WO2001035968A1 - Procedes permettant d'induire la chondrogenese et de produire du cartilage de novo in vitro - Google Patents

Procedes permettant d'induire la chondrogenese et de produire du cartilage de novo in vitro Download PDF

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WO2001035968A1
WO2001035968A1 PCT/US2000/031607 US0031607W WO0135968A1 WO 2001035968 A1 WO2001035968 A1 WO 2001035968A1 US 0031607 W US0031607 W US 0031607W WO 0135968 A1 WO0135968 A1 WO 0135968A1
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cartilage
cells
chondrogenesis
cell
pleuripotent
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PCT/US2000/031607
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English (en)
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Samy Ashkar
Samer Zawaideh
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Children's Medical Center Corporation
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Priority to AU16182/01A priority Critical patent/AU1618201A/en
Publication of WO2001035968A1 publication Critical patent/WO2001035968A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0655—Chondrocytes; Cartilage
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/90—Polysaccharides
    • C12N2501/905—Hyaluronic acid
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C12N2533/80—Hyaluronan

Definitions

  • Cartilage is a hypocellular tissue consisting of extracellular matrix molecules, water, and a relatively small population of chondrocytes.
  • Caltilage provides elasticity at the junction of bones of the mammalian skeletature (e.g., among the bones of the rib cage), provides structure to superficial organs such as the nose and ears, and plays a critical role in absorbing shock and minimizing stress on the bones.
  • There are at least three prominent types of cartilage including elastic cartilage, fibrocartilage. and articular or hyaline cartilage, the latter being archetypic for the appendicular skeleton and the vertebral column.
  • Hyaline cartilage arises from pluripotent mesenchymal ancestor cells that remain morphologically undifferentiated prior to a localized cell condensation in specific regions destined to undergo chondrogenesis. Injuries to articular cartilage can result in numerous clinical symptoms, such as pain and decreased functional levels.
  • the limited reparative capabilities of hyaline cartilage often results in the generation of repair tissue that lacks the structure and biomechanical properties of normal cartilage.
  • chondrocytes are unable to adequately proliferate, migrate, and synthesize high-quality repair tissue.
  • therapies for the treatment of articular cartilage injuries or degradation rely in great part on the ability to culture chondrocytes in culture.
  • the available treatments depend on the isolation of chondrocytes from one area of a patient and autologous implantation of those cells at the site of injury.
  • Various permeations of this technique involve culturing the chondrocytes in collagen gels or matrices to provide mechanical strength.
  • entire autologous cartilage grafts can be used, for example, in reconstruction procedures.
  • a significant drawback of these procedures is the paucity of the graftable material. Accordingly, there exists a need for methods of synthesizing de novo cartilage from cultured cells for implantation and reconstruction.
  • Systems previously described for the cultivation of cartilage cells include both monolayer or organoid (high density) cultures.
  • chondrocytes are grown at low density in serum-containing media, however, after a short culture period, the cells dedifferentiate, are fragmented to fibroblast-like cells (Grundmann, et al, 1980; Quarto et al, 1990; Shakibaei, 1995; Shakibaei et al, 1996), form collagen type I (Benya et al, 1977; von der Mark et al, 1977; von der Mark, 1980), change the pattern of their proteoglycan synthesis (Kim and Conrad, 1977; Benya and Shaffer, 1982) and that of their surface receptors (Shakibaei, 1995), or they are overgrown by fibroblast-like cells. Longer periods of culture are needed for the observation of certain aspects of chondrogenesis (e.g. , maturation or mineralization).
  • chondrogenesis e.g. , maturation or mineralization
  • chondrocytes are either transformed to fibroblast-like cells after a 2- to 3- week culture period (Norby et al, 1977; Schr ⁇ ter-
  • Another disadvantage of these methods is that they do not yield pure cartilage cultures but cultures with a perichondrium and unspecific connective tissue between the cartilage nodules (Zimmermann et al, 1992; Shakibaei et al, 1993a; b; 1995).
  • the murine mesenchymal 10T1/2 cells is a pleuripotent cell line capable of differentiating originally into the three distinct lineages of muscle, fat, and cartilage upon 5-azacytidine addition (Taylor and James, 1979).
  • Cartilage cells account for about 0.1-1% of the total number of differentiated cells, with the majority of cells (25-50%) differentiating into the muscle lineage.
  • confluent monolayers of 10T1/2 fibroblasts stably transfected with BMP-2 or BMP-4 differentiate into the three lineages of bone, fat and cartilage (Ahrens et al, 1993).
  • the present invention is based at least, in part, on the discovery that pleuripotent mesenchymal cells can be cultured in vitro, in the presence of specified glycosaminoglycans, such that chondrogenesis is achieved. Further development of the cultures results in the formation of implantable cartilaginous cultures for use in repairing cartilage injury or degeneration as well as reconstructing cartilage in vivo.
  • the glycosaminoglycan, hyaluronic acid is sufficient to promote expression of the chondrogenic phenotype and that chondrocytes so-generated are capable of being grown into implantable cultures.
  • the present invention relates to methods for promoting chondrogenesis and/or making cartilage in vitro. More specifically, the invention relates to methods for promoting chondrogenesis in culture, which includes proliferating (e.g., expanding) a population of pleuripotent mesenchymal cells in the presence of a glycosaminoglycan until chondrocytic differentiation is promoted.
  • proliferating e.g., expanding
  • a population of pleuripotent mesenchymal cells in the presence of a glycosaminoglycan until chondrocytic differentiation is promoted.
  • the population of pleuripotent mesenchymal stem cells (“PMCs") is cultured until the cells become contact inhibited.
  • the cells are cultured until at least one chondrocytic or cartilage marker is exhibited.
  • the invention features a method of producing cartilage in vitro which includes culturing a population of CD44-positive cells (e.g., PMCs) under conditions such that cartilage is produced.
  • the present invention further features methods of promoting chondrocytic differentiation (e.g., of a PMC) and methods of promoting chondrogenesis which include treating cells with a chondrogenesis-promoting agent (e.g., a glycosaminoglycan) such that differentiation or chondrogenesis are promoted or enhanced.
  • a chondrogenesis-promoting agent e.g., a glycosaminoglycan
  • the present invention also features implantable cartilaginous cultures as well as methods of producing such cultures.
  • the methods include culturing PMCs in the presence of a chondrogenesis-promoting agent (e.g., a glycosaminoglycan) such that chondrogenesis is promoted and/or cartilage is produced.
  • a chondrogenesis-promoting agent e.g., a glycosaminoglycan
  • Preferred glycosaminoglycans include but are not limited to chondroitin sulfate, dermatan sulfate, keratan sulfate and hyaluronic acid (e.g. , low molecular weight hyaluronic acid).
  • the methods of the invention are useful in the reconstruction of cartilage, and/or methods of repairing bone-related injury, methods of repairing cartilage injury and methods of promoting normal bone growth. Moreover, the methods of the present invention can be used to replace or augment existing cartilage tissue and to join together biological tissues or structures.
  • the present invention relates to methods for promoting chondrogenesis and/or making cartilage in vitro. More specifically, the invention relates to methods for promoting chondrogenesis in culture, which includes proliferating (e.g., expanding) a population of pleuripotent mesenchymal cells in the presence of a glycosaminoglycan until chondrocytic differentiation is promoted.
  • proliferating e.g., expanding
  • a population of pleuripotent mesenchymal cells in the presence of a glycosaminoglycan until chondrocytic differentiation is promoted.
  • the population of pleuripotent mesenchymal stem cells (“PMCs") is cultured until the cells become contact inhibited.
  • the cells are cultured until at least one chondrocytic or cartilage marker is exhibited.
  • the invention features a method of producing cartilage in vitro which includes culturing a population of CD44-positive cells (e.g., PMCs) under conditions such that cartilage is produced.
  • the present invention further features methods of promoting chondrocytic differentiation (e.g., of a PMC) and methods of promoting chondrogenesis which include treating cells with a chondrogenesis-promoting agent (e.g., a glycosaminoglycan) such that differentiation or chondrogenesis are promoted or enhanced.
  • a chondrogenesis-promoting agent e.g., a glycosaminoglycan
  • the present invention also features implantable cartilaginous cultures as well as methods of producing such cultures.
  • the methods include culturing PMCs in the presence of a chondrogenesis-promoting agent (e.g., a glycosaminoglycan) such that chondrogenesis is promoted and/or cartilage is produced.
  • a chondrogenesis-promoting agent e.g., a glycosaminoglycan
  • Glycosaminoglycans include but are not limited to chondroitin sulfate, dermatan sulfate, keratan sulfate and hyaluronic acid (e.g., low molecular weight hyaluronic acid).
  • a preferred glycosaminoglycan is hyaluronic acid (e.g., low molecular weight hyaluronic acid).
  • cartilage is evidenced by the detection of at least one chondrocytic marker or cartilage marker.
  • Chondrocytic markers include but are not limited to chondroitin-4-sulfate. chondroitin-6-sulfate, a chondrocyte- specific sulfated proteoglycan, type II collagen and aggrecan.
  • Production of cartilage can further be evidenced microscopically and/or ultrastructurally.
  • the cartilage produced by the herein-described methodologies is hyaline cartilage.
  • the methods of the invention are useful in the reconstruction of cartilage, and/or methods of repairing bone-related injury, methods of repairing cartilage injury and methods of promoting normal bone growth.
  • the methods of the present invention can be used to replace or augment existing cartilage tissue and to join together biological tissues or structures.
  • the present invention features a method of repairing cartilage in vivo which includes producing cartilage in vitro according to one of the herein-described methodologies and implanting the cartilage in a patient at a site in need of augmentation.
  • the invention features a method of augmenting cartilage in vivo which includes producing cartilage in vitro according to one of the herein-described methodologies and implanting the cartilage in a patient at a site in need of augmentation.
  • PMC pleuripotent mesenchymal cell
  • a PMC is further defined by its ability to undergo an unlimited number of mitotic cell divisions in order to propagate its own phenotype.
  • a PMC has the potential to generate progeny which are referred to herein as "lineage-committed” or “lineage-immature” cells, i.e., cells which are incapable of undergoing multiple mitotic cell divisions and which will eventually differentiate into a specific cell type (e.g., myocytes, osteoblasts, adipocytes, chondrocytes, or fibroblasts).
  • lineage-committed or “lineage-immature” cells, i.e., cells which are incapable of undergoing multiple mitotic cell divisions and which will eventually differentiate into a specific cell type (e.g., myocytes, osteoblasts, adipocytes, chondrocytes, or fibroblasts).
  • cartilage is art-recognized and generally refers to a dense connective tissue containing cells embedded in an extracelllular matrix. Cartilage consists primarily of fibrils or type II collagen in the form of arcade-like structures that are distended by the presence of highly-charged proteoglycans. Naturally occurring cartilage (i.e , cartilage existing in an animal, for example, a human, or alternatively isolated form an animal) can also be characterized by the presence of, at least, type IX collagen, type X collagen, type XI collagen, proteoglycans and hyaluronic acid or hyaluronate.
  • Naturally occurring cartilage contains about 10% cells (e.g., chondrocytes) and about 90% extracellular matrix (Hammerman and Schubert, 1962) comprising mainly type II collagen fibers, proteoglycan macromolecules and water. Water constitutes about 75% of cartilage
  • the proteoglycans are present in variable amounts (Stockwell,1979) and can account for up to 10% of the wet weight of cartilage (or about a quarter of the dry weight).
  • “Hyaline” cartilage is sparse in collagen fibers and accordingly, appears bluish-white, glassy, and translucent both macroscopically and microscopically.
  • “Fibrocartilage” has a higher collagen and lower proteoglycan content, therefore its fibers are macroscopically visible.
  • Elastic cartilage contains elastic fibers in the extracellular matrix in addition to the collagen and proteoglycans.
  • Hyaline cartilage is well suited to withstand compressive forces and is found on the articular surfaces of most long bones (e.g., in the joints), while fibrocartilage is found where shearing forces are applied (Ogden et al, 1975).
  • Hyaline cartilage is also found in costal cartilages (e.g., cartilage connecting the ribs and bones), in septum of nose, as well as in the larynx and trachea.
  • proteoglycan includes polymers having repeating units made of polysaccharide (about 95%) covalently attached to a polypeptide backbone or "core protein” (about 5%).
  • the polysaccharide portions also referred to as glycosaminoglycan
  • the polysaccharide portions are made up of disaccharide repeating units including, but not limited to, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratan sulfate, or hyaluronic acid.
  • the ground substance of cartilage is proteoglycan.
  • hyaluronic acid include polymers which are composed of repeated disaccharide units of hyalobiuronic acid.
  • HA hyaluronic acid
  • cartilage in vivo e.g., naturally-occurring cartilage
  • hyaluronic acid exists as an elongated filament to which the core proteins of proteoglycans are covalently attached. This interaction is promoted by small link proteins in vivo.
  • hyaluronic acid or hyaluronate can be purified from in vivo sources and thus can exist in an "isolated" form.
  • Isolated HA refers to HA which has been purified from tissue sources or from medium of hyaluronic acid-producing cells (e.g., hyaluronic acid- producing bacteria). Alternatively, HA can be chemically synthesized. Accordingly, “isolated” HA is substantially free of contaminating materials (e.g., proteins) from the cell or tissue source from which it is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language “substantially free of contaminating material” includes preparations of HA which are separated from components of the cells or tissue from which it is isolated or bacterially produced.
  • contaminating materials e.g., proteins
  • the language "substantially free of contaminating material” includes preparations of HA having less than about 30% (by weight) of contaminating materials, more preferably less than about 20% of contaminating materials, still more preferably less than about 10% of contaminating materials, and most preferably less than about 5% contaminating materials.
  • the HA is bacterially produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of HA which are separated from chemical precursors or other chemicals which are involved in the synthesis of the polymer.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of HA having less than about 30% (by dry weight) of chemical precursors or non-HA chemicals, more preferably less than about 20% chemical precursors or non- HA chemicals, still more preferably less than about 10% chemical precursors or non-HA chemicals, and most preferably less than about 5% chemical precursors or non-HA chemicals.
  • chondrogenesis includes the differentiation of pleuripotent mesenchymal cells, followed by expression of cartilage-specific genes producing extracellular matrix proteins, and maturation of chondroblasts into chondrocytes which occurs concomitant with suppression of alternative phenotypes.
  • CD44 refers to a plasma membrane glycoprotein which acts as the principal receptor for hyaluronic acid.
  • CD44 is involved in cell-cell and cell- matrix adhesion and is expressed by hematopoetic cells and by other tissues such as the lung, brain, smooth muscle, connective tissue and proliferating epithelial cells. Its expression is particularly high in osteocytes, osteoclasts and periosteal cells. It is not, however, expressed by osteoblasts (Hughes, 1994).
  • CD44 is expressed in the condensing limb bud mesenchyme prior to the cartilage-generating chondrogenic process. Recent evidence indicates that PMC transiently express a specific CD44 isoform on their surface prior to their migration and condensation, termed CD44v.
  • condensation includes the controlled aggregation of PMCs in a manner such that chondrogenesis occurs in the absence of cellular hypertrophy.
  • chondrogenesis occurs early in the development of the cartilaginous skeleton when PMCs migrate from the notochord (a region low in hyaluronic acid) toward strips of hyaluronate-rich lateral plate mesoderm called the Wolffian Ridges, forming regions of high cell density (Hall and Miyale, 1995).
  • Condensation in vivo precedes the differentiation of mesenchymal cells into chondrocytes producing cartilage matrix (Fell, 1925; Searls et al, 1972).
  • aggregation includes an accumulation of cells (e.g., PMCs) in an uncontrolled manner such that cells within the aggregate undergo hypertrophy, as compared to differentiation. Aggregation can result, for example, from cells being plated in culture at too high a density or not being plated as a monodisperse suspension. As used herein, “aggregation” is a non-desirable phenomenon by contrast to condensation, which is desirable and necessary for chondrogenesis.
  • PMCs pleuripotent mesenchymal cells
  • one aspect of the invention features PMCs isolated from animals, preferably from human, more preferably from donor-matched humans, and most preferably from the patient in need of cartilage reconstruction or repair or in need of a cartilage implant.
  • PMCs are derived from the periosteum of an animal, preferably a human.
  • periostum refers to the fibrous membrane localized at the surface of bones (e.g., ileum, scapula, tibia, fibula, femur, etc.).
  • periosteal tissue can be excised arthroscopically by removing a portion of, for example, the rib, tibia, or femur of a patient in advance of the surgery scheduled to reconstruct, repair or implant the cartilage cultures.
  • the periostial tissue is harvested and incubated with collagenase in the appropriate medium (e.g., Ham's F12 media) for about 3 hours at about 37°C, preferably with agitation.
  • the appropriate medium e.g., Ham's F12 media
  • PMCs are derived from the skin of an animal, preferably a human.
  • skin can be cleaned with betadine and rinsed several times with sterile PBS.
  • Skin biopsies can be taken (e.g., lmm ⁇ biopsies) and the cells dissociated with a collagenase-dispase mixture (e.g., 1 mg/ml collagenase-dispase). Dispase is included to minimize fibrocyte contamination of primary skin cultures.
  • Tissue samples can then be digested (e.g., at 37 C for 1 h) and cells resuspended at a concentration sufficient for plating (e.g., 1-5 x 10*5 cells/ml).
  • Yet another aspect of the invention features genetically engineered cells as a source of PMCs.
  • a "genetically engineered cell” includes a cell which has been genetically manipulated to overexpress a level of gene product greater than that expressed prior to manipulation of the cell or in a comparable cell which has not been manipulated.
  • Genetic manipulation can include, but is not limited to, altering or modifying regulatory sequences or sites associated with expression of a particular gene (e.g., by adding strong promoters, inducible promoters or multiple promoters or by removing regulatory sequences such that expression is constitutive), modifying the chromosomal location of a particular gene, altering nucleic acid sequences adjacent to a particular gene, increasing the copy number of a particular gene, modifying proteins (e.g., regulatory proteins, suppressors, enhancers, transcriptional activators and the like) involved in transcription of a particular gene and/or translation of a particular gene product, or any other conventional means of increasing expression of a particular gene which is routine in the art.
  • altering or modifying regulatory sequences or sites associated with expression of a particular gene e.g., by adding strong promoters, inducible promoters or multiple promoters or by removing regulatory sequences such that expression is constitutive
  • modifying the chromosomal location of a particular gene altering nucleic acid sequence
  • overexpressed includes expression of a gene product at a level greater than that expressed prior to manipulation of a cell or in a comparable cell which has not been manipulated.
  • the genetically engineered cell has been genetically manipulated to overexpress CD44 (e.g., a fibroblastic cell overexpressing CD44).
  • Yet another aspect of the present invention features established cell lines as a source of pleuripotent mesenchymal cells.
  • HA for use in the present invention is an isolated preparation of HA, or isolated HA.
  • isolated HA is derived from a tissue source (e.g., from vitreous bodies, for example, vitreous bodies of bovine eyes, from nasal septum, from umbilical cords, from cartilage, for example, cetacean cartilages, from skin or from fowl crests).
  • isolated HA is derived from a cellular source (e.g., from the culture broth of HA-producing bacteria).
  • HA is synthesized chemically.
  • a preferred HA for use in the present invention is low molecular weight HA.
  • low molecular weight HA is HA having a molecular weight of about 5- 200 kDa, preferably about 10-100 kDa, more preferably about 20-80 kDa, and even more preferably 30-50 kDa.
  • high molecular weight HA is HA having a molecular weight of greater than 200 kDa, preferably greater than 500 kDa, and more preferably greater than 1000, 1500, 2500 kDa.
  • high molecular weight HA with the methods of the present invention is sufficient to promote chondrogenesis in vitro.
  • cells e.g., PMCs
  • the present inventor has observed that cells (e.g., PMCs) which have been treated with high molecular weight HA may attempt to clear or process the HA from the extracellular environment during the proliferative or expansion phase of generating a cartilage culture.
  • the cells e.g., PMCs
  • Use of high molecular weight HA has also been observed to slow the differentiation of cells (e.g., PMCs).
  • the slowing of proliferation of the cells within the implant may be undesirable, one of skill in the art will appreciate that the effect may have the beneficial side effect of slowing the proliferation of potentially invading cells, for example, macrophages.
  • Potentially invading cells may be detrimental to cartilaginous implants used in joint-specific treatments (e.g., the treatment of arthritis).
  • the present invention features implantable cartilage cultures for use in the treatment of a wide variety of cartilage disorders (e.g., degenerative disorders, genetic disorders) and for use in a variety of reconstructive and/or augmentative procedures.
  • cartilage disorders e.g., degenerative disorders, genetic disorders
  • the pleuripotent mesenchymal cells as described above are cultured in vitro under conditions such than chondrogenesis is promoted (i. e. , the chondrocytic phenotype is enhanced) and/or cartilage is produced.
  • the methods of the present invention include a step involving proliferation or expansion of the pleuripotent mesenchymal cells.
  • the proliferation or expansion step is carried out in the presence of hyaluronic acid.
  • HA as described above can be diluted to a concentration of about 10 ⁇ g/ml -100 mg/ml soluble HA with an aqueous solution (e.g., diluted with buffer, for example carbonate buffer or phosphate-buffered saline) and added to dishes for a period of time sufficient to achieve deposition of the HA from the solution onto the surface of the dish.
  • aqueous solution e.g., diluted with buffer, for example carbonate buffer or phosphate-buffered saline
  • soluble HA is added to the dishes at a concentration of about 10 ⁇ g/ml to 100 ⁇ g/ml, preferably at a concentration of about 100 ⁇ g/ml to 1 mg/ml, more preferably at a concentration of about 1 mg/ml to 10 mg/ml, even more preferably at a concentration of about 10 mg/ml to 100 mg/ml. It has been observed that when using high molecular weight HA, the lower concentrations of soluble HA may be preferable, for example, from about 10 ⁇ g/ml to 10 mg/ml.
  • the soluble HA is added to the dishes and incubated for at least about 1-2 hours, preferably about 2-4 hours, more preferably about 4-16 hours, and even more preferably about 16-24 hours. In another embodiment, the soluble HA is incubated on the dishes at about 4°C, preferably at about 25°C, and more preferably about 37°C.
  • the solution can be aspirated from the HA- coated dishes and the dishes can be dried (e.g., dried in a laminar flow hood in a sterile environment). In another embodiment, the solution can be aspirated and the dishes can be washed with media or buffer prior to plating PMCs on the coated dishes.
  • isolated HA as described above can be diluted to a concentration of about 10 ⁇ g/ml -100 mg/ml soluble HA with an aqueous solution (e.g., diluted with media) and the cells can be suspended in the HA-containing media prior to plating on tissue culture dishes.
  • the soluble HA is made at a concentration about 10 to 100X that desired in the final media and added to cells which have been plated in non-HA containing media.
  • the HA-containing media is replaced at least about every 72 hours and preferably about every 48 or 24 hours. It is also within the scope of the present invention to grow, culture or expand cells (e.g., PMCs) on HA-coated dishes further in the presence of HA-containing media.
  • PMCs e.g., PMCs
  • alternative preferred embodiments include growing, culturing or expanding cells in the presence of chondroitin sulfate (e.g., chondriotin-4 sulfate or chondroitin-6 sulfate), dermatan sulfate or keratan sulfate.
  • the methods of the present invention further provide for expanding cells (e.g.,
  • PMCs chondrocytic phenotype
  • contact inhibition is desirable such that the differentiated chondrocytic phenotype may be achieved.
  • plate cells at a density such that proliferation and differentiation proceed at a controlled pace. For example, cells (e.g., PMCs) plated at too low a cell density are slow to reach adequate cell numbers for forming cartilage cultures or proliferate and/or differentiate in localized populations with vast spaces of tissue culture dish surface remaining between populations. Alternatively, plating cells at too high a cell density results in uncontrolled aggregation, rather that the desired condensation.
  • cells are plated at a density of about 1000 to 30,000 cells/cm 2 , more preferably about 2000 to 25,000 cells/cm 2 , even more preferably about 5000 to 20,000 cells/cm 2 , and even more preferably about 7500 to 10,000 cells/cm 2
  • cells are suspended at about 25,000 to 250,000 cells/ml and 1 ml cells are plated per well in a 6-well tissue culture dish (9.6 cm 2 ) (see for example, Example 7).
  • cells can be plated in 12-well dishes (-3.8 cm 2 ), 24-well dishes (-2.0 cm 2 ), 48-well dishes (-0.75 cm 2 ), or 96-well dishes (-0.32 cm 2 ).
  • cells can be plated in 35mm dishes (-9.6 cm 2 ), 60mm dishes (-19-21 cm 2 ), 100mm dishes (-58 cm 2 ), or 150mm dishes (-152 cm 2 ).
  • Plating cells at the above-described densities is favorable to promote condensation and subsequent chondrocytic differentiation and generation of cartilage.
  • cells e.g., PMCs
  • cells are plated at a density sufficient to achieve confluence within one week. Further culture of confluent cells results in the deposition of matrix.
  • cultures containing cells and cartilage result from an addition.
  • the cultures are to be used for implantation in vivo, it preferable to obtain the PMCs from the patient's own tissue (e.g., skin or periosteum) or from the tissue of a biologically related subject or, in the alternative, form a fetal source.
  • the patient's own tissue e.g., skin or periosteum
  • the tissue of a biologically related subject e.g., from the tissue of a biologically related subject or, in the alternative, form a fetal source.
  • the implantable culture may further comprise one or more other components, including, for example, selected antibiotics, anesthetics, anti-inflammatory compounds to reduce the risk of rejection if the implant, or immunosuppressants.
  • the growth of implantable cartilaginous cultures may be further enhanced by including other bioactive materials (e.g., growth factors including, but not limited to, TGF- ⁇ s, BMPs (e.g., BMP- 2, BMP- 12, BMP- 13) or IGFs).
  • the growth of implantable cartilaginous cultures can further be supplemented by the addition of extracellular matrix components.
  • the extracellular matrix surrounding the mesenchymal cells is primarily composed of type I and type III collagen, fibronectin and proteoglycans (von der Mark et al, 1976; Dessau et al, 1980; Silver et al, 1981; Kulyk et /., 1989b; Shinomura et al.. 1990).
  • matrix molecules which are components of basement membrane, such as laminin and type IV collagen are found throughout the developing limb bud, including the regions in which condensation and chondrogenesis occur (Solursh and Jensen, 1988).
  • preferred additional matrix components which can be included in growing, implantable, cartilatagenous cultures include, but are not limited to type I, type III collagen, fibronectin, laminin and/or type IV collagen.
  • cytochalasin D can be added to PMCs to enhance the production of cartilage cultures.
  • Cytochalasin D is a microfilament-disrupting agent which can cause cells to become round. Cytochalasin D has been shown to modify the cytoskeleton of dedifferentiated chondrocytes, causing them to round up and this change in the cytoskeleton also stimulated chondrogenesis (Loty, 1995).
  • the cartilage cultures can be implanted by a number of means routinely practiced in the art.
  • the cultures can be implanted surgically into patients (e.g., arthroscopic surgery).
  • Preferred patients are human patients although the methods of the present invention are equally useful to other mammals in need of repair or augmentation including, but not limited to horses, dogs, sheep, pigs, and/or cattle.
  • An advantage of the present invention is in the reconstruction of many structures including the nose, nasal septum, ear, respiratory system or parts of the rib cage.
  • Cartilage repair or augmentation is also advantageous in the joins of patients, for example, when cartilage is damaged by inflammation, trauma, or aging, and to repair cartilage which is congenitally defective.
  • a particularly advantageous use of the implantable cartilage cultures of the present invention is the instance where a bone is fractured or broken (e.g., a femoral or tibial fracture), particularly one of the long bones of a child.
  • a bone is fractured or broken (e.g., a femoral or tibial fracture), particularly one of the long bones of a child.
  • chondrogenesis is characterized by the transition from pluripotent mesenchymal stem cell to committed chondroprogenitor cell (i.e., mesenchymal cells which are committed to the chondrocytic differentiation pathway yet are undifferentiated, i.e., "lineage-committed cells"), to chondroblast, to chondrocyte, and these cells can be distinguished from one another based primarily on their relative maturity (Hall, 1983).
  • the various stages can be differentiated, for example, by the presence or expression of various markers or marker proteins.
  • chondrocytes are characterized by the extracellular matrix that they secrete, i.e., chondroitin-4- and chondroitin-6- sulfates are produced in high quantities by chondrocytes, and their presence has served as a useful criterion in studies of chondrogenic differentiation (Levitt and Dorfman, 1973; Kosher and Lash, 1975; Kosher et ⁇ l, 1979a,b; Kosher and Savage, 1980).
  • a marker of the chondrocytic phenotype or "cartilage marker” includes chondroitin-4- or chondroitin-6- sulfate and determination of said chondrocytic marker or "cartilage marker” includes determination of the presence of and/or levels of chondroitin-4- and/or chondroitin-6- sulfate produced by the cells of a culture (e.g., as determined by detection of chondroitin-4- and/or chondroitin-6- sulfate synthesis, detection of glycosaminoglycan levels, and/or detection of glycosaminoglycan secretion, for example, detection of the presence or levels of the glycosaminoglycan in the medium of the culture).
  • proteoglycans synthesized by embryonic chick chondrocytes can be separated into two major fractions by molecular sieve chromatography on either agarose (Palmoski and Goetinck, 1972; Levitt and Dorfman, 1974) or controlled-pore glass beads (Lever and Goetinck, 1976).
  • the larger of these fractions makes up about 90% of the total proteoglycan produced by embryonic chondrocytes and consists of aggregated and unaggregated proteoglycan monomers (Goetinck et al, 1974; Lever and Goetinck. 1976).
  • Sucrose density gradient has also been used to demonstrate that embryonic chondrocytes synthesize a large proteoglycan species produced neither by precartilagenous mesenchymal cells nor by a variety of nonchondrogenic cell types (Okayama et al, 1976; Kitamura and Yamagata, 1976). More compelling evidence supporting the existence of a cartilage-specific proteoglycan molecule is derived from immunologic studies. Antibodies prepared against the large proteoglycan monomers of 4- week old chick sternal cartilage do not cross-react with the proteoglycans produced by precartilaginous limb mesenchymal cells (Royal et al, 1980) or chick skin (Sparks et al, 1980).
  • the antibodies do, however, cross-react with proteoglycans produced by embryonic chick and quail sternal cartilage, chick Meckel's cartilage and chick limb cartilage (Sparks et al, 1980; Royal et al, 1980).
  • antibodies prepared against epiphyseal cartilage proteoglycan monomer from which most of the chondroitin sulfate glycosaminoglycan chains have been removed by hyaluronidase treatment show little, if any, cross-reactivity with the proteoglycans of precartilaginous limb mesenchymal cells (Ho et al, 1977).
  • a marker of the chondrocytic phenotype or "cartilage marker” includes chondrocyte-specific sulfated proteoglycans and determination of said chondrocytic marker or "cartilage marker” includes determination of the presence of and/or levels of said chondrocyte-specific sulfated proteoglycan produced by the cells of a culture (e.g., as determined by detection of chondrocyte- specific sulfated proteoglycan synthesis, detection of chondrocyte-specific sulfated proteoglycan levels, e.g., using antibodies, molecular sieve chromatography and/or density gradient ultracentrifugation). and/or detection of chondrocyte-specific sulfated proteoglycan secretion, for example, detection of the presence or levels of the chondrocyte-specific sulfated proteoglycan in the medium of the culture).
  • a second major macromolecule in the extracellular matrix of cartilage is collagen.
  • the predominant collagenous component of cartilage matrix is type II collagen (Miller and Matukas, 1969; Miller, 1976), although small amounts of other collagen species including type V may be present (Rhodes and Miller, 1978; Burgeson and Hollister, 1979; Shellswell et al, 1980).
  • Type II collagen is a triple helical molecule consisting of three identical polypeptide chains (called alphal (II) chains). The chains are distinct gene products differing in primary structure from the alpha chains of the other known collagens. It is found in the extracellular matrix of hyaline cartilages from a variety of sources and is absent from tissues such as bone, tendon and skin (Miller, 1976).
  • precartilaginous mesenchymal cells produce type I collagen (Linsenmayer et al, 1973a; von der Mark et al, 1976; von der Mark and von der Mark, 1977; Bailey et al, 1979; Dessau et al, 1980).
  • type II collagen is not produced by myogenic and connective tissue cells that differentiate in association with limb cartilage (Linsenmayer et al, 1973a; von der Mark et al, 1976; von der Mark and von der Mark, 1977; Bailey et al, 1979; Dessau et al, 1980).
  • Type II collagen is also not produced by limb mesenchymal cells whose differentiation into cartilage in vitro has been inhibited by 5-bromodeoxyuridine (Levitt and Dorfman, 1974) or by low-density culture conditions (von der Mark and von der Mark, 1977).
  • a marker of the chondrocytic phenotype or "cartilage marker” includes type II collagen and determination of said chondrocytic marker or "cartilage marker” includes determination of the presence of and/or levels of type II collagen produced by the cells of a culture (e.g., as determined by detection of type II collagen synthesis synthesis, detection of type II collagen levels, e.g., using Northern blot analysis, western blot analysis, ELISA and the like), and/or detection of type II collagen, for example, detection of the presence or levels of type II collagen in the medium of the culture or matrix of the cells of the culture).
  • nonchondrogenic tissues are capable of producing type II collagen and because of this it cannot be described as being a cartilage-specific molecule. It is produced by the embryonic notochord (Linsenmayer et al, 1973b; von der Mark et al, 1976; Miller and Matthews, 1974), embryonic chick corneal epithelium (Linsenmayer et al, von der Mark et al, 1977), and neural retina (Newsome et al, 1976; Smith et al, 1976; Linsenmayer and Little, 1978).
  • Type II collagen can however, be considered to be cartilage-characteristic because it is produced by only a limited number of nonchondorgenic cell types and it is not produced by precartilaginous mesenchymal cells or the fibroblastic and myogenic cells that differentiate in association with cartilage. Accordingly, it is within the scope of the present invention to include chondrocyte- specific markers as well as chondrocyte-characteristic markers within the meaning of the phrase "chondrocytic marker" or "cartilage marker”. In yet another embodiment, the presence of aggrecan can be a marker of chondrocytic differentiation or cartilage marker. IV. Determination of the Presence of Hyaline Cartilage
  • hyaline cartilage matrix is characterized by numerous densely staining granules and thin unhanded or faintly banded fibrils representing the proteoglycan and collagenous components of the matrix (Matukas et al, 1967; Anderson and Sadjera, 1971; Searls et al, Minor, 1973; Levitt et al, 1975; Pennypacker and Goetinck, 1976).
  • hyaline cartilage appears homogeneous and structureless, and is characterized by its ability to stain metachromatically in toluidine blue, or positively in Alcian blue.
  • differentiated chondrocytes are typically rounded or polygonal cells with scalloped borders. Their cytoplasm contains an extensive network of rough endoplasmic reticulum and large Golgi complexes and secretory vacuoles, suggesting that the cells are actively synthesizing and secreting extracellular matrix components (Godman and Porter, 1960; Goel, 1970; Searls et al, 1972; Thorogood and Hinchliffe, 1975).
  • any of the above-described visual, histologic and/or microscopic characteristics can be utilized as markers of the chondrocytic phenotype or "cartilage markers".
  • EXAMPLE 1 INDUCTION OF CHONDROGENESIS IN PLEURIPOTENT MESENCHYMAL STEM CELLS
  • This Example describes the isolation of primary skin and periosteal cells and demonstrates the stimulation of condensation with hyaluronic acid and initiation of differentiation. This example also demonstrates that hyaluronic acid-treated cells express CD44(v3) and co-expressed integrin ⁇ 3 subunit.
  • Hyaluronic Acid-Treated Cells lOO ⁇ l aliquots of cells were placed in 96- well plates plated on either hyaluronate (HA)-coated dishes (10 mg/ml high molecular weight HA or 100 mg/ml low molecular weight HA) or plates on plastic tissue culture dishes.
  • Hyaluronate induced the aggregation of periosteal cells (CD44 positive, see below) within 1 hour after plating, whereas cells cultured on uncoated dishes remained as single cells.
  • Mouse periosteal cells were cultured for 14 days on either HA-coated or uncoated culture dishes. Cells were fixed with 1% paraformaldehyde, 1% gluteraldehyde in 0.1 M cacodylic acid (pH 7.3). CD44 was visualized by indirect immunofluorescent microscopy using Rhodamine optics under 200 X magnification.
  • Periosteal cells or skin cells were cultured for two weeks either HA-coated or uncoated culture dishes. Cells were fixed and stained for ⁇ 3 subunit of integrin. Original magnification was 200 X.
  • CD44 refers to all CD44. ⁇ 1% of the population contains exon3. CD44v3 ⁇ 3 cells represent less than 0.5% of total skin cells.
  • EXAMPLE 2 INDUCTION OF CHONDROGENESIS IN CD44-EXPRESSING FIBROBLASTS
  • GAG-Induced Condensation of a CD44 Negative Cell Line (A31 Control Cell Line) Aggregation Assay Only aggregates of 4 or more cells are considered positive for aggregation. Only the wells which display at least 50% of the cells in aggregate are considered to score positive for aggregation.
  • A31 cells were investigated for the production of aggregate. 1 x 10 ⁇ A31 cells were incubated in calcium- and magnesium-free PBS with 5mg/ml low molecular weight hyaluronate (HAL) or high molecular weight hyaluronate (HAH) or 5mg/ml chondroitin sulfate C (CS) for 5 h at room temperature then incubated at 37°C in media up to 24 hours. At the 0 minute interval, all of the A31 control cells were isolated but they maintained an aggregation of 5% between the 0 minute and 5 hour period. At the 1 hour interval, 5% of the A31 cells which were exposed to high and low molecular weight HA and chondroitin sulfate C formed aggregates.
  • HAL low molecular weight hyaluronate
  • HAH high molecular weight hyaluronate
  • CS chondroitin sulfate C
  • the A31 cells consisted of 10% of its cells in aggregates when low and high molecular weight HA and chondroitin sulfate C were added. Cultures of A31 cells without any ligand did not aggregate. Since none of the fields showed more than 50% of cells in aggregates, it was concluded that GAGs did not induce cell aggregation of A31 cells.
  • CD44-positive cells (1 x 10 ⁇ cells) were incubated in calcium- and magnesium- free PBS with 5mg/ml low molecular weight hyaluronate (HAL) or high molecular weight hyaluronate (HAH) or 5mg/ml chondroitin sulfate C (CS) as described above. Approximately 200 cells per well were counted for each group and were scored as positive when more than 50% of the cells were involved in aggregates of four or more cells. At the 0 minute interval, 10% of the CD44 + cells had aggregated. 60% of the CD44 + cells formed aggregates after 1 hour in low or high molecular weight HA or in chondroitin sulfate C.
  • HAL low molecular weight hyaluronate
  • HAH high molecular weight hyaluronate
  • CS chondroitin sulfate C
  • CD44 + cells which were exposed to low molecular weight hyaluronic acid formed aggregates.
  • 70% scored positive for aggregation.
  • Cultures of CD44 + cells without any ligand did not aggregate. Since more than 50% of the cells per field where found in aggregates of 4 or more, it was concluded that GAGs induced the aggregation of CD44 + cells.
  • CD44-Positive Cells Display Phenotypic Characteristics of Differentiated Chondrocytes. This example demonstrates that CD44-positive, but not control cells, display markers and shape characteristic of the differentiated chondrocyte phenotype.
  • CD44 + cells which were exposed to high or low molecular weight hyaluronic acid or to chondroitin sulfate C were round and stained positively for CD44.
  • the CD44 + cells which were not exposed to the ligands (control) appeared flat and polygonal in shape, and 80% stained positively at the cell surface.
  • the CD44 + cells which were exposed to chondroitin sulfate C were flat and polygonal and 70% of the cells stained positively at the cell surface.
  • the A31 cells which were exposed to high or low molecular weight hyaluronic or to chondroitin sulfate C and the control (no ligand) were flat and polygonal and stained intracellularly with safronin O but there was no extracellular staining, suggesting that the cells did not synthesize or secrete new proteoglycans into the surrounding extracellular matrix.
  • the A31 cells demonstrated the same pattern of staining as the one day group.
  • the aggregated CD44 + 10T1/2 cells which were incubated with high or low molecular weight hyaluronic acid or with chondroitin sulfate C stained positively after one day.
  • the control CD44 + cells (no ligand) also exhibited positive intracellular staining after one day. This suggests that the cells did not synthesize or secrete new proteoglycans into the surrounding extracellular matrix.
  • the CD44 + cells which were exposed to high or low molecular weight hyaluronic acid, were round in shape. These cells stained positively with more intense staining around the aggregates.
  • the control cells (no ligand) and the cells exposed to chondroitin sulfate C were also round and stained positively on the periphery of the aggregates. This positive staining suggests that the differentiated cells synthesized proteoglycans which were secreted into the surrounding extracellular matrix.
  • This Example describes the mechanism by which CD44 aggregation induces markers characteristic of the chondrogenic phenotype.
  • A31.C1 or A31.mlv cells were treated with 1 mg/ml PBS and incubated at 4°C. After 1 h, the media was removed and replaced with DME containing 10% FBS. The cells were then incubated at 37°C in a humidified atmosphere of 5% CO2- After 24h, RNA was extracted from the cultures and Northern blot analysis performed.
  • A31.cl cells up regulate colll expression in response to hyaluronic acid (HA), however neither the mock transfected cells, A31.MLV, nor the untreated A31.C 1 express significant levels of colll ( ⁇ l).
  • A31.C 1 cells were examined for Map kinase activation.
  • Treatment of A31.C 1 with hyaluronic acid results in rapid activation of erk- 1 and erk-2 as judged by immunoprecipitation of 32p labeled erk-1 and erk-2.
  • neither untreated cells nor mock transfected cells activate Map kinase in response to HA.
  • EXAMPLE 5 Enhancement of the Chondrogenic Phenotype by Activation of Sox 9 and/or Mutation of Chondrocyte-Specific Silencers
  • Sox9 is a member of a family of genes which encodes transcription factors with a high mobility group (HMG)-type DNA binding domain. Heterozygous mutations in Sox9 in humans leads to campomelic dysplasia (CD), a severe dwarfism syndrome characterized by anomalies in all skeletal elements derived from cartilages (Forster et al, 1994).
  • HMG high mobility group
  • Sox9 is expressed in mesenchymal cell condensations in the embryo before and during cartilage deposition, but its expression ceases after the cartilage matrix is laid down. This short period of expression suggests that Sox9 has a role during initiation and maintenance of chondrogenesis and is no longer required once chondrogenesis is complete. When chondrocytes undergo dedifferentiation in culture, the expression of Sox9 is lost, indicating that the expression of Sox9 is necessary for the maintenance of the chondrocytic phenotype.
  • Whole mount in situ hybridization studies in mouse embryos have shown that Sox9 is highly expressed during gonadal development as well as during chondrogenesis (Wright, 1995).
  • Sox-9 is a gene which mediates the expression of ColIIal (the gene which expresses type II collagen) by activating chondrocyte-specific enhancers on the ColIIal gene (Bell, 1997; Lefebvre, 1997; Zhao, 1997). Sox9 inactivates the CIIS1 and CIIS2 silencers of aggrecan, a gene essential in chondrogenic progression. Moreover, the c-fos gene is expressed by PMC in the developing embryo only after they begin to undergo condensation and it plays a role in the transition of mesenchymal cells to the chondrogenic phenotype (Edwall-Arvidsson and Wroblewski, 1996).
  • CD44/HA bond releases intracellular calcium when capped CD44 binds to ankyrin, causing disruption of the cytoskeleton.
  • MAP kinase becomes phosphorylated and it activates the c-fos gene.
  • Sox-9 gene is also activated but by an alternate pathway and the ColIIal gene is expressed.
  • the migrating PMC already express Sox-9, condensation activates the c-fos gene and together both genes upregulate the ColIIal gene which is expressed in chondrocyte-like cells.
  • the cells Example 1 are derived from embryonic mesenchyme and may already be encoded to express the Sox-9 gene.
  • Sox9 expression it may be desirable to induce Sox9 expression in cells such as those used in Examples 2-3 or, alternatively, to mutate silencers of chondrocytic genes (e.g., CIIS1 and/or CIIS2 in aggrecan.
  • Induction of Sox9 expression can be achieved, for example, by transiently or stably expressing the gene in mammalian cells or by contacting cells with a compound which induced expression.
  • Mutation of chondrocytic silencers can be achieved, for example, by mutating a base within one or both of CIIS 1 and/or CIIS2.

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Abstract

La présente invention concerne des procédés permettant de produire du cartilage in vitro, ainsi que des procédés permettant de stimuler la différenciation des chondrocytes, des procédés permettant de stimuler la chondrogenèse et des procédés permettant de produire des structures cartilagineuses implantables. Les procédés de l'invention permettent, en particulier, de cultiver des cellules souches mésenchymateuses multipotentes. Les procédés de l'invention sont utilisés, en particulier, dans la réparation du cartilage ainsi que dans l'augmentation du cartilage in vivo, par exemple chez un patient qui a besoin d'une réparation ou d'une augmentation du cartilage. L'invention se rapporte également à des cultures cartilagineuses implantables.
PCT/US2000/031607 1999-11-19 2000-11-17 Procedes permettant d'induire la chondrogenese et de produire du cartilage de novo in vitro WO2001035968A1 (fr)

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