter 23:pp. 90-92; Aulthouse et al. (1989) JEn Vitro Cellular and Developmental Biology 25:659-668; Delbruck et al. (1986) Connective Tissue Res. 15
  • the resulting cartilage remains embedded within a gel matrix making it unsuitable for implantation into mammals.
  • chondrocytes may be cultured as colonies in suspension culture. See for example, Franchimont et al. (1989) J_ ⁇ Rheumatol. 1 ⁇ :5-9; and Bassleer et a_l. (1990) in "Methods and Cartilage Research", Academic Press Ltd., Chapter 24.
  • the resulting particles containing synthetic cartilage-like material may be small and of undefined shape thus making the particles unsuitable for implantation and repair of a pre-determined articular cartilage defect.
  • primary cultures of chondrogenic cells isolated from primary tissue may be grown as monolayers attached to the surface of a cell culture flask. See for example: Yoshihashi (1983) J. Jpn. Ortho. Assoc. 5_8:629-641; and U.S. Pat. No. 4,356,261.
  • the primary cells derived directly from explant tissue remain capable of producing and secreting extracellular components characteristic of natural cartilage, specifically, type II collagen and sulfated proteoglycans.
  • the proliferated but undifferentiated chondrogenic cells may be cultured under conditions that stimulate the secretion of extracellular components characteristic of cartilage.
  • the invention comprises a method for preparing in vitro a synthetic cartilage patch for the repair of a cartilage defect in a mammal.
  • the method includes: (1) seeding denuded chondrogenic cells, proliferated ex vivo, into a pre-shaped well having a cell contacting, cell abhesive surface; and (2) culturing the proliferated chondrogenic cells in the well for a time sufficient to permit the cells to secrete an extracellular matrix thereby to form a three-dimensional, multi cell-layered patch of synthetic cartilage.
  • the resulting synthetic cartilage preferably synthetic articular cartilage, contains chondrogenic cells dispersed within an endogenously produced and secreted extracellular matrix.
  • the resulting synthetic cartilage patch may be used subsequently for the repair of an articular cartilage defect in a mammal.
  • synthetic cartilage is understood to mean any cartilage tissue produced in vitro that contains chondrogenic cells dispersed within an endogenously produced and secreted extracellular matrix.
  • the extracellular matrix is composed of collagen fibrils (predominantly fibrils of type II collagen), sulfated proteoglycans, for example, chondroitin-6-sulfate and keratan sulfate, and water.
  • synthetic articular cartilage is understood to mean any cartilage tissue produced in vitro that biochemically and morphologically resembles the cartilage normally found on the articulating surfaces of mammalian joints.
  • cartilage cell is understood to mean any cell which, when exposed to an appropriate stimuli, may differentiate into a cell capable of producing and secreting components characteristic of cartilage tissue, for example, fibrils of type II collagen, and the sulfated proteoglycans, chondroitin-6-sulfate and keratan sulfate.
  • the term "denuded cell” is understood to mean any cell that has been isolated from a disaggregated tissue containing such a cell.
  • the tissue of interest may be enzymatically and/or mechanically disaggregated in order to release the denuded cells.
  • the cells are cultured in a pre-shaped well having a cell contacting, cell abhesive surface.
  • the cell abhesive surface discourages the chondrogenic cells from attaching to the cell contacting surface of the well.
  • the use of such a well having a cell contacting, cell abhesive surface is a critical aspect of the instant invention.
  • chondrogenic cells expanded by serially passaging the cells as monolayers usually lose their ability to secrete type II collagen and sulfated proteoglycans. It now has been discovered that the undifferentiated, proliferated chondrogenic cells when cultured in such a well redifferentiate and once again start to secrete cartilage specific type II collagen and sulfated proteoglycans.
  • the actual dimensions of the well may be pre-determined when the actual size and shape of the cartilage defect to be repaired is known.
  • the well may be dimensioned such that the resulting cartilage may interfit directly into the cartilage defect.
  • the synthetic cartilage patch may be "trimmed" mechanically to the appropriate size and shape by the surgeon prior to insertion into the defect during a surgical procedure.
  • synthetic cartilage patches prepared in such a manner have the additional advantage over patches prepared as anchorage-dependent primary explant cultures in that the patches may be easily removed from the well obviating the use of enzymatic or other mechanical procedures. Such procedures may affect deleteriously the biochemical and/or biomechanical properties of the resulting cartilage patch.
  • Cell abhesive surfaces may be prepared by coating the cell contacting surface of a well with a reagent that discourages cell attachment.
  • Preferred reagents include, but are not limited to, silicon based reagents, for example, dichlorodimethylsilane or polytetrafluoroethylene based reagents, for example, Teflon ⁇ .
  • the well may be cast in a material that naturally discourages the attachment of chondrogenic cells.
  • Preferred materials include, but are not limited to, agarose, glass, untreated cell culture plastic and polytetrafluoroethylene, for example. Teflon ⁇ . It is contemplated that any biocompatible material or coating capable of discouraging the attachment of chondrogenic cells may be useful in the practice of the instant invention.
  • Chondrogenic cells useful in the practice of the invention may be isolated from essentially any tissue containing chondrogenic cells.
  • the chondrogenic cells may be isolated directly from pre ⁇ existing cartilage tissue, for example, hyaline cartilage, elastic cartilage, or fibrocartilage.
  • chondrogenic cells may be isolated from articular cartilage (from either weight bearing or non- weight bearing joints), costal cartilage, nasal cartilage, auricular cartilage, tracheal cartilage, epiglottic cartilage, thyroid cartilage, arytenoid cartilage and cricoid cartilage. Methods for isolating chondrogenic cells from such tissues are set forth hereinbelow.
  • chondrogenic cells may be isolated from bone marrow. See for example, U.S. Pat. Nos. 5,197,985 and 4,642,120, and Wakitani et al. (1994) J. Bone Joint Surg. 2_5 :579_591 > the disclosures of which are incorporated by reference herein.
  • chondrogenic cells Once chondrogenic cells have been isolated from the pre-existing tissue they are proliferated ex vivo in monolayer culture using conventional techniques well known in the art. See for example, Pollack (1975) in “Readings in Mammalian Cell Culture", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, the disclosure of which is incorporated by reference herein. Briefly, the population of chondrogenic cells is expanded by culturing the cells as monolayers and by serially passaging the cells. The chondrogenic cells are passaged after the cells have proliferated to such a density that they contact one another on the surface of the cell culture plate. During the passaging step, the cells are released from the substratum.
  • the proteolytic enzyme hydrolyzes proteins which anchor the cells on the substratum.
  • the resulting cells now in suspension, are diluted with culture medium and replated into a new tissue culture dish at a cell density such that the cells do not contact one another.
  • the cells subsequently reattach onto the surface of the tissue culture and start to proliferate once again.
  • the cells in suspension may be cryopreserved for subsequent use using techniques well known in the art. See for example. Pollack (supra) .
  • the cells are repeatedly passaged until enough cells have been propagated to prepare a piece of synthetic cartilage of pre-determined size.
  • a population containing a small number of chondrogenic cells originally isolated from a biopsy sample may be expanded iri vitro thereby to generate a large number of chondrogenic cells for subsequent use in the practice of the invention.
  • the chondrogenic cells are enzymatically released from the substratum to provide a suspension of cells.
  • the cells in suspension then are diluted by the addition of cell culture medium to give a cell density of about Ixl0 5 -lxl0 9 proliferated chondrogenic cells per ml, or more preferably about Ixl0 6 -5xl0 8 cells per ml, and most preferably about 3xl0 6 -2xl0 8 cells per ml.
  • the cells then are seeded into the pre-shaped well having a cell contacting, cell abhesive surface.
  • Ixl0 3 -lxl0 7 cells preferably Ixl0 -lxl0 6 cells, and most preferably about 5x10 -5xl0 5 cells produce a piece of synthetic cartilage 1 mm 3 in volume.
  • the artisan may produce a patch of synthetic cartilage of pre-determined size by seeding an appropriate number of chondrogenic cells into a pre-shaped well.
  • the cells subsequently are cultured in the well under conventional cell culture conditions well known in the art from 1 to 90 days, preferably 5 to 60 days, and most preferably 10 to 30 days thereby to induce the production and secretion of extracellular matrix.
  • the present invention therefore enables the production of large quantities of synthetic cartilage patches from small pieces of biopsied tissue.
  • the chondrogenic cells once proliferated ejc vivo, may be seeded into a pre- shaped well dimensioned to determine the volume of the. resulting cartilage tissue. Therefore, using the methodologies described herein, one skilled in the art may prepare synthetic cartilage of pre-determined volume for the repair of articular cartilage defects of pre-determined volume.
  • polypeptide growth factors may be added to the chondrogenic cells in the pre-shaped well to enhance or stimulate the production of cartilage specific proteoglycans and/or collagen.
  • Preferred growth factors include, but are not limited to, transforming growth factor- ⁇ (TGF- ⁇ ), insulin-like growth factor (IGF), platelet derived growth factor (PDGF), epidermal growth factor (EGF), acidic or basic fibroblast growth factor (aFBF or bFBF), hepatocytic growth factor (HGF), keratinocyte growth factor (KGF) the bone morphogenic factors (BMPs) including : BMP-1; BMP-2; BMP-3; BMP-4; BMP-5; and BMP-6 and the osteogenic proteins (OPs) including: OP-1; OP-2; and OP-3.
  • TGF- ⁇ transforming growth factor- ⁇
  • IGF insulin-like growth factor
  • PDGF platelet derived growth factor
  • EGF epidermal growth factor
  • aFBF or bFBF acid
  • ascorbate may be added to the chondrogenic cells in the pre- shaped well to enhance or stimulate the production of cartilage specific proteoglycans and collagen.
  • these particular compounds are not limiting. Any compound or composition capable of stimulating or inducing the production of cartilage specific proteoglycans and collagen may be useful in the practice of the instant invention.
  • the invention also provides methodologies for effecting the repair of an articular cartilage defect at a pre-determined site in a mammal.
  • the method comprises the steps of: (1) surgically implanting at the pre-determined site a piece of synthetic cartilage prepared by the methodologies described herein; and (2) permitting the synthetic cartilage to integrate into the pre-determined site.
  • the synthetic cartilage patch may be fixed in place during the surgical procedure. This may be effected by surgically fixing the patch with sutures and/or by applying a biocompatible, bioadhesive to the surface interfacing the cartilage patch and the defect. In some instances, defective cartilage tissue may be removed prior to implantation.
  • the shape of the synthetic cartilage may be dimensioned to interfit with the cartilage defect, in specific instances, for example, when the defect is large, it is contemplated that a plurality of synthetic cartilage patches may be surgically implanted into the defect.
  • the resulting synthetic cartilage patch is preferably allogenic, and more preferably autogenic in nature.
  • synthetic allogenic cartilage may be prepared from biopsy tissue isolated from a mammal belonging to the same species as the recipient.
  • Synthetic autogenic cartilage may be prepared from biopsy tissue derived from the intended recipient.
  • the invention provides synthetic articular cartilage for the repair articular cartilage defects in humans.
  • chondrogenic cells may be isolated from human cartilage tissue,i.e., human articular cartilage (from weight bearing and non-weight bearing joints), human costal cartilage, human nasal cartilage, human auricular cartilage, human tracheal cartilage, human epiglottic cartilage, human thyroid cartilage, human arytenoid cartilage and human cricoid cartilage.
  • the chondrogenic cells useful in the practice of the invention may be derived from human bone marrow.
  • Figure 1 shows a flow chart summarizing the steps in the preparation of large amounts of synthetic cartilage from small samples of biopsy tissue for the repair of cartilage defects in a mammal.
  • tissue containing chondrogenic cells is disaggregated to release denuded chondrogenic cells.
  • the isolated, cells then are proliferated by serially culturing and passaging the cells in monolayer culture.
  • monolayer culture the chondrogenic cells dedifferentiate and lose their ability to secrete cartilage specific extracellular matrix.
  • the proliferated cells are seeded into a pre-shaped well having a cell contacting, cell abhesive surface.
  • the chondrogenic cells are cultured in the well for a time sufficient to allow them to redifferentiate and secrete a cartilage specific extracellular matrix thereby to form synthetic cartilage in vitro.
  • Figures 2a and 2b provide a schematic plan view and a cross-sectional illustration, respectively, of a patch of synthetic cartilage prepared in a pre-shaped well in accordance with the invention. Detailed Description of the Invention
  • chondrogenic cells sampled from a mammal and proliferated in monolayer culture ex vivo may be cultured further in a pre-shaped well having a cell contacting, cell abhesive surface thereby to generate a three-dimensional, multi cell- layered patch of synthetic cartilage.
  • synthetic cartilage patches of pre-determined volume for use in the surgical replacement of damaged articular cartilage and subsequent integration into the joint of the recipient may be prepared in accordance with the invention.
  • chondrogenic cells useful in the practice of the instant invention may be isolated from a variety of pre-existing tissues, for example, cartilage tissue and perichondrial tissue or alternatively from bone marrow.
  • FIG. 1 A flow chart summarizing the steps associated with the preparation of three-dimensional, multi cell- layered patches of synthetic cartilage is shown in Figure 1. All of the steps described hereinbelow are preferably performed under aseptic conditions.
  • tissue (10) containing chondrogenic cells (12) is disaggregated to release denuded chondrogenic cells (16) from their extracellular matrix (14).
  • the denuded cells then are isolated and proliferated as monolayers in an undifferentiated state e _ vivo (18):
  • the passaging procedure may be repeated multiple times (n), for example up to about 7 to 10 passages until enough cells have been propagated to prepare a piece of cartilage of pre-determined size.
  • the proliferated but undifferentiated chondrogenic cells (20) then are seeded into a pre-shaped well (24) having a cell contacting, cell abhesive surface (22).
  • the cell abhesive surface prevents chondrogenic cells cultured in the well from attaching to the surface of the well.
  • the cells deprived of anchorage, interact with one another and coalesce within hours to generate a cohesive plug of cells.
  • the chondrogenic cells then begin to differentiate, as characterized by the production and secretion of cartilage-specific markers, i.e., type II collagen and sulfated proteoglycans. Type II collagen is found specifically in cartilage.
  • the chondrogenic cells then are cultured in the well for time sufficient to permit the formation of a three- dimensional, multi cell-layered patch of synthetic cartilage (26).
  • the resulting synthetic cartilage patch comprises chondrogenic cells (20) dispersed with a new, endogenously produced and secreted extracellular matrix (28).
  • the extracellular matrix deposited during this procedure is biochemically and morphologically similar to the extracellular matrix found in natural cartilage.
  • the synthetic matrix comprises fibers of type II collagen, and sulfated proteoglycans such as chondroitin sulfate and keratan sulfate.
  • Figure 2a is a schematic top plan view of a patch of synthetic cartilage (26) prepared in a pre-shaped well (24) in accordance with the invention.
  • Figure 2b is a schematic cross-sectional view of the patch of cartilage in the well of Figure 1 taken at lines 2-2. Particulars of methods for making and using the synthetic cartilage are set forth in detail below.
  • Chondrogenic cells useful in the practice of the instant invention may be sampled from a variety of sources in a mammal that contain such cells, for example, pre-existing cartilage tissue, perichondrial tissue or bone marrow.
  • articular cartilage from either weight bearing or non-weight bearing joints
  • Biopsy samples of articular cartilage may be readily isolated by a surgeon performing arthroscopic or open joint surgery. Procedures for isolating biopsy tissues are well known in the art and so are not described in detailed herein. See for example, "Operative Arthroscopy” (1991) by McGinty et al. ,; Raven Press, New York, the disclosure of which is incorporated by reference herein.
  • Perichondrial tissue is the membranous tissue that coats the surface of all types of cartilage, except for articular cartilage. Perichondrial tissue provides nutrients to the chondrocytes located in the underlying unvascularized cartilage tissue. Perichondrial tissue sampled from costal (rib) cartilage of patients suffering from osteoporosis provides a source of of chondrogenic cells when the normal articular cartilage is diseased or unavailable. Biopsy samples of perichondrial tissue may be isolated from the surface of costal cartilage or alternatively from the surface of auricular cartilage, nasal cartilage and cricoid cartilage using simple surgical procedures well known in the art. See for example: Skoog et a ' (1990) Scan. J.
  • chondrogenic cells specifically mesenchymal cells
  • useful in the practice of the invention may be isolated from bone marrow. Surgical procedures useful in the isolation of bone marrow are well known in the art and so are not described in detailed herein. See for example, Wakitani et al. (1994) J. Bone Joint Surg. 76: 579-591, the disclosure of which is incorporated by reference herein.
  • Protocols for preparing denuded chondrogenic cells from cartilage tissue, perichondrial tissue, and bone marrow are set forth below.
  • Articular cartilage both loaded (weight bearing) and unloaded (non-weight bearing), maybe be subjected to enzymatic treatment in order to disaggregate the tissue and release denuded chondrogenic cells from the extracellular matrix.
  • Solutions containing proteolytic enzymes for example, chondroitinase ABC, hyaluronidase, pronase, collagense, or trypsin may be added to articular cartilage tissue in order to digest the extracellular matrix. See for example, Watt & Dudhia (1988) Differentiation 318:140-147, the disclosure of which is incorporated herein by reference.
  • articular cartilage is initially cut into pieces of about 1 mm in diameter, or less. This is routinely performed using a sterile scalpel. The minced tissue then is disaggregated enzymatically, for example, by the addition of a solution containing 0.1% collagenase (Boehringer Mannheim GmbH, Germany). Approximately 1 ml of collagenase is added per 0.25 ml equivalents of minced tissue. The sample is then mixed and incubated overnight (up to 16 hours) at 37°C, with agitation.
  • the residual pieces of tissue are harvested by centrifugation, the supernatant removed, and the remaining cartilage pieces redigested by the addition of a solution containing, for example, 0.25% collagenase and 0.05% trypsin (Sigma Chemical Co., St. Louis). Approximately 1 ml of 0.25% collagenase, 0.05% trypsin is added per 0.25 ml equivalents of residual tissue. The sample then is mixed and incubated for 2-4 hours at 37°C, with agitation. Any remaining tissue is pelleted by centrifugation and the cell suspension harvested. The collagenase, trypsin step is repeated 2-4 times or until the tissue is completely disaggregated.
  • a solution containing, for example, 0.25% collagenase and 0.05% trypsin (Sigma Chemical Co., St. Louis). Approximately 1 ml of 0.25% collagenase, 0.05% trypsin is added per 0.25 ml equivalents of residual tissue. The sample then is mixed and incubated
  • tissue culture medium supplemented with approximately 10% fetal bovine serum (FBS) (Hyclone, Logan, Utah) .
  • FBS fetal bovine serum
  • a preferred cell culture medium includes, for example, Dulbecco's minimal essential medium (DMEM) (Sigma Chemical Co., St. Louis) supplemented with 10% FBS.
  • DMEM Dulbecco's minimal essential medium
  • An alternative cell culture medium includes a 1:1 (vol/vol) mixture of Medium 199 (Sigma Chemical Co., St. Louis) and Molecular Cell Developmental Biology Medium 202 (MCDB 202) (Sigma Chemical Co., St. Louis), respectively, supplemented with 10% FBS.
  • another cell culture medium useful in the practice of the invention includes a 3:1 (vol/vol) mixture of DMEM and Ham's F-12 (F12) (Sigma Chemical Co., St. Louis), respectively, supplemented with 10% FBS. Fractions containing denuded chondrogenic cells are combined, and the cells inoculated into a cell culture dish at a plating density of about Ixl0 2 -5xl0 5 cells/cm 2 , preferably about 5xl0 2 -lxl0 5 cells/cm 2 , and most preferably about 1x10 -lxlO 4 cells/cm 2 , for cell expansion and testing.
  • Chondrocytes may be isolated from costal cartilage, nasal cartilage, auricular cartilage, tracheal cartilage, epiglottic cartilage, thyroid cartilage, arytenoid cartilage and cricoid cartilage using the aforementioned procedure.
  • Denuded chondrogenic cells preferably are isolated from perichondrial tissue using the same procedure as described in section II A, hereinabove.
  • the tissue is minced into pieces of about 1 mm in diameter, or less.
  • the minced tissue is repeatedly digested with proteolytic enzymes, for example, trypsin and collagenase.
  • proteolytic enzymes for example, trypsin and collagenase.
  • the resulting denuded cells are inoculated into a cell culture dish at a plating density of about 1x10 -5x10 cells/cm , preferably about 5xl0 2 to lxlO 5 cells/cm 2 , and most preferably about Ixl0 3 -lxl0 4 cells/cm 2 for cell expansion and testing.
  • a non-destructive procedure may be used to isolate chondrogenic cells from perichondrial tissue.
  • intact explant tissue is placed in a cell culture dish and incubated in growth medium.
  • the chondrogenic cells located within the tissue migrate out of the tissue and onto the surface of the tissue plate where they begin to proliferate.
  • a preferred cell culture medium comprises DMEM supplemented with 10% FBS.
  • the explant tissues are incubated at 37°C, 5% C0 2 for 3-7 days. During this time the chondrogenic cells migrate out of the explant tissue and onto the surface of the tissue culture dish. After reattaching to the surface of the plate, the cells start to proliferate again.
  • Chondrogenic cells may be isolated from samples of bone marrow. Procedures useful for the isolation of mesenchymal cells from bone marrow are well known in the art, see for example: U.S. Pat. No. 5,197,985; U.S. Patent No. 4,642,120; and Wakitani et_ al- (1994, supra) .
  • a plug of bone marrow may be removed surgically from the mammal of interest and added to cell culture medium.
  • Preferred complete growth media are disclosed in U.S. Pat. No. 5,197,985.
  • the mixture then is vortexed to break up the plug of tissue.
  • the resulting suspension is centrifuged to separate bone marrow cells from large pieces of particulate matter i.e., bone fragments.
  • the cells then are dissociated to give a single cell suspension by forcing the cells through a syringe fitted with a series of 16, 18, and 20 gauge needles.
  • the cells then are plated out into a tissue culture plate at a cell density of about 1x10 5-1x106 cells/cm2 for selectively separating and/or isolating bone marrow derived mesenchymal cells from the remaining cells present in the suspension.
  • Chondrogenic cells isolated from cartilage tissue, perichondrial tissue, or bone marrow using the methods described in section II hereinabove may be placed in monolayer culture for proliferative expansion.
  • the process enables one to amplify the number of isolated chondrogenic cells.
  • the artisan may produce essentially an unlimited number of chondrogenic cells and therefore essentially an unlimited amount of synthetic cartilage. It is appreciated, however, that during proliferative expansion the chondrogenic cells dedifferentiate and lose their ability to secrete cartilage specific extracellular matrix. A procedure to assay whether the undifferentiated cells still retain their chondrogenic potential is described hereinbelow.
  • Protocols for proliferating cells by monolayer culture are well known in the art, see for example, Pollack (supra) , and so are not described in detail herein.
  • monolayer cultures are initiated by inoculating primary chondrogenic cells, isolated from either cartilage tissue or perichondrial tissue, into a cell culture dish at a plating density density of about 1x10 -5x10 cells/cm 2 , more preferably about 5xl0 2 - 1x10 cells/cm and most preferably about Ixl0 3 -lxl0 4 cells/cm .
  • Chondrogenic cells that have undergone one or more cycles of passaging are also plated out at the same plating densities.
  • Primary chondrogenic cells isolated from bone marrow are plated out into a tissue culture plate at a cell density of about Ixl0 5 -lxl0 6 cells/cm .
  • Chondrogenic cells from bone marrow that have undergone one or more cycles of passaging are plated out at plating densities of about 1x10 -5x10 cells/cm , more preferably about 5x10 -1x10 cells/cm and most preferably about Ixl0 3 -lxl0 4 cells/cm .
  • the chondrogenic cells subsequently are cultured at 37°C, 5% CO ⁇ in cell culture medium.
  • a preferred cell culture medium comprises DMEM supplemented with 10% FBS.
  • a cell culture medium comprising a 1:1 (vol/vol) mixture of Medium 199 and MCDB 202, respectively, supplemented with 10% FBS may be used.
  • Still another cell culture medium useful in the practice of the invention comprises a 3:1 (vol/vol) mixture of DMEM and F12, respectively, supplemented with 10% FBS.
  • the cells are passaged and inoculated into a new plate. This may be accomplished by initially removing the cell culture medium overlaying the cells monolayer by aspiration, and washing the cell monolayer with phosphate buffered saline (PBS). The PBS is removed, by aspiration, and a solution containing a proteolytic enzyme, i.e., 0.1% trypsin, then is poured onto the monolayer. The proteolytic enzyme hydrolyzes proteins that anchor the cells onto the surface of the plate thereby releasing the cells from the surface of the plate.
  • PBS phosphate buffered saline
  • the proteolytic enzyme in the cell suspension then is inactivated by adding FBS to the suspension to give a final concentration of 10% (vol/vol).
  • the density of cells in the suspension then is estimated and the cells re-plated into a new cell culture plate at a density of about Ixl0 2 -5xl0 5 cells, more preferably about 5x10 - 1x10 cells, and most preferably about 1x10 -10 cells per cm 2 .
  • the passaging procedure may be repeated multiple times, for example up to about 7 to 10 times. until enough cells have been propagated to prepare a piece of cartilage of pre-determined size.
  • suspensions of proliferated cells may be cryopreserved indefinitely using techniques well known in the art. See for example. Pollack (supra) . Accordingly, populations of chondrogenic cells may be stored for subsequent use whenever a necessity arises.
  • Undifferentiated chondrogenic cells expanded in monolayer culture, may be assayed to determine whether they still retain their chondrogenic potential. This may be performed by culturing the cells in a semi-solid medium in a process called agarose culture. This procedure is described in Benya et a2- (1982) Cell 3_0:215-224, the disclosure of which is incorporated by reference herein.
  • proliferated chondrogenic cells are seeded into a solution of warm 2% low melting temperature agarose (LT agarose) (BioRad, Richmond, CA) .
  • LT agarose low melting temperature agarose
  • the use of LT agarose permits cells to be seeded into the agarose without thermal damage to the cells.
  • the agarose is cooled to about 39-41°C prior to the addition of cells.
  • Approximately lxlO 3 - 1x10 cells are seeded into 1 ml of the liquid agarose.
  • the cells are cultured subsequently at 37°C, 5% C0 2 for 3-4 weeks in a cell culture medium preferably containing DMEM supplemented with 10% FBS. During this time, the chondrogenic cells replicate to from colonies which start to secrete an extracellular matrix. The resulting colonies have the appearance of small "nodules" embedded within the agarose. The colonies may be counted and the chondrogenic proportion of cells determined histochemically and immunohistochemically using procedures well known in the art.
  • agarose gels containing the cells are fixed with 10% formalin in PBS.
  • the samples then are sectioned into 8-18 ⁇ m sections with a cryo-cut microtome (American Optical).
  • General cellular morphology and tissue phenotype may be assessed by staining the section by the hematoxylin-eosin method well known in the art. Briefly, the resulting section is incubated in a stain comprising hematoxylin dissolved in 5% ethanol for 10 minutes. The section then is washed with water and incubated subsequently in an stain containing 1% eosin in 70% ethanol for 45 seconds. The sections then are washed with 95% ethanol. The nodules of extracellular matrix stain purple under these experimental conditions.
  • Sulfated proteoglycans in the extracellular matrix may be visualized by incubating the agarose particles in a stain comprising 1% alcian blue in 0.1N hydrochloric acid for 15-30 minutes.
  • proteoglycans may be visualized by incubating the agarose particles in a stain comprising 0.2% safranin O/fast green in 1% acetic acid for 15-30 minutes. The stained particles then are washed with water.
  • Sulfated proteoglycans present in the extracellular matrix stain blue and orange, by the two methods, respectively. I ⁇ utmnohistochemical Staining.
  • the agarose particles containing the chondrogenic cells are sectioned into 8-18 ⁇ m sections with a cryo- cut microtome (American Optical). The sections then are enzymatically digested in order to expose antigenic epitopes present on the extracellular matrix.
  • a preferred enzyme includes the proteolytic enzyme protease type XIV (Sigma Chemical Co., St. Louis). Briefly, the agarose sections are incubated for 90 minutes at room temperature in Tris buffered saline, pH 7.4 (TBS) containing 0.4 mg/ml of protease type XIV. The resulting section then is washed twice with TBS.
  • link protein 8A4 from Hybridoma Bank, Baltimore, MD
  • type I collagen AB745 and MAB1340 from Chemicon International, Timacula, CA
  • type II collagen PS48 from SanBio Inc., Amsterdam, Holland
  • CIICI Hybridoma Bank
  • chondroitin-6-sulfate may be used to detect the presence of these extracellular components in the agarose particles. Immunostaining may be performed using the VECTASTAIN ABC-AP kit (Vector
  • the chondrogenic cells still having chondrogenic potential are cultured in an anchorage-independent manner, i.e., in a well having a cell contacting, cell abhesive surface, in order to stimulate the secretion of cartilage-specific extracellular matrix components.
  • chondrogenic cells proliferatively expanded in an anchorage- dependent manner usually dedifferentiate and lose their ability to secrete cartilage-specific type II collagen and sulfated proteoglycan (Schlitz et al. , supra; Mayne et al. , supra; Mayne et aJL. , supra; Okayama et al_. , supra; Pacifici & Holtzer, supra; Pacifici et al. , supra; West et al . « , supra; von der Mark, supra; Oegama & Thompson, supra; Benya & Schaffer, supra) .
  • undifferentiated chondrogenic cells when seeded into, and cultured in a well having a cell contacting surface that discourages adhesion of cells to the cell contacting surface, redifferentiate and start to secrete cartilage-specific collagen and sulfated proteoglycans thereby to form a patch of synthetic cartilage ij vitro.
  • the cell contacting surface of the well may be coated with a molecule that discourages adhesion of chondrogenic cells to the cell contacting surface.
  • Preferred coating reagents include silicon based reagents i.e., dichlorodimethylsilane or polytetrafluoroethylene based reagents, i.e.. Teflon ⁇ . Procedures for coating materials with silicon based reagents, specifically dichlorodimethylsilane, are well known in the art. See for example, Sambrook et al.
  • the well may be cast from a pliable or moldable biocompatible material that does not permit attachment of cells per se.
  • Preferred materials that prevent such cell attachment include, but are not limited to, agarose, glass, untreated cell culture plastic and polytetrafluoroethylene, i.e., Teflorr ⁇ .
  • Untreated cell culture plastics i.e., plastics that have not been treated with or made from materials that have an electrostatic charge are commercially available, and may be purchased, for example, from Falcon Labware, Becton-Dickinson, Lincoln Park, N.J.
  • the aforementioned materials are not meant to be limiting. It is appreciated that any other other pliable or moldable biocompatible material that inherently discourages the attachment of chondrogenic cells may be useful in the practice of the instant invention.
  • the size and shape of the well may be determined by the size and shape of the articular cartilage defect to to be repaired.
  • the well may have a cross-sectional surface area of 25 cm 2 . This is the average cross-sectional surface area of an adult, human femoral chondyle. Accordingly, it is anticipated that a single piece of synthetic cartilage may be prepared in accordance with the invention in order to resurface the entire femoral chondyle.
  • the depth of the well is preferably greater than about 0.3 cm and preferably about 0.6 cm in depth.
  • the thickness of natural articular cartilage in an adult articulating joint is usually about 0.3 cm. Accordingly, the depth of the well should be large enough to permit a cartilage patch of about 0.3 cm to form. However, the well should also be deep enough to contain growth medium overlaying the cartilage patch.
  • a large piece of cartilage prepared in accordance with the invention may be "trimmed" to a pre-selected size and shape by a surgeon performing surgical repair of the damaged cartilage. Trimming may be performed with the use of a sharp cutting implement, i.e., a scalpel, a pair of scissors or an arthroscopic device fitted with a cutting edge, using procedures well known in the art.
  • the pre-shaped well preferably is cast in a block of agarose gel under aseptic conditions. Agarose is an economical, biocompatible, pliable and moldable material that can be used to cast pre-shaped wells, quickly and easily. As mentioned above, the dimensions of the well may dependent upon the size of the resulting cartilage plug that is desired.
  • a pre-shaped well may be prepared by pouring a hot solution of molten LT agarose (BioRad, Richmond, CA) into a tissue culture dish containing a cylinder.
  • the cylinder having dimensions that mirror the shape of the well to be formed.
  • the size and shape of the well may be chosen by the artisan and may be dependent upon the shape of the articular cartilage defect to be repaired.
  • the cylinder is carefully removed with forceps.
  • the surface of the tissue culture dish that is exposed by the removal of the cylinder is covered with molten agarose. This seals the bottom of the well and provides a cell abhesive surface at the base of the well.
  • the resulting pre-shaped well is suitable for culturing, and stimulating the redifferentiation of proliferated chondrogenic cells. It is appreciated, however, that alternative methods may be used to prepare a pre-shaped well useful in the practice of the invention.
  • Proliferated chondrogenic cells in suspension (from section III A, hereinabove) subsequently are seeded into and cultured in the pre-shaped well.
  • the cells are diluted by the addition of cell culture medium to a cell density of about Ixl0 5 -lxl0 9 proliferated chondrogenic cells per ml, or more preferably about 1x10 -5xl0 8 cells per ml, and most preferably about 3x10 -2x10 cells per ml.
  • a preferred cell culture medium comprises DMEM supplemented with 10% FBS.
  • a cell culture medium comprising a 1:1 (vol/vol) mixture of Medium 199 and MCDB 202, respectively, supplemented with 10% FBS may be used.
  • Still another cell culture medium useful in the practice of the invention comprises a 3:1 (vol/vol) mixture of DMEM and F12, respectively, supplemented with 10% FBS.
  • Ixl0 3 -lxl0 7 cells preferably Ixl0 4 -lxl0 6 cells, and most preferably about 5xl0 4 -5xl0 cells produce a piece of synthetic cartilage 1 mm in volume. Accordingly, the artisan may produce a patch of synthetic cartilage of pre-determined size by seeding an appropriate number of chondrogenic cells into a pre- shaped well.
  • the cells subsequently are cultured at 37°C, 5% C0 2 , for 1 to 90 days, preferably 5 to 60 days, and most preferably 10 to 30 days in order to permit secretion of cartilage-specific type II collagen and sulfated proteoglycans thereby to form of synthetic cartilage ii vitro.
  • the cell culture medium is removed from the well and replaced with fresh cell culture medium every other day in order to maintain optimal viability of the cells.
  • the cells coalesce to form a cohesive plug of cells.
  • the cells in the cohesive plug initially secrete type I collagen.
  • the cells start to secrete cartilage- specific sulfated proteoglycans and type II collagen.
  • the level of type I collagen synthesis decreases.
  • the collagen expressed by the chondrogenic cells in the well is predominantly type II collagen. It is contemplated however, that the cohesive plug of cells formed within four hours may be removed from the well and surgically implanted into the cartilage defect. It is anticipated that the undifferentiated chondrogenic cells subsequently may redifferentiate _in situ thereby to form synthetic cartilage within the joint.
  • the resulting synthetic cartilage tissue formed in the pre-shaped well may be assayed, biochemically or morphologically, using the procedures described hereinabove prior to implantation into the joint. Briefly, the synthetic cartilage may be sectioned into 8-18 ⁇ m sections using a cryo-microtome (American Optical) and resulting sections stained using the procedures described in section III B.
  • a cryo-microtome American Optical
  • polypeptide growth factors may be added to the chondrogenic cells in the pre- shaped well to enhance or stimulate the production of articular cartilage specific proteoglycans and/or collagen (Luyten & Reddi (1992) in "Biological Regulation of the Chondrocytes", CRC Press, Boca Raton, Ann Arbor, London, and Tokyo, p.p. 227-236).
  • Preferred growth factors include, but are not limited to transforming growth factor- ⁇ (TGF- ⁇ ), insulin-like growth factor (IGF), platelet derived growth factor (PDGF), epidermal growth factor (EGF), acidic fibroblast growth factor (aFBF), basic fibroblast growth factor (bFBF), hepatocytic growth factor, (HGF) keratinocyte growth factor (KGF), the bone morphogenic factors (BMPs) i.e., BMP-1, BMP-2, BMP-3, BMP-4, BMP-5 and BMP-6 and the osteogenic proteins (OPs), i.e. OP-1, OP-2 and OP-3.
  • TGF- ⁇ transforming growth factor- ⁇
  • IGF insulin-like growth factor
  • PDGF platelet derived growth factor
  • EGF epidermal growth factor
  • aFBF acidic fibroblast growth factor
  • bFBF basic fibroblast growth factor
  • HGF keratinocyte growth factor
  • BMPs bone morphogenic factors
  • Preferred concentrations of TGF- ⁇ , IGF, PDGF, EGF, aFBF, bFBF, HGF, and KGF range from about 1 to 100 ng/ml, more preferably from about 5 to about 50 ng/ml, and most preferably from about 10 to about 20 ng/ml.
  • Preferred concentrations of the BMP's and OP's range from about 1 to about 500 ng/ml, more preferably from about 50 to about 300 ng/ml, and most preferably from about 100 to about 200 ng/ml.
  • these particular growth factors are not limiting. Any polypeptide growth factor capable of stimulating or inducing the production of cartilage specific proteoglycans and collagen may be useful in the practice of the instant invention.
  • ascorbate may be added to the chondrogenic cells in the pre-shaped well to enhance or stimulate the production of cartilage specific proteoglycans and collagen.
  • concentrations of ascorbate range from about 1 to about 1000 ⁇ g/ml, more preferably from about 20 to about 500 ⁇ g/ml, and most preferably from about 50 to about 100 ⁇ g/ml.
  • Cartilage defects in mammals are readily identifiable visually during arthroscopic examination or during open surgery of the joint. Cartilage defects may also be identified inferentially by using computer aided tomography (CAT scanning), X-ray examination, magnetic resonance imaging (MRI), analysis of synovial fluid or serum markers or by any other procedures known in the art. Treatment of the defects can be effected during an arthroscopic or open surgical procedure using the methods and compositions disclosed herein.
  • CAT scanning computer aided tomography
  • X-ray examination X-ray examination
  • MRI magnetic resonance imaging
  • Treatment of the defects can be effected during an arthroscopic or open surgical procedure using the methods and compositions disclosed herein.
  • the defect may be treated by the following steps of (1) surgically implanting at the pre-determined site, a piece of synthetic articular cartilage prepared by the methodologies described herein, and (2) permitting the synthetic articular cartilage to integrate into pre ⁇ determined site.
  • the synthetic cartilage patch optimally has a size and shape such that when the patch is implanted into the defect, the edges of the implanted tissue contact directly the edges of the defect.
  • the synthetic cartilage patch may be fixed in placed during the surgical procedure. This can be effected by surgically fixing the patch into the defect with biodegradable sutures, i.e., (Ethicon, Johnson & Johnson) and/or by applying a bioadhesive to the region interfacing the patch and the defect.
  • Preferred bioadhesives include, but are not limited to: fibrin- thrombin glues similar to those disclosed in Fr. Pat. No. 2 448 900; Fr. Pat. No. 2 448 901 and EP.S.N.
  • damaged articular cartilage maybe surgically excised prior the to implantation of the patch of synthetic cartilage.
  • adhesion of the synthetic cartilage patch to the articular cartilage defect may be enhanced by treating the defect with transglutaminase (Ichinose et al. (1990) J_ ⁇ Biol. Chem. 2-__.(3):13411-13414; Najjar et al. (1984) in "Transglutaminases", Boston, Martinuse- Nijhoff).
  • the cartilage defect is dried, for example by using cottonoid, and filled with a solution of transglutaminase.
  • the solution is subsequently removed, for example, by aspiration, leaving a film containing transglutaminase upon the cartilage.
  • the synthetic cartilage patch is implanted subsequently into the defect by the methods described above.
  • the synthetic cartilage patches preferably are allogeneic, and most preferably autogenic nature. Accordingly, synthetic allogeneic cartilage may be prepared from biopsy tissue isolated from a mammal belonging to the same species as the intended recipient. Synthetic autogenic cartilage may be prepared from biopsy tissue derived from the intended recipient.
  • chondrogenic cells may be isolated from: human cartilage tissue,i.e, human articular cartilage (from weight-bearing and non-weight bearing joints), human costal cartilage, human nasal cartilage, human auricular cartilage, human tracheal cartilage, human epiglottic cartilage, human thyroid cartilage, human arytenoid cartilage and human cricoid cartilage; from human perichondrial tissue, i.e., perichondrial tissue sampled from the surface of human costal cartilage, human nasal cartilage, human auricular cartilage, human tracheal cartilage, human epiglottic cartilage, human thyroid cartilage, human arytenoid cartilage and human cricoid cartilage; or from human bone marrow.
  • human cartilage tissue i.e, human articular cartilage (from weight-bearing and non-weight bearing joints)
  • human costal cartilage i.e, human nasal cartilage, human auricular cartilage, human tracheal carti
  • HPT human perichondrial tissue
  • DPT dog perichondrial tissue
  • HAC human articular cartilage
  • DAC dog articular cartilage
  • the tissues were minced finely and incubated overnight in a buffer solution containing 0.1% collagenase at 37°C, with agitation. Following the overnight digestion, the residual pieces of tissue were harvested, and digested further with a solution containing 0.25% collagenase and 0.05% trypsin for 2-4 hours 37°C.
  • the collagenase, trypsin step was repeated for a total of three times and the fractions containing denuded chondrogenic cells combined, and plated out into a cell culture plate at a density of about 1x10 - lxlO 4 cells per cm 2 of the plate.
  • the cells were cultured at 37 C, 5% C0 2 in a medium containing DMEM supplemented with 10% FBS. When the cellular monolayers became confluent, the cells were washed three times with PBS and removed from the surface of the plate by adding a solution containing 0.05% trypsin to the monolayer. The trypsin was inactivated by the addition of 10% FBS to the suspension of cells. The number of cells in the single cell suspension were counted, re-plated, proliferated and repassaged until use.
  • the proliferated chondrogenic cells were shown to maintain their chondrogenic potential by culturing them in semi-solid agarose medium (Benya et al ⁇ - (1982), supra) .
  • the proliferated cells were seeded into 2% LT agarose at a cell density of about 1x10 -1x10 cells per ml of liquid agarose. Then, the cells were cultured at 37°C for 21-35 days, as specified below, in a medium containing DMEM and 10% FBS. The colonies gave the appearance of small "nodules" in agarose.
  • the controls were chondrogenic cells that were grown as monolayers but not cultured in agarose culture.
  • the composition of the resulting particles was assayed by histochemical staining.
  • the resulting particles were first fixed with 10% formalin in PBS.
  • the cellular morphology and tissue phenotype was assessed by staining a section of the agarose gel with hematoxylin/eosin.
  • the presence of sulfated proteoglycans in the extracellular matrix was assayed by staining the particles with 1% alcian blue in hydrochloric acid.
  • type I and type II collagen in the particles was assayed immunohistochemically using the: anti-type I collagen monoclonal antibodies AB745 and MAB1340; and the anti-type II collagen monoclonal antibodies PS48 and CIICI in combination with the VECTASTAIN ABC-AP immunodetection kit (Vector Laboratory).
  • chondrogenic cells can be isolated from mammalian perichondrial tissue and cartilage. The cells also maintain their chondrogenic potential following proliferation as monolayers.
  • the size of the well may be dependent upon the size of the piece of cartilage required.
  • the wells prepared herein were formed in LT agarose. 2ml of a hot 2% agarose solution (not containing any cells) was poured into a tissue culture dish (35mm in diameter) having a cylinder (3-5 mm, outside diameter) resting in the center of the dish. After the agarose solidified around the cloning cylinder (5-10 minutes), the cylinder was carefully removed with forceps. The exposed surface of the tissue culture dish resulting from the removal of the cloning cylinder was completely covered by the addition of a further 50-100 ⁇ l of hot, liquid 2% agarose.
  • Example II Another batch of proliferated chondrogenic cells prepared in accordance with Example I were subsequently seeded as replicate samples into wells prepared as described in Example II. The cells were either passaged twice (2°) or three times (3°), as specified below, prior to seeding them into agarose wells. Approximately, lxlO 6 proliferated chondrogenic cells were seeded into the pre-shaped wells. The cells were cultured for 14 days at 37°C, 5% C0 2 in growth medium containing DMEM supplemented with 10% FBS.
  • the cells seeded into the agarose well were unable to attach to the surface of the well.
  • the chondrogenic cells deprived of anchorage, interacted with one another and coalesced within about four hours to generate a cohesive plug of cells.
  • the chondrogenic cells began to differentiate, as judged by the production of articular cartilage specific markers. After 14 days in culture, the cohesive plugs of cells were assayed morphologically and histochemically.
  • the controls were chondrogenic cells that were grown as monolayers but not cultured in the pre-shaped wells.
  • the resulting patches and cellular monolayers were first fixed with 10% formalin in PBS.
  • the cellular morphology and tissue phenotype were assessed by staining a section of the patch with hematoxylin/eosin.
  • the presence of sulfated proteoglycans in the extracellular matrix was assayed by staining the remaining sections of the patches with either 1% alcian blue in hydrochloric acid or 0.2% safranin O/fast green.
  • type I and type II collagen in the particles was assayed immunohistochemically using the: anti-type I collagen monoclonal antibodies AB745 and MAB1340; and the anti-type II collagen monoclonal antibodies PS48 and CIICI in combination with the VECTASTAIN ABC-AP immunodetection kit (Vector Laboratory) .
  • chondrogenic cells derived from human and dog articular cartilage and from human perichondrial tissue cartilage as prepared in Example I were seeded into pre-shaped wells as prepared in Example II.
  • the cells were cultured for four days at 37°C, 5% C0 2 in growth medium containing DMEM supplemented with 10% FBS.

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Abstract

L'invention concerne des procédés ainsi que des compositions destinés à la réparation de défauts du cartilage articulaire chez un mammifère. On fait proliférer des cellules chondrogènes dénudées Si(ex vivo) sous la forme de cultures monocouches afin de développer la masse de cellules chondrogènes disponibles. Pendant la prolifération, les cellules chondrogènes cessent de secréter les constituants de matrice extra-cellulaire, le collagène de type II et des protéoglycanes sulfatées. Les cellules ayant proliféré sont ensuite ensemencées dans un puits préformé présentant une surface abhésive de contact cellulaire. Les cellules mises en culture dans le puits se rédifférencient et commencent à secréter à nouveau une matrice extra-cellulaire spécifique au cartilage. Par conséquent, des quantités sensiblement illimitées de cartilage synthétique peuvent être préparées à partir de petits échantillons de tissu de biopsie. L'invention concerne également des procédés de réparation chirurgicale de défauts du cartilage articulaire chez des mammifères à l'aide du cartilage synthétique préparé selon l'invention.
PCT/US1995/005609 1994-05-05 1995-05-04 Procedes et compositions de reparation de defauts de cartilage articulaire chez les mammiferes WO1995030742A1 (fr)

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Cited By (11)

* Cited by examiner, † Cited by third party
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DE19540487A1 (de) * 1995-10-20 1997-04-24 Olaf Schultz Zellinteraktionssystem zur Induktion künstlicher Gewebe
EP0812351A1 (fr) * 1994-06-06 1997-12-17 Advanced Tissue Sciences, Inc. Cultures de cartilage tridimensionnelles
WO1999047186A1 (fr) * 1998-03-18 1999-09-23 University Of Pittsburgh Matieres composites a base de chitosane contenant du glycosaminoglycane pour la reparation du cartilage
CN1061812C (zh) * 1996-04-01 2001-02-14 胡杰 制备皮肤移植体的方法
EP1085082A1 (fr) * 1999-09-16 2001-03-21 Universite Pierre Et Marie Curie Paris Vi Méthodes et compositions pour la conservation de chondrocytes et utilisations
FR2801317A1 (fr) * 2000-11-16 2001-05-25 Univ Paris Curie Methodes et compositions pour la conservation de chondrocytes et utilisations
EP1181908A1 (fr) * 1996-08-30 2002-02-27 VTS Holdings, Ltd. Méthode, instruments et kit pour greffe autologue
EP1360948A1 (fr) * 2002-05-06 2003-11-12 Karl Storz GmbH & Co. KG Dispositif pour former un greffon de cartilage
WO2005012512A1 (fr) 2003-08-01 2005-02-10 Norimasa Nakamura Tissu synthetique 3 d auto-organise sans structure
WO2007083504A1 (fr) * 2003-08-01 2007-07-26 Norimasa Nakamura Tissu synthétique en 3d auto-organisé sans échafaudage
CH703968A1 (de) * 2010-10-28 2012-04-30 Christian Herzog Diagnose und Mittel zur Behandlung von Knorpelzellendefekten.

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US5632745A (en) * 1995-02-07 1997-05-27 R&D Biologicals, Inc. Surgical implantation of cartilage repair unit
US6756358B2 (en) 1994-08-19 2004-06-29 Sumitomo Pharmaceuticals Co., Ltd. Therapeutic agent for cartilaginous diseases
US5932459A (en) * 1995-10-20 1999-08-03 Sittinger; Michael Artificial tissues, methods for the production and the use thereof
US6569172B2 (en) 1996-08-30 2003-05-27 Verigen Transplantation Service International (Vtsi) Method, instruments, and kit for autologous transplantation
US5866415A (en) * 1997-03-25 1999-02-02 Villeneuve; Peter E. Materials for healing cartilage and bone defects
DE69714035T2 (de) 1997-08-14 2003-03-06 Sulzer Innotec Ag Zusammensetzung und Vorrichtung zur Reparatur von Knorpelgewebe in vivo bestehend aus Nanokapseln mit osteoinduktiven und/oder chondroinduktiven Faktoren
IL141308A0 (en) 1998-08-14 2002-03-10 Verigen Transplantation Serv Methods, instruments and materials for chondrocyte cell transplantation
EP1204385A1 (fr) * 1999-08-02 2002-05-15 Verigen Transplantation Service International (VTSI) AG Trousses de transplantation de cellules chondrocytes
EP1764117A1 (fr) 2005-09-20 2007-03-21 Zimmer GmbH Implant pour la réparation de défauts cartilagineux et son procédé de préparation

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US4904259A (en) * 1988-04-29 1990-02-27 Samuel Itay Compositions and methods for repair of cartilage and bone
US5206023A (en) * 1991-01-31 1993-04-27 Robert F. Shaw Method and compositions for the treatment and repair of defects or lesions in cartilage

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Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0812351A1 (fr) * 1994-06-06 1997-12-17 Advanced Tissue Sciences, Inc. Cultures de cartilage tridimensionnelles
EP0812351A4 (fr) * 1994-06-06 1998-07-08 Advanced Tissue Sciences Inc Cultures de cartilage tridimensionnelles
DE19540487A1 (de) * 1995-10-20 1997-04-24 Olaf Schultz Zellinteraktionssystem zur Induktion künstlicher Gewebe
CN1061812C (zh) * 1996-04-01 2001-02-14 胡杰 制备皮肤移植体的方法
EP1181908A1 (fr) * 1996-08-30 2002-02-27 VTS Holdings, Ltd. Méthode, instruments et kit pour greffe autologue
EP1459709A1 (fr) * 1996-08-30 2004-09-22 Verigen AG Méthode, instruments et kit pour greffe autologue
EP1384452A1 (fr) * 1996-08-30 2004-01-28 Verigen Transplantation Service International (VTSI) AG Méthode, instruments et kit pour greffe autologue
WO1999047186A1 (fr) * 1998-03-18 1999-09-23 University Of Pittsburgh Matieres composites a base de chitosane contenant du glycosaminoglycane pour la reparation du cartilage
EP1085082A1 (fr) * 1999-09-16 2001-03-21 Universite Pierre Et Marie Curie Paris Vi Méthodes et compositions pour la conservation de chondrocytes et utilisations
FR2798671A1 (fr) * 1999-09-16 2001-03-23 Univ Paris Curie Compositions de chondrocytes, preparation et utilisations
WO2001019964A1 (fr) * 1999-09-16 2001-03-22 G P H Compositions de chondrocytes, preparation et utilisations
US6365405B1 (en) 1999-09-16 2002-04-02 Universite Pierre Et Marie Curie (Paris Iv) Compositions of chondrocytes, preparation and utilization
WO2001019965A1 (fr) * 1999-09-16 2001-03-22 G P H Methodes et compositions pour la conservation de chondrocytes et utilisations
EP1085084A1 (fr) * 1999-09-16 2001-03-21 Universite Pierre Et Marie Curie Paris Vi Compositions de chondrocytes, préparation et utilisations
FR2801317A1 (fr) * 2000-11-16 2001-05-25 Univ Paris Curie Methodes et compositions pour la conservation de chondrocytes et utilisations
EP1360948A1 (fr) * 2002-05-06 2003-11-12 Karl Storz GmbH & Co. KG Dispositif pour former un greffon de cartilage
WO2005012512A1 (fr) 2003-08-01 2005-02-10 Norimasa Nakamura Tissu synthetique 3 d auto-organise sans structure
WO2007083504A1 (fr) * 2003-08-01 2007-07-26 Norimasa Nakamura Tissu synthétique en 3d auto-organisé sans échafaudage
US9370606B2 (en) 2003-08-01 2016-06-21 Two Cells, Co., Ltd. Scaffold-free self-organized 3D synthetic tissue
US9694107B2 (en) 2003-08-01 2017-07-04 Two Cells, Co., Ltd. Scaffold-free self-organized 3D synthetic tissue
CH703968A1 (de) * 2010-10-28 2012-04-30 Christian Herzog Diagnose und Mittel zur Behandlung von Knorpelzellendefekten.

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