WO2002083878A1 - Methode de transplantation autologue - Google Patents

Methode de transplantation autologue Download PDF

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Publication number
WO2002083878A1
WO2002083878A1 PCT/US2002/011497 US0211497W WO02083878A1 WO 2002083878 A1 WO2002083878 A1 WO 2002083878A1 US 0211497 W US0211497 W US 0211497W WO 02083878 A1 WO02083878 A1 WO 02083878A1
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WIPO (PCT)
Prior art keywords
cells
cartilage
collagen
membrane
matrix
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PCT/US2002/011497
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English (en)
Inventor
Bruno M. Giannetti
Samuel S. Asculai
Ahmed Idouraine
Original Assignee
Verigen Transplantation Service International (Vtsi) Ag
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Verigen Transplantation Service International (Vtsi) Ag filed Critical Verigen Transplantation Service International (Vtsi) Ag
Priority to EP02726736A priority Critical patent/EP1390471A4/fr
Priority to IL15837102A priority patent/IL158371A0/xx
Priority to HU0401450A priority patent/HUP0401450A3/hu
Priority to CA002444004A priority patent/CA2444004A1/fr
Priority to MXPA03009312A priority patent/MXPA03009312A/es
Priority to BR0208879-7A priority patent/BR0208879A/pt
Priority to SK1389-2003A priority patent/SK13892003A3/sk
Priority to JP2002582217A priority patent/JP2005502390A/ja
Publication of WO2002083878A1 publication Critical patent/WO2002083878A1/fr
Priority to NO20034581A priority patent/NO20034581L/no

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3817—Cartilage-forming cells, e.g. pre-chondrocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/30—Joints
    • A61F2/30756—Cartilage endoprostheses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3629—Intestinal tissue, e.g. small intestinal submucosa
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
    • A61L27/3645—Connective tissue
    • A61L27/3654—Cartilage, e.g. meniscus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A61L27/3843—Connective tissue
    • A61L27/3852—Cartilage, e.g. meniscus
    • 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
    • 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
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin

Definitions

  • the present invention relates to the field of chondrocyte cell transplantation, bone and cartilage grafting, healing, joint repair and the prevention of arthritic pathologies.
  • the present invention is directed to new methods and instruments for chondrocyte cell transplantation and cartilage regeneration.
  • a method for regeneration- treatment of cartilage would be most useful, and could be performed at an earlier stage of a joint damage, thus reducing the number of patients needing artificial joint replacement surgery. With such preventative methods of treatment, the number of patients developing osteoarthritis would also decrease.
  • the articular chondrocytes are specialized mesenchymal derived cells found exclusively in cartilage.
  • Cartilage is an avascular tissue whose physical properties depend on the extracellular matrix produced by the chondrocytes.
  • endochondral ossification chondrocytes undergo a maturation leading to cellular hypertrophy, characterized by the onset of expression of type X collagen (Upholt, W. B. and Olsen, R. R.) CRC Boca Raton 1991, 43; Reichenberger, E. et al., Dev. Biol. 1991, 148:562; Kirsch, T. et al., Differentiation, 1992, 52:89; Stephens, M. et al., J. Cell Sci. 1993, 103:1111).
  • the teachings of the instant invention provide for effective, and efficient means of promoting the transplantation of cartilage and/or chondrocytes into a defect in an articulating joint whereby cartilage is regenerated to fix the defect.
  • the present invention provides an implantable article including a support matrix which can support the growth and attachment of cells thereto, and a method of implanting such an article to regenerate cells at an implantation location.
  • the present invention provides a method for the effective treatment of articulating joint surface cartilage in an animal by the transplantation of an implantable article including chondrocyte cells retained on an absorbable support matrix.
  • chondrocyte cells are retained only on an edge of the matrix.
  • the support matrix is a covering matrix made from collagen, and the chondrocyte cells are autologous or homologous.
  • the support matrix is made from collagen and elastin, or collagen and one or more other resorbable materials.
  • the support matrix is made from small intestine submucosa from animal sources.
  • the support matrix is made from pericardium. In a different embodiment, the support matrix is made from collagen and one or more other materials related to polyesters.
  • the implantable article preferably is secured to the transplantation site by an adhesive or mechanical retention means.
  • the present invention also is directed to an instrument for placing and manipulating the implantable article at the site of implantation, and a retention device for securing the implantable article to the site of implantation.
  • the present invention is also directed to an implantable article for cartilage repair in an animal, the implantable article including chondrocyte cells retained to an absorbable support matrix, and a method of making same.
  • the present invention is directed to a method for treating an articulating joint surface cartilage including the placing chrondrocytes upon a surface to be treated, and covering the surface to be treated with a covering matrix. The covering matrix is secured to the area of cartilage surrounding the defect.
  • FIG. 1A is a drawing showing a typical articulating end of a bone.
  • the bone material is covered on the articulating surface with a cartilaginous cap (shown by stripping labeled cartilage).
  • a cartilaginous cap shown by stripping labeled cartilage.
  • the defective site can be treated directly, or enlarged slightly by surgical procedures.
  • a hemostatic barrier (numbered 1) may be placed within the defect in the cartilage cap to inhibit or prevent vascularization into regenerating cartilage from the underlying bone (FIG. IC), when necessary, for example in defects that extend into or below the subchondral layer.
  • the chondrocytes to be implanted into the defect cavity are then layered on top of this hemostatic barrier, or directly on top of the defect.
  • FIG. 2 is a drawing showing the treated defect (gap in stippling area) in the cartilaginous cap (stippling area) covered by a matrix which is used to form a cap/patch or bandage over the defect site.
  • This cap is fixed in place, either sutured or glued to the edge of the cavity to healthy cartilage or otherwise attached.
  • This cap is covering the defective area of the joint into which the cultured chondrocytes have been transplanted.
  • FIG. 3 A shows a typical articulating end of a bone in a knee joint, having an articulating surface with a cartilaginous cap.
  • FIG. 3B shows a cartilage defect or injury to a cartilaginous cap of an articulating end of a bone.
  • FIG. 4 shows one embodiment of an implantable article according to the present invention.
  • FIG. 5 shows how the implantable article of Fig. 4 may be disposed for implantation in an arthroscopic introducer such as that shown in Fig. 6.
  • FIG. 6 shows an arthroscopic introducer for implanting the implantable article at the site of implantation, according to the present invention.
  • FIG. 7 is a drawing schematically illustrating the placement of the implantable article of Fig. 5 at the site of defect or injury in the cartilaginous cap using two access channels which can accommodate arthroscopic instruments.
  • FIG. 8 is a schematic cross section of cartilage with a defect or injury which does not extend into the subchondral layer, and an implantable article according to the present invention secured by adhesive to the site of defect or injury.
  • FIG. 9 is a schematic cross section of cartilage with a defect or injury which does not extend into the subchondral layer, and an implantable article secured to the site of defect or injury by a mechanical retainer.
  • FIG. 10 illustrates one embodiment of the mechanical retainer used to secure the implantable article to the site of defect or injury.
  • FIG. 11 is a schematic cross section of cartilage with a defect or injury which extends into the subchondral layer, and an implantable article according to the present invention secured by adhesive to the site of defect or injury.
  • FIG. 12 is a schematic cross section of cartilage with a defect or injury which extends into the subchondral layer, and an implantable article secured to the site of defect or injury by a mechanical retainer.
  • FIG. 13 A is a black and white copy of a color microphotograph of histological specimen of a solid support matrix at the beginning of chondrocyte cell growth thereon.
  • FIG. 13AA is the digitized microphotograph of Fig. 13 A.
  • FIG. 13B is a black and white copy of a color microphotograph showing the support matrix of Fig. 13 A loaded with chondrocyte cells after three weeks of chondrocyte cell growth thereon.
  • FIG. 13BB is the digitized microphotograph of Fig. 13B.
  • FIG. 13C is a photograph showing a support matrix formed of collagen having chondrocyte cells grown thereon, shown by immunohistochemical staining.
  • FIG. 13D is a photograph showing a support matrix formed of collagen, and having chondrocyte cells grown thereon in a bioreactor system, shown by immunohistochemical staining.
  • FIG. 14 is a photomicrograph showing chondrocyte cells 24 on the DePuy support matrix 22.
  • FIG. 15 is a graph depicting total cell numbers in control (black shaded), Chondro-Gide membrane group (stippled), and DePuy membrane group (gray shaded) at 3 days, 2 weeks, and 6 weeks.
  • FIG. 16 is a graph depicting cell viability in control (black shaded), Chondro-Gide membrane group (stippled), and DePuy membrane group (gray shaded) at 3 days, 2 weeks, and 6 weeks.
  • the present invention concerns the use of certain products that inhibit the formation of vascular tissue, for instance such as capillary loops projecting into the cartilage being established, during the process of autologous transplantation of chondrocytes into defects in the cartilage.
  • vascular tissue for instance such as capillary loops projecting into the cartilage being established, during the process of autologous transplantation of chondrocytes into defects in the cartilage.
  • Such products are useful in repairing cartilage defects in bones where the defects extend into or below the subchondral layer, sometimes referred to as a full thickness defect.
  • the formation of vascular tissue from the underlying bone will tend to project into the new cartilage to be formed leading to the appearance of cells other than the mesenchymal specialized chondrocytes desired.
  • the contaminating cells introduced by the vascularization may give rise to encroachment and over-growth into the cartilage to be formed by the implanted chondrocytes.
  • Surgicel ® (Ethicon Ltd., UK) which is absorbable after a period of 7-14 days. This is contrary to the normal use of a hemostatic device, such as Surgicel ® , as described in a product insert from Ethicon Ltd.
  • a hemostatic material can be used and will act as a gel like artificial coagulate.
  • Fig. IC describes such a hemostatic barrier (numbered 1) that can be used to inhibit the re-vascularization in a cartilage defect. If red blood cells should be present within a full-thickness defect of articular cartilage that is covered by such a hemostatic barrier, these blood cells will be chemically changed to hematin and thus not be able to induce vascular growth.
  • a hemostatic product used as a re-vascularization inhibitory barrier with or without fibrin adhesives such as for example Surgicel ®
  • a matrix such as a cell-free matrix or another matrix described below, is used as a patch covering or inserted into the defective area of the joint into which the cultured chondrocytes are being transplanted using, for example, autologous chondrocytes for the transplantation.
  • the present invention may also utilize allogeneic chondrocytes or xenogeneic chondrocytes for the repair of a cartilage defect.
  • the instant invention teaches methods for effective repair or treatment of cartilage defects in articular joint bone surfaces which comprises administering an agent or device to block vascular invasion of the cartilage site to be repaired, and also providing for a matrix which will isolate the repair site and keep transplanted cells in place.
  • the instant invention also provides for a kit, comprising an optional hemostatic component for insertion into the site to be repaired such that there is an effective inhibition of vascularization into the site to be repaired; and once the chondrocytes to be transplanted are placed into the site to be repaired, a matrix 2 is capped over the repair site such that the transplanted chondrocytes are held in place, but are still able to gain access to nutrients.
  • Certain aspects of the invention have been exemplified using an in vitro system to study the behavior of the chondrocytes when in contact with a certain product or a combination of certain products that will inhibit the formation of vascular tissue.
  • This in vitro testing predicts the ability of certain tested materials to support chondrocyte cell growth thereon, tests matrices for use as implants having chrondrocytes held thereon, and tests each matrix's ability to inhibit vascularization, as will occur in vivo where capillary loops project into the cartilage being established during the process of autologous transplantation of chondrocytes into defects in the cartilage.
  • Suitable hemostatic products will be characterized by having the ability to inhibit the growth, or invasion of vascular tissue, osteocytes, fibroblasts, etc. into the developing cartilage.
  • a suitable hemostatic material will achieve the goal of the methods of the instant invention in that vascular and cellular invasion into developing cartilage should be prevented in order to optimize the formation of cartilage and achieve repair of the full thickness of any defects in the articular cartilage.
  • the hemostatic barrier will be stable for an extended period of time sufficient to allow for full cartilage repair, and then be able to be absorbed or otherwise broken down over time.
  • One material identified as suitable is called
  • Surgicel ® W1912 Lit GG3DH, Ethicon Ltd. UK
  • is an absorbable hemostat such as oxidized regenerated sterile cellulose.
  • the present invention also includes a cartilage repair implant and implantation method and apparatus for such an implant.
  • the implant comprises a support matrix and autologous or homologous chondrocyte cells retained thereon.
  • the chondrocyte cells are retained only on one or more edges or layers of the matrix.
  • the support matrix is a material which will support chondrocyte cell growth and which, over time will be absorbed in a body of a patient receiving the implant.
  • the transplantation procedure may be by arthroscopic, minimally invasive or open surgery techniques.
  • the method of the invention also contemplates the use of suitable allogenic and xenogenic chondrocyte cells for the repair of a cartilage defect.
  • an implantable article 20 includes a support matrix 22 having chondrocyte cells 24 retained thereon.
  • a suitable support matrix 22 will be a solid or gel-like scaffold characterized by being able to hold a stable form for a period of time to enable the growth of chondrocytes cells thereon, both before transplant and after transplant, and to provide a system similar to the natural environment of the chondrocyte cells to optimize chondrocyte cell growth differentiation.
  • Support matrix 22 will be stable for a period of time sufficient to allow full cartilage repair and then be absorbed by the body over time, for example, within two to three months without leaving any significant traces and without forming toxic degradation products.
  • support matrix 22 preferably is a physiologically absorbable, nonantigenic membrane-like material.
  • support matrix 22 preferably is in a sheet like form having one relatively smooth side 21 and one relatively rough side 23. Rough side 23 typically faces cartilage defect 18 and promotes chondrocyte cell ingrowth, while the smooth side 21 typically faces away from cartilage defect 18 and impedes tissue ingrowth.
  • support matrix 22 has two smooth sides of similar porosity.
  • support matrix 22 is formed of polypeptides or proteins.
  • the polypeptides or proteins are obtained from natural sources, e.g., from mammals. Artificial materials, however, having physical and chemical properties comparable to polypeptides or proteins from natural sources, may also be used to form support matrix 22. It is also preferred that support matrix 22 is reversibly deformable as it is handled by the user so implantable article 20 can be manipulated and then returns to its original shape as described below, during one aspect of the present invention.
  • a preferred material from which support matrix 22 is formed is collagen such as that obtained from equine, porcine, bovine, ovine, and chicken.
  • Suitable materials from which support matrix 22 can be formed include ChondroCell ® (a commercially available type II collagen matrix pad, Ed. Geistlich Sohne, Switzerland), and Chondro-Gide ® (a commercially available type I collagen matrix pad, Ed. Geistlich Sohne, Switzerland).
  • a support matrix 22 formed of collagen Type I is somewhat stiffer than a support matrix formed from collagen
  • Type II collagen matrices may also be used.
  • Another preferred material from which support matrix is formed is a cross-linked or uncross-linked form of PermacolTM (Tissue Science Laboratories, UK).
  • collagen is obtained from a marine sponge as described in Swatschek, et al., 2002, Eur. J. of Pharmaceutics and
  • An implantable article as described above may be made, for example, by culturing chondrocyte cells on this support matrix as described in more detail below.
  • a cartilage biopsy first is harvested by arthroscopic technique from a non- weight bearing area in a joint of the patient and transported to the laboratory in a growth media containing 20% fetal calf serum.
  • the cartilage biopsy is then treated with an enzyme such as trypsin ethylenediaminetetraacetic acid (EDTA), a proteolytic enzyme and binding agent, to isolate and extract cartilage chondrocyte cells.
  • the extracted chondrocyte cells are then cultured in the growth media from an initial cell count of about 50,000 cells to a final count of about 20 million chondrocyte cells or more.
  • the growth media is exchanged for a transplant media which contains 10% autologous serum (that is, serum extracted from the patient's blood as described below). Then, the cultured chondrocyte cells in the transplant media are soaked into and penetrate one or more layers of support matrix 22, and continue multiplying to form implantable article 22. Preferably, chondrocyte cells are adhered only to one edge or an outer layer of support matrix 22. Implantable article 22 is then implanted at a site of cartilage defect 18 in the patient.
  • autologous serum that is, serum extracted from the patient's blood as described below.
  • defect or injury 18 can be treated directly, enlarged slightly, or sculpted by surgical procedure prior to implant such as described in U.S. Patent Application No. 09/320,246, to accommodate implantable article 20.
  • the culturing procedure as well as the growth and transplant medias are described by way of example, in detail below, starting first with a description of a laboratory procedure used to process the harvested cartilage biopsy and to culture the chondrocyte cells according to the present invention.
  • the growth media used to treat the cartilage biopsy during the culturing process and to grow the cartilage chondrocyte cells is prepared by mixing together 2.5 ml gentomycin sulfate (concentration 70 micromole/liter), 4.0 ml amphotericin (concentration 2.2 micromole/liter; tradename Fungizone , an antifungal available from Squibb), 15 ml 1 -ascorbic acid (300 micromole/liter), 100 ml fetal calf serum (final concentration 20%), and the remainder DMEM/F 12 media to produce about 400 ml of growth media.
  • the growth media used to treat the cartilage biopsy during the culturing process and to grow the cartilage chondrocyte cells is prepared by mixing together 2.5 ml gentomycin sulfate (concentration 70 micromole/liter), 4.0 ml amphotericin (concentration 2.2 micromole/liter; tradename Fungizone , an antifungal available from Squibb), 15
  • cartilage biopsy (The same growth media is also used to transport the cartilage biopsy from the hospital to the laboratory in which the chondrocyte cells are extracted and multiplied.) Blood obtained from the patient is centrifuged at approximately 3,000 rpm to separate the blood serum from other blood constituents. The separated blood serum is saved and used at a later stage of the culturing process and transplant procedure. Cartilage biopsy previously harvested from a patient for autologous transplantation is shipped in the growth media described above to the laboratory where it will be cultured. The growth media is decanted to separate out the cartilage biopsy, and discarded upon arrival at the laboratory. The cartilage biopsy is then washed in plain DMEM/F 12 at least three times to remove the film of fetal calf serum on the cartilage biopsy.
  • the cartilage biopsy is then washed in a composition which includes the growth media described above, to which 28 ml trypsin EDTA (concentration 0.05%) has been added. In this composition it is incubated for five to ten minutes at 37°C, and 5% CO 2 . After incubation, the cartilage biopsy is washed two to three times in the growth media, to cleanse the biopsy of any of the trypsin enzyme. The cartilage is then weighed. Typically, the minimum amount of cartilage required to grow cartilage chondrocyte cells is about 80-100 mg. A somewhat larger amount, such as 200 to 300 mg, is preferred.
  • the cartilage After weighing, the cartilage is placed in a mixture of 2 ml collagenase (concentration 5,000 enzymatic units; a digestive enzyme) in approximately 50 ml plain DMEM/F12 media, and minced to allow the enzyme to partially digest the cartilage. After mincing, the minced cartilage is transferred into a bottle using a funnel, and approximately 50 ml of the collagenase and plain DMEM/F 12 mixture is added to the bottle. The minced cartilage is then incubated for 17 to 21 hours at 37°C, and 5% CO 2 .
  • collagenase concentration 5,000 enzymatic units; a digestive enzyme
  • the incubated minced cartilage is then strained using a 40 ⁇ mesh, centrifuged (at 1054 rpm, or 200 times gravity) for 10 minutes, and washed twice with growth media.
  • the chondrocyte cells are then counted to determine their viability, following which the chondrocyte cells are incubated in the growth media for at least two weeks at 37°C, and 5% CO 2 , during which time the growth media was changed three to four times.
  • the chondrocyte cells are removed by trypsinization and centrifugation from the growth media, and transferred to a transplant media containing 1.25 ml gentamycin sulfate (concentration 70 micromole/liter), 2.0 ml amphotericin (concentration 2.2 micromole/liter; tradename Fungizone ® , an antifungal available from Squibb), 7.5 ml 1 -ascorbic acid (300 micromole/liter), 25 ml autologous blood serum (final concentration 10%), and the remainder DMEM/F12 media to produce about 300 ml of transplant media.
  • gentamycin sulfate concentration 70 micromole/liter
  • 2.0 ml amphotericin concentration 2.2 micromole/liter
  • tradename Fungizone ® an antifungal available from Squibb
  • 7.5 ml 1 -ascorbic acid 300 micromole/liter
  • 25 ml autologous blood serum final concentration 10%
  • Support matrix 22 is then cut to a suitable size fitting into the bottom of a well in a NUNCLONTM cell culture tray, and then placed under aseptic conditions on the bottom of the well with 1-2 ml transplant media.
  • a sufficient number of cultivated cartilage chondrocyte cells e.g. 3-10 million chondrocyte cells
  • support matrix 22 is then soaked into support matrix 22, and incubated approximately 72 hours at 37°C, and 5% CO 2 to allow the chondrocyte cells to continue to grow.
  • the chondrocyte cells arrange in clusters and adhere to support matrix 22.
  • the chondrocyte cells are adhered to only one outer layer of one side of support matrix 22.
  • support matrix 22 supports the growth and retention of chondrocyte cells thereon in a sufficient number to form implantable article 20, without significant loss of the biomechanical properties of support matrix 22.
  • Support matrix 22 also provides an environment to support continued growth of chondrocyte cells after implantation of the implantable article at the site of the cartilage defect.
  • the chondrocyte cells are transferred to the transplant media and then grown directly on support matrix 22 as described above for a period of at least two weeks. It has been found that implantable article 20 temporarily can be deformed without mechanical destruction or loss of the chondrocyte cells adhered to support matrix 22. This deformation is completely reversible once implantable article 20 is introduced into the joint or is placed on the surface to be treated, as described below.
  • support matrix 22 onto which chondrocyte cells are grown or loaded in a sufficient number temporarily can be deformed in a way that allows its introduction into the working device of an arthroscope without mechanical destruction or loss of its chondrocyte cell load.
  • this matrix can be secured by adhesive or mechanical retention means, to the cartilage defect area without impairing the further in situ differentiation of the chondrocytes and the regeneration of the natural cartilage matrix material.
  • FIG. 5 shows how implantable article 20 can be rolled across the diameter thereof to form a spirally cylindrical transplant so that implantable article 20 can be delivered to an implantation site through a working channel 26 of an arthroscopic introducer 28.
  • a suitable arthroscopic introducer is depicted in Fig. 6.
  • an arthroscopic introducer 30 includes a working channel 32 having a diameter and length suitable to enter the joint of interest and to deliver the desired dimension of implantable article 20.
  • the diameter of working channel 32 is approximately 8-20 mm, and the length is approximately 30-60 cm.
  • an injection channel 34 accommodating a retractable and removable needle 36.
  • Injection channel 32 is attached to a handle 38 which is telescopically depressible at least partially into working channel 32. Needle 36 extends the length of injection channel 34 and allows fluids to pass therethrough to the site of implantation.
  • Injection channel 34 is moved within working channel 32 by telescopically moving handle 38 toward or away from the implantation site.
  • Introducer 30 also includes a cap 40 made of rubber or other suitable material, slideably engaged on introducer 30.
  • cap 40 surrounds the site of the cartilage defect and excludes fluids, such as blood and other natural fluids, from flowing into the site of the cartilage defect.
  • Introducer 30 also has two or more outwardly biased gripping elements 42 attached to handle 38, for grasping, introducing and placing implantable article 20 at the implantation site.
  • gripping elements 42 engage the inside of working channel 32 and are moved toward each other in a gripping manner (as handle 38 is moved toward the user), and away from each other to release the grip (as handle 38 is moved away from the user).
  • Such telescopic movement may be controlled by a biasing element (not shown) disposed within handle 38 which allows injection channel 34 and gripping elements 42 to be slideably advanced and retracted within working channel 42.
  • Figs. 7-9 show a typical arthroscopic procedure for implanting implantable article 22 at a site of implantation such as knee joint 10.
  • Defective cartilage 18 is removed from the site of the defect, preferably to a depth above subchondral layer 44 leaving a well 46 (See Figs. 8-9).
  • the defect site is prepared to receive implantable article 22. If the subchondral layer has been disturbed to the point that bleeding occurs at the implantation site, the site optionally may first be covered with any absorbable material which acts as a hemostatic barrier. Otherwise, site preparation may include injection of a biocompatible glue through needle 36 into well 46. Such a biocompatible glue, seen as adhesive 48 in Fig.
  • Implantable article 20 previously cut to the desired dimension, and rolled into a spiral cylindrical shape as shown in Fig. 7 is then gripped by gripping elements 42 and held within the end of arthroscopic introducer 30.
  • Arthroscopic introducer 30 holding implantable article 20 within its end is then advanced to the site of implantation through an access channel 33, released from gripping elements 42, and unrolled using gripping elements 42 or allowed to unroll as it exits working channel 32.
  • Access channel 33 includes one or more channels that allow instruments such as introducer 30 and visualization instruments, to access the transplantation site.
  • implantable article 20 is manipulated such that the side holding chondrocytes therein, in this embodiment rough side 23 of implantable article 20, faces well 46 and is gently held in place in well 46 to allow adhesive 48 to harden and bind implantable article 20 in well 46.
  • a second access channel having one or more channels may be used to allow access of instruments to the site of implantation to assist in placement of the implantable article, adhesive and/or mechanical retention means, or to allow for access of visualization instruments to the site of implantation.
  • Such a separate access channel may also be used to perform one or more of the functions described in relation to arthroscopic introducer 30 or other arthroscopic instruments.
  • Suitable pins 50 include Ortho-PinTM (a commercially available lactide co-polymer pin, Ed. Geistlich Sohne, Switzerland).
  • Fig. 10 shows one embodiment of absorbable pin 50.
  • pin 50 includes head 52, intramedullar channel 54 within shaft 56, and one or more retention rings 58.
  • the dimensions of pin 50 will vary with the particular use, but, typically, pin 50 is about 10-15 mm in length, head 52 is about 4 mm in diameter, intramedullar channel 54 is approximately 1.2 mm in diameter, shaft 56 is approximately 2 mm in diameter, and retention rings 58 are about 2.5 mm in diameter.
  • Retention rings 58 serve to anchor pin 50 into healthy cartilage surrounding the cartilage defect.
  • Pin 50 is formed from any material that will not harm the body and can be absorbed or otherwise broken down by the body after a period of time.
  • pin 50 may be made from polylactide.
  • a combination of adhesive 48 and mechanical retention means such as pins 50 to secure implantable article 20 in well 46.
  • the above procedure is modified optionally to include placement of a hemostatic barrier 62 in well 46 prior to placement of implantable article 20.
  • a physician optionally can use hemostatic barrier 62 to inhibit the growth or invasion of vascular tissue, osteocytes, fibroblasts, etc. into developing cartilage. This is believed to allow hyaline cartilage to grow at the transplantation site.
  • Suitable hemostatic barriers will inhibit vascularization and cellular invasion into the developing cartilage to optimize formation of cartilage and to achieve growth of the full thickness of cartilage at the defect site.
  • the hemostatic barrier is stable for an extended period of time to allow full cartilage repair, and then will be absorbed or otherwise broken down by the body over time.
  • a suitable hemostatic barrier is Surgicel ® W1912 (Ethicon, Ltd., United Kingdom), an absorbable hemostat formed of oxidized regenerated sterile cellulose.
  • Support matrix 22 or covering matrix 2 of the present invention is formed of a collagen membrane, as manufactured by the process described in U.S. Patent No. 5,028,695 (assigned to Chemokol Deutschen Zurtechnik von Kollagen arean), which is hereby incorporated by reference.
  • the collagen membrane can be prepared from collagen raw material from cattle or pig as follows: the collagen raw material is freed of fatty acid residues, washed with water, treated with an alkali, washed with water, treated with an acid, washed with water, treated again with a strong alkali, treated with an acid, which causes swelling, treated with an inorganic salt to cause shrinkage, the material is squeezed off to a dry weight of 40-50% by weight, the water retained within the material is removed by the addition of a solvent, the material can be cross-linked if necessary, and then dried in stretched form.
  • Support matrix 22 or covering matrix 2 can also be formed of a membrane including collagen, such as Type I or Type II collagen, and elastin, such as the membrane described in U.S. Patent No. 5,397,353 (assigned to Oliver, et al., University of Dundee), which is hereby incorporated by reference.
  • the collagen of the collagen/elastin membrane may be obtained from equine, porcine, bovine, ovine, and chicken sources.
  • support matrix 22 or covering matrix 2 is a collagen/elastin porcine dermis that undergoes numerous organic extraction stages to remove the fat content of the dermis. Once the fat has been removed, the sections undergo numerous enzymatic extractions to remove all cellular material.
  • Patent No. 5,397,353 is presumed to be PermacolTM and various cross-linked versions of PermacolTM.
  • Other membranes similar to PermacolTM such as the Rapi-Seal Patch (Fusion Medical Technologies, Inc., Fremont, CA) and the Tissue Repair Patch (Glycar Vascular Inc., Dallas, TX), may also be used in the present invention.
  • the collagen/elastin membrane may also be up to 20% cross-linked with polyisocyanates, preferably hexamethylene diisocyanate (HMDI).
  • HMDI hexamethylene diisocyanate
  • the collagen/elastin membrane used as support matrix 22 or covering matrix 2 can be in the form of a sheet having two smooth sides and homogenous pore size and texture.
  • the collagen/elastin membrane has the following specifications: a thickness of 0.75 mm, a length of 4.8-5.2 cm, a width of 4.8-5.2 cm, a collagen content of >79% and a fat content of 0.4%.
  • Chondrocyte cells can be cultured on this support as described previously above to form an implantable article.
  • the implantable article can be placed into or over the cartilage defect site.
  • support matrix 22 can be formed of a Small Intestine Submucosa ("SIS").
  • SIS Small Intestine Submucosa
  • the method of preparing the SIS from a segment of small intestine is detailed in U.S. Patent No. 4,902,508, which is hereby incorporated by reference.
  • a segment of intestine, preferably harvested from porcine, ovine or bovine species, is first subject to abrasion using a longitudinal wiping motion to remove both the outer layers (particularly the tunica serosa and the tunica muscularis) and the inner layers (at least the luminal portions of the tunica mucosa).
  • the small intestinal submucosa is rinsed with saline and optionally stored in a hydrated or dehydrated state until used as described below.
  • a plurality of superimposed layers of intestinal submucosa tissue is then compressed, secured to one another and shaped to provide a multi-layered reconstructive structure, as described in U.S. Patent Nos. 5,788,625, 5,922,028 and 6,176,880 (all assigned to DePuy Orthopaedics (Warsaw, IN)), which are hereby incorporated by reference.
  • the multi-layered structure are provided with a sufficient number of submucosal layers to form a reconstructive tissue graft structure having the desired thickness for the replacement of the endogenous cartilaginous structure.
  • Other SIS membranes which are useful in the present invention include the Suspend SlingTM from Mentor Corporation (Santa Barbara, CA), Staple StripsTM from Glycar Vascular, Inc.
  • Another membrane that can be employed for support matrix 22 or covering matrix 2 according to the present invention is the resorbable collagen membrane described in U.S. Patent No. 5,837,278 (assigned to Ed Geistlich Sohne AG), which is hereby incorporated by reference.
  • This resorbable collagen membrane can be derived from naturally occurring membranes, such as sections of hide with a grain side, tendons, and various animal membranes.
  • This collagen membrane has a fibrous side to promote cell growth thereon and a smooth side to inhibit cell adhesion thereon.
  • the membrane is prepared by treatment with an alkali to saponify fats and degrade alkali sensitive substances, treated with acid to degrade acid sensitive substances, washed, dried, degreased and cross-linked if necessary.
  • membranes suitable for use as support matrix 22 or covering matrix 2 include CollaTec membrane from Colla-Tec, Inc. (Plainsboro, NJ), Collagraft from NeuColl (Campbell, CA), BioMend from Integra Life Sciences Corporation (Plainsboro, NJ), and BioMend ® Absorbable Collagen Membrane from Collagen Matrix, Inc. (Franklin Lakes, NJ). Biosynthetic Surgical Mesh from Colla-Tec, Inc. (Plainsboro, NJ), Collagraft from NeuColl (Campbell, CA), BioMend from Integra Life Sciences Corporation (Plainsboro, NJ), and BioMend ® Absorbable Collagen Membrane from Collagen Matrix, Inc. (Franklin Lakes, NJ). Biosynthetic Surgical Mesh from Colla-Tec, Inc. (Plainsboro, NJ), Collagraft from NeuColl (Campbell, CA), BioMend from Integra Life Sciences Corporation (Plainsboro, NJ), and BioMend ® Absorbable Collagen
  • support matrix 22 and/or covering matrix 2 can be formed of collagen fibrils which are cross-linked to each other via a reducing sugar or a derivative of a reducing sugar, for example as disclosed in U.S. Patent
  • sugars can include, but are not limited to, a ketone or aldehyde mono sugar, ribose, glycerose, threose, erythrose, lyxose, xylose, arabinose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose, or any other diose, triose, tetrose, pentose, hexose, septose, octose, nanose, or decose, and combinations of one or more of the same.
  • Support matrix 22 and/or covering matrix 2 can also include antimicrobial agents which have a therapeutic effect during cartilage regeneration.
  • antimicrobials include penicillin, cephalosporins, tetracyclines, streptomycin, gentamicin, sulfonamides, antifungals, such as myconazole, and anti-inflammatories, such as cortisone, and combinations of one or more of the above.
  • Factors having tissue inductive properties such as fibroblast growth factor, platelet derived growth factors, transforming growth factors, differentiating growth factors, and the like, are also included in support matrix 22.
  • Other collagen materials useful in the present invention are disclosed in U.S. Patent Nos.
  • support matrix 22 or covering matrix 2 can be formed of a sponge of collagen fibers containing antibacterial substances taurolidine and/or taurultam, as described in EP 446,262 (Geistlich Soehne AG, Wolhusen, Switzerland), which is hereby incorporated by reference.
  • the collagen sponge may be obtained from commercial sources, such as from Pentapharm AG of Basel, Switzerland, from Dr. Otto Suwelak GmgH of Billerbeck, West Germany or from Ed Geistlich Sohne A.G. of Wolhusen, Switzerland.
  • the collagen sponge can also be made by conventional methods. For example, bovine skin may be chemically and mechanically treated to separate the epidermis from the underlying associated fat.
  • the layer can then be treated with mild alkali, followed by treatment with acid, and then washed.
  • a proteolytic enzyme may be used to separate collagen from other proteins and a lipase may be used to remove residual fat.
  • the collagen product can then be treated with an oxidizing agent, homogenized, and lyophilized.
  • the incorporation of the taurolidine or taurultam may be effected prior to lyophilization or by redissoving lyophilized collagen in a solution of the taurolidine or taurultam and relyophilizing.
  • support matrix 22 or covering matrix 2 can be formed according to the method for producing porous structures described in WO99/27315 (Heshcel Ingo Dipl Ing, Germany), which is hereby incorporated by reference.
  • the porous structures are formed from a liquid or pasty mixture of substances that have at least partially solidified by cooling the mixture between two interspersed surfaces which can be tempered and have varying temperatures. During solidification an ordered structure is formed. The partially solidified product is then freeze-dried to create a homogeneous porous structure.
  • Support matrix 22 or covering matrix 2 may also be formed of one or more bioabsorbable polymers, such as collagen, fibrin, laminin and fibronectin, having large interconnected pores according to the method described in U.S. Patent No.
  • This bioabsorbable polymer can be cross-linked, for example, with hexamethylene diisocyanate (HMDI) prior to freezing.
  • HMDI hexamethylene diisocyanate
  • Support matrix 22 or covering matrix 2 can be formed of a collagen sponge such as InstatTM (Johnson & Johnson) as described in Johnson & Johnson brochure entitled “Instat Collagen Absorbable Hemostat", Sept. 1985, which is hereby incorporated by reference.
  • support matrix 22 or covering matrix 2 can be formed of a bioabsorbable sponge according to the method described in U.S. Patent No. 5,700,476 (assigned to Johnson & Johnson Medical Inc., NJ), which is hereby incorporated by reference.
  • the sponge comprises a matrix structure and at least one substructure and at least one pharmacologically active agent.
  • the matrix and substructure may be made of bioabsorbable materials such as collagen, laminin, elastin, and fibronectin, among others.
  • the sponge matrix may also comprise one or more proteins or one or more polysaccharides, or mixtures thereof.
  • the pharmacological agent may include an antimicrobial, a cytokine, a growth factor, or an antibody, among others.
  • support matrix 22 or covering matrix 2 is formed from collagen fibers as described in WO96/25961 (Geistlich Soehne AG), which is hereby incorporated by reference.
  • the matrix may further contain a hydrogel-like material comprising glycosaminoglycans, such as chondroitin sulphate, keratan sulphate, dermatan sulphate and hyaluronic acid, and growth factors.
  • glycosaminoglycans such as chondroitin sulphate, keratan sulphate, dermatan sulphate and hyaluronic acid, and growth factors.
  • An example of such a matrix is described in U.S. Patent No. 5,489,304 to Orgill, et al, hereby incorporated by reference in its entirety.
  • Support matrix 22 and covering matrix 2 can also be formed of a multi-layer membrane comprising a porous matrix layer predominantly of collagen
  • the matrix layer has an open sponge-like texture and the barrier layer has a close relatively impermeable texture.
  • the matrix layer may further contain glycosaminoglycans, such as hyaluronic acid, chondroitin sulphate, keratan sulphate and dermatan sulphate, among others.
  • the matrix layer may also contain laminin, fibronectin calcium alginate or anchorin II and growth factors.
  • the barrier may be made of collagen I and III or synthetic materials such as polyesters, polyglycolic and polylactic acids homopolymers and copolymers, glycolide and lactide copolymers, polyorthoesters and polycaprolactones.
  • membranes incorporating synthetic materials such as polyesters are included in the present invention and are exemplified by Parietex Mesh and Parietex ® Composite Mesh from Sofradim Production (Trevoux, France), SepraMeshTM from Genzyme Corporation (Framingham, MA), and ComposixTM
  • support matrix 22 and/or covering matrix 2 are formed of pericardium.
  • membranes formed from pericardium which are useful in the present invention include Tutopatch ® from Tutogen Medical, Inc. (Parsipanny, NJ), Peri-Guard Series of membranes and the BioVascular Sling from BioVascular (St. Paul, MN).
  • Certain aspects of the invention have been exemplified by using an in vitro system to study the behavior of chondrocyte cells when in contact with different support matrices. This in vitro testing predicts the ability of certain materials to mechanically withstand the arthroscopic procedure and also provides information as to chondrocyte cell growing behavior.
  • the Surgicel ® In order for the Surgicel ® to be used according to our invention in preventing development of blood vessels into autologous implanted cartilage or chondrocytes, we treated the Surgicel ® with a fixative, such as glutaric aldehyde; we have chosen 0.6% glutaric aldehyde treatment of the Surgicel ® for 1 minute, followed by washings to eliminate glutaric aldehyde residues that may otherwise be toxic to tissue.
  • the Surgicel ® was treated with the fibrin adhesive called Tisseel ® (Immuno AG, Vienna, Austria)), prior to treatment with glutaric aldehyde as described in example 2.
  • Tisseel ® fibrin adhesive
  • Surgicel ® is resorbed in a period between 7 and 14 days. This time would be too short, because a longer time is needed for preventing the development of blood vessels or vascularization as such from the bone structure into the implanted cartilage before the implanted chondrocytes have grown into a solid cartilage layer getting its nutrition requirements from the neighboring cartilage. In other words sufficient inhibition of the vascularization is needed for a longer time such as for instance one month. Therefore, the product should not be absorbed significantly prior to that time. On the other hand resorption is needed eventually.
  • the organic material used as an inhibiting barrier shall have these capabilities, and we have found that the Surgicel treated in this manner provides that function.
  • the Surgicel ® was also coated with an organic glue, and in this case we have used Tisseel ® as a glue. This product, together with the Surgicel ® produces a useable barrier for our particular purpose. Any other hemostatic or vascular inhibiting barrier could be used.
  • the Tisseel ® was mixed as described below. The Surgicel® was then coated with Tisseel ® by spraying Surgicel ® on both sides until soaked. The Tisseel (a fibrin glue) was then allowed to solidify at room temperature. Immediately prior to completed solidification, the Surgicel ® was then placed in 0.6% glutaric aldehyde for 1 minute and then washed with sterile physiological (0.9%) saline. The pH was then adjusted by PBS and/or with NaOH until the pH was stable at 7.2 to 7.4. Afterwards the thus treated Surgicel® was then washed in tissue culture medium such as minimum essential medium/F12 with 15 mM Hepes buffer.
  • Tisseel ® as the fibrin adhesive to coat the Surgicel ® .
  • fibrin adhesive or glue may also be applied directly on the bottom of the lesion towards the bone, on which the
  • the fibrin adhesive can be any adhesive which together with the fibrin component will produce a glue that can be tolerated in humans (Diara, N, et al., Burns Incl. Therm. Inj., 1984, 10:396).
  • the invention also anticipates any other glue component that can be used in lieu of the fibrin adhesive.
  • Tisseel ® or Tissucol ® Immuno AG, Vienna, Austria.
  • the Tisseel ® kit consists of the following components:
  • Tisseel ® a lyophilized, virus-inactivated Sealer, containing clottable protein, thereof: fibrinogen, Plasmafibronectin (CIG) and Factor XIII, andPlasminogen Aprotinin Solution (bovine)
  • Thrombin 500 (bovine)
  • the Tisseel ® kit contains a DUPLOJECT ® Application System.
  • the fibrin adhesive or the two-component sealant using Tisseel ® Kit is combined in the manner according to Immuno AG product insert sheet.
  • Chondrocytes were grown in minimal essential culture medium containing HAM F12 and 15 mM Hepes buffer and 5 to 7.5% autologous serum in a CO 2 incubator at 37° C. and handled in a Class 100 laboratory at Verigen Europe A/S, Symbion Science Park, Copenhagen, Denmark. Other compositions of culture medium may be used for culturing the chondrocytes.
  • the cells were trypsinized using trypsin EDTA for 5 to 10 minutes and counted using Trypan Blue viability staining in a Burker-Turk chamber. The cell count was adjusted to 7.5 x 10 5 cells per ml.
  • One NUNCLONTM plate was uncovered in the Class 100 laboratory.
  • the Surgicel ® hemostatic barrier was cut to a suitable size fitting into the bottom of the well in the NUNCLONTM tissue culture tray. In this case a circle, of a size of approximately 4 cm (but could be of any possible size) and placed under aseptic conditions on the bottom in well in a NUNCLONTM tissue culture tray. Delta 6 well sterile disposable plate for cell research work (NUNC (InterMed) Roskilde, Denmark).
  • NUNC InterMed
  • a small amount of the cell culture medium containing serum was applied to be absorbed into the hemostatic barrier and at the same time keeping the hemostatic barrier wet at the bottom of the well.
  • a number of approximately 10 6 cells in 1 ml culture medium were placed directly on top of the hemostatic barrier, dispersed over the surface of the hemostatic barrier, pre-treated with 0.4% glutaraldehyde as described above.
  • the plate was then incubated in a CO 2 incubator at 37° C for 60 minutes.
  • An amount of 2 to 5 ml of tissue culture medium containing 5 to 7.5% serum was carefully added to the well containing the cells avoiding splashing the cells by holding the pipette tip tangential to the side of the well when expelling the medium.
  • the medium was decanted and cold refrigerated 2.5% glutaraldehyde containing 0. 1M sodium salt of dimethylarsinic acid, also called sodium cacodylate, pH is adjusted with HCI to 7.4, was added as fixative for preparation of the cell and supporter (hemostatic barrier) for later preparation for electron microscopy.
  • Chondrocytes were grown in minimal essential culture medium containing HAM F12 and 15 mM Hepes buffer and 5 to 7.5% autologous serum in a CO 2 incubator at 37° C. and handled in a Class 100 laboratory at Verigen Europe A/S, Symbion Science Park, Copenhagen, Denmark. Other compositions of culture medium may be used for culturing the chondrocytes.
  • the cells were trypsinized using trypsin EDTA for 5 to 10 minutes and counted using Trypan Blue viability staining in a Buurker-Turk chamber. The cell count was adjusted to 7.5 x 10 5 cells per ml.
  • One NUNCLONTM plate was uncovered in the Class 100 laboratory.
  • the Surgicel ® (for use as a hemostatic barrier) was treated with 0.6% glutaric aldehyde for one minute as described in Example 1, and washed with 0.9% sterile sodium chloride solution or, preferably, with a buffer such as a PBS buffer or the culture medium such as MEM/F12, because pH after the glutaric aldehyde treatment is 6.8 and should preferably be 7.0 to 7.5.
  • the Tisseel ® was applied on both side of the Surgicel ® using the DUPLOJECT ® system, thus coating both sides of the Surgicel ® , the patch intended to be used, with fibrin adhesive.
  • the glue is left to dry under aseptic condition for at least 3 to 5 minutes.
  • the "coated" hemostatic barrier was placed on the bottom of the well in a NUNCLONTM Delta 6- well sterile disposable plate for cell research work (NUNC (InterMed) Roskilde, Denmark).
  • NUNC InterMed
  • a small amount of tissue culture medium containing serum was applied to be absorbed into the hemostatic barrier.
  • a number of approximately 10 6 cells in 1 ml tissue culture medium containing serum was placed directly on top of the hemostatic barrier, dispersed over the surface of the hemostatic barrier.
  • the plate was then incubated in a CO 2 incubator at 37° C. for 60 minutes. An amount of 2 to
  • the plate was incubated for 3 to 7 days with medium change at day 3. At the end of the incubation period the medium was decanted and cold refrigerated 2.5% glutaraldehyde containing 0.1M sodium salt of dimethylarsinic acid, also called sodium cacodylate, pH is adjusted with HCI to 7.4, was added as fixative for preparation of the cell and supporter (hemostatic barrier) for later preparation for electron microscopy.
  • Chondrocytes were grown in minimal essential culture medium containing HAM F12 and 15 mM Hepes buffer and 5 to 7.5% autologous serum in a CO 2 incubator at 37° C. and handled in a Class 100 laboratory at Verigen Europe A/S, Symbion Science Park, Copenhagen, Denmark.
  • the cells were trypsinized using trypsin EDTA for 5 to 10 minutes and counted using Trypan Blue viability staining in a B ⁇ rker-T ⁇ rk chamber. The cell count was adjusted to 7.5 x 10 5 to 2 x 10 6 cells per ml.
  • One NUNCLONTM plate was uncovered in the Class 100 laboratory.
  • the Bio-Gide ® is a resorbable bilayer membrane which will be used as the patch or bandage covering the defective area of the joint into which the cultured chondrocytes are being transplanted by autologous transplantation.
  • the Bio-Gide® is a pure collagen membrane obtained by standardized, controlled manufacturing processes by E. D. Geistlich Sohne AG, CH-61 10 Wolhusen. The collagen is extracted from veterinary certified pigs and is carefully purified to avoid antigenic reactions, and sterilized in double blisters by gamma-i ⁇ adiation.
  • the bilayer membrane has a porous surface and a dense surface. The membrane is made of collagen type I and type III without further crosslinking or chemical treatment. The collagen is resorbed within 24 weeks.
  • the membrane retains its structural integrity even when wet and it can be fixed by sutures or nails.
  • the membrane may also be "glued” using fibrin adhesive such as Tisseel ® to the neighboring cartilage or tissue either instead of sutures or together with sutures.
  • the Bio-Gide ® was uncovered in a class 100 laboratory and placed under aseptic conditions on the bottom of the wells in a NUNCLONTM tissue culture tray. Delta 6 well sterile disposable plate for cell research work (NUNC (InterMed) Roskilde, Denmark), either with the porous surface of the bilayer membrane facing up or with the dense surface facing up. Approximately 10 6 cells in 1 ml tissue culture medium containing serum was placed directly on top of the Bio-Gide ® , dispersed either over the porous or the dense surface of the Bio-Gide ® .
  • the plate was then incubated in a CO 2 incubator at 37° C for 60 minutes. An amount of 2 to 5 ml of tissue culture medium containing 5 to 7.5% serum was carefully added to the well containing the cells avoiding splashing the cells by holding the pipette tip tangential to the side of the well when expelling the medium. On day 2 after the chondrocytes were placed in the well containing the Bio-Gide ® , the cells were examined in a Nikon Invert microscope. It was noticed that some chondrocytes had adhered to the edge of the Bio-Gide ® . It was of course not possible to be able to look through the Bio-Gide itself using this microscope. The plate was incubated for 3 to 7 days with medium change at day
  • Chondrocytes were grown in minimal essential culture medium containing HAM F12 and 15 mM Hepes buffer and 5 to 7.5% autologous serum in a CO 2 incubator at 37° C. and handled in a Class 100 laboratory at Verigen Europe A/S, Symbion Science Park, Copenhagen, Denmark.
  • the cells were trypsinized using trypsin EDTA for 5 to 10 minutes and counted using Trypan Blue viability staining in a B ⁇ rker-T ⁇ rk chamber. The cell count was adjusted to 7.5 x 10 5 to 2 x 10° cells per ml.
  • One NUNCLONTM plate was uncovered in the Class 100 laboratory.
  • the Bio-Gide ® used as a resorbable bilayer membrane may also be used together with an organic glue such as Tisseel ® with additional, significantly higher content of Aprotinin than normally found in the Tisseel ® , as described in the product insert.
  • an organic glue such as Tisseel ®
  • the resorption of the material will be delayed by weeks instead of the normal time span of days.
  • the Tisseel is applied to the bottom of the well of the NUNCLONTM plate, and allowed to solidify incompletely.
  • a collagen patch such as a Bio-Gide ® is then applied over the Tisseel ® and glued to the bottom of the well.
  • This combination of Bio-Gide ® and Tisseel ® is designed to be a hemostatic barrier that will inhibit or prevent development or infiltration of blood vessels into the chondrocyte transplantation area.
  • This hybrid collagen patch can now be used for both as a hemostatic barrier at the bottom of the lesion (most proximal to the surface to be repaired) but also as a support for cartilage formation because the distal surface can be the porous side of the collagen patch and thus encourage infiltration of chondrocytes and cartilage matrix.
  • this hybrid collagen patch can also be used to cover the top of the implant with the collagen porous surface directed towards the implanted chondrocytes and the barrier forming the top.
  • the hybrid collagen patch with an elevated Aprotinin component may also be used without any organic glue such as Tisseel ® and placed within the defect directly, adhering by natural forces.
  • the collagen patch can be used both as the hemostatic barrier, and the cell-free covering of the repair/transplant site, with the porous surfaces of the patches oriented towards the transplanted chondrocytes/cartilage.
  • Another variant would use a collagen patch which consists of Type II collagen (Geistlich Sohne AG, CH-61 10 Wolhusen).
  • the instant invention provides for a hybrid collagen patch where said patch is a collagen matrix with elevated levels of aprotinin component, preferably about 25,000 KlU/ml, in association with organic matrix glue, where the collagen component is similar to the Bio-Gide ® resorbable bilayer material or Type II collagen, and the organic glue is similar to the Tisseel ® material.
  • the hybrid collagen patch does not use any organic glue to adhere to the site of repair.
  • kits as envisioned, will allow for the convenient practice of the method of the instant invention.
  • a kit of the invention will provide sterile components suitable for easy use in the surgical environment, and will provide a suitable hemostatic barrier, suitable covering patch, and if needed organic glue.
  • a kit of the invention may also provide sterile, cell-free matrix material for supporting autologous chondrocytes that are to be implanted into an articular joint surface defect.
  • a kit of the invention contains a Surgicel hemostatic barrier and a Bio-Gide ® covering patch with suitable coating of Tisseel ® organic glue, where the Surgicel ® and Bio-Gide ® have been treated according to the teachings of the invention to increase the time till resorption.
  • the Tisseel ® is supplemented with additional Aprotinin to increase time of resorption.
  • the hemostatic barrier and covering patch are both a semi-permeable collagen matrix which is treated to extend the time of resorption of the material.
  • Tisseel ® glue in enhanced form as a separate component to be applied as needed because of the inherent variability and unique circumstances every repair/transplantation procedure will encounter. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments and examples are, therefore, to be considered in all respects as illustrative and not restrictive.
  • EXAMPLE 8 Chondrocyte cells were grown for three weeks in the growth media described above in a CO 2 incubator at 37° C and handled in a Class 100 laboratory at Verigen Transplantation Service ApS, Copenhagen, DK or at University of Liibeck, Lubeck, Germany. [Note that other compositions of growth media may also be used for culturing the chondrocyte cells.] The cells were trypsinized using trypsin EDTA for 5 to 10 minutes and counted using Trypan Blue viability staining in a Biirker-Turk chamber. The cell count was adjusted to 7.5 x 10 5 chondrocyte cells per milliliter. One NUNCLONTM plate was uncovered in the Class 100 laboratory.
  • a support matrix mate ⁇ al specifically a Chondro-Gide collagen membrane (identical to Bio-Gide ® except Chondro-Gide ® has a larger width and length than Bio-Gide ® ; both available from Ed Geistlich Sohne, Garlich Pharma AG, Wolhusen, Switzerland), was cut to a suitable size to fit into the bottom of a well in a NUNCLONTM cell culture tray. In this case a circle of a size of approximately 4 cm was placed under aseptic conditions on the bottom of the well.
  • chondrocyte cells were transferred from the growth media to the transplant media described above, and approximately 5 x 10 6 chondrocyte cells in 5 ml transplant media were placed directly on top of the support matrix and dispersed over the surface thereof.
  • the plate was incubated in a CO 2 incubator at 37° C for 3 days. After this period the chondrocyte cells had arranged in clusters and started to grow on the support matrix, and could not be removed from the support matrix by rinsing it with medium or even by mechanically exerting mild pressure on the matrix.
  • the transplant media was decanted and the support matrix holding chondrocyte cells grown thereon was cold refrigerated in 2.5 % glutaraldehyde containing 0.1 M sodium salt of dimethylarsinic acid, added as fixative.
  • the support matrix was stained with
  • Fig. 13 A A black and white copy of a color microphotograph thereof is shown in Fig. 13 A.
  • a color version of the microphotograph is also submitted as Fig. 13AA to better illustrate the features of the microphotograph.
  • EXAMPLE 9 Chondrocytes were grown for three weeks in the growth media described above in a CO 2 incubator at 37° C and handled in a Class 100 laboratory at Verigen Transplantation Service ApS, Copenhagen, DK or at University of L ⁇ beck, Germany. The cells were trypsinized using trypsin EDTA for 5 to 10 minutes and counted using Trypan Blue viability staining in a B ⁇ rker-Turk chamber. The chondrocyte cell count was adjusted to 5 x 10 5 chondrocyte cells per milliliter.
  • One NUNCLONTM plate was uncovered in the Class 100 laboratory.
  • the Chondro-Gide ® support matrix, as in Example 1 was cut to a suitable size fitting into the bottom of a well in the NUNCLONTM cell culture tray.
  • the chondrocyte cells were transferred from the growth media to the transplant media described above, and approximately 5x 10 5 cells in 5 ml transplant media were placed directly on top of the support matrix and dispersed over the surface of the support matrix.
  • the plate was incubated in a CO 2 incubator at 37° C for 3 weeks.
  • the transplant media was decanted, and the support matrix holding the chondrocyte cells thereon was cold refrigerated in 2.5% glutaraldehyde containing 0.1 M sodium salt of dimethylarsinic acid, added as fixative.
  • the support matrix was stained with Safranin O for histological evaluation.
  • the chondrocyte cells were observed to have grown and multiplied on the support matrix building clusters in the center of the carrier and lining up along the surface.
  • the chondrocyte cells were trypsinized using trypsin EDTA for 5 to 10 minutes and counted using Trypan Blue viability staining in a B ⁇ rker-T ⁇ rk chamber. The chondrocyte cell count was adjusted to 5 x 10 5 chondrocyte cells per milliliter.
  • One NUNCLONTM plate was uncovered in the Class 100 laboratory.
  • the chondrocyte cells were transferred from the growth media to the transplant media described above, and approximately 5 x 10 6 cells in 5 ml transplant media were placed directly on top of the support matrix and dispersed over the surface of the support matrix.
  • the plate was incubated in a CO 2 incubator at 37° C for 3 weeks.
  • the support matrix holding the grown chondrocyte cells was then incubated with collagenase for 16 hours.
  • the support matrix holding the chondrocyte cells was then centrifuged. Cells were seeded on a NUNCLONTM plate and an aliquot counted using Trypan Blue viability staining in a B ⁇ rker-T ⁇ rk chamber. A microphotograph thereof is shown in Fig. llC. The total calculated cell number was found to be 6 x 10 6 and the viability was >95%.
  • EXAMPLE 11 The present example describes a test of the toxicity and biocompatability of a membrane prepared according to U.S. Patent No. 4,902,508;
  • the DePuy membrane was tested with the Chondro-Gide ® membrane as positive control, and a negative control using the same method but without any membrane.
  • Previously frozen human chondrocyte cells 14 million cells were thawed and washed, and the cell number and viability were determined. 3.2 million cells were recovered after thaw at 87% viability.
  • 1.6 million cells were added to each of two tissue culture flasks at a concentration of 5.3 x 10 4 cells per milliliter, and incubated at 37° C for three days. The resulting cell number and viability for the flasks were 3.5 million cells, with 98% viability and 3.6 million cells, with 93% viability, respectively.
  • Cell count and viability were analyzed for six samples of each membrane, and six samples of the control group without any membrane.
  • the membranes were analyzed at three days, two weeks, and six weeks.
  • the samples of the DePuy and Chondro-Gide ® membranes were cut into one inch squares.
  • Chondrocytes were trypsinized and cell viability and cell number were determined as indicated above. The cells were pelleted by centrifugation and resuspended to a concentration of 1 million cells per milliliter.
  • the DePuy and Chondro-Gide membranes were washed twice with phosphate buffered saline (PBS), having a pH of 7.17. Each membrane was inserted into a well of a culture dish. One hundred microliters of a chondrocyte cell suspension at a concentration of 1 million cells per milliliter was applied to each piece of membrane and in the bottom of six wells with no membrane. Additional culture medium (3 milliliters) was added to each well. The culture plates were incubated for at least three days at 37° C.
  • PBS phosphate buffered saline
  • Figure 14 illustrates chondrocyte cells adhered to the DePuy membrane. Cells were harvested and counted at three days, two weeks, and six weeks.
  • the DePuy and Chondro-Gide ® membranes were treated with an enzyme solution containing a mixture of 2 milliliters of 0.25% trypsin and 1 milliliter of collagenase (a total of 5000 units) to dissolve the membranes so cell count and viability could be determined. Once dissolved, the chondrocyte cells were harvested by centrifugation and counted. The length of treatment with the enzyme solution varies with the type of membrane. For the DePuy membrane, collagenase digestion was much longer than for the Chondro-Gide ® . The DePuy membrane was not completely dissolved after 2 hours of digestion while the Chondro-Gide ® membrane completely dissolved in about 1 to 1.5 hours. To avoid cell stress, collagenase digestion should not proceed for more than 2 hours.
  • the control group of chondrocytes were trypsinized, washed, and pelleted by centrifugation. The cells were then resuspended in DMEM medium and the final cell number was ascertained.
  • control cells amounted to about 72,000 cells while Chondro- Gide ® cells were about 109,167 and the DePuy cells amounted to about 169,583 cells on the support matrices.
  • control cells proliferated about eight times to 575,167, while Chondro-Gide ® cells nearly quadrupled in number to 427,500 and the DePuy cells nearly tripled in number to 494,167.
  • the number of control cells decreased slightly to about 528,333 while the number of Chondro-Gide ® cells and the DePuy cells decreased dramatically to 153,333 and 100,833, respectively.

Abstract

La présente invention concerne diverses matrices de support auxquelles des cellules peuvent adhérer et sur lesquelles elles peuvent proliférer. De telles matrices de support sont utiles dans le cadre d'une implantation dans une plaie, afin de favoriser la guérison et la régénération du tissu atteint. L'invention concerne en outre un article qui comporte une membrane dont au moins une couche présente une surface poreuse et comprend du tissu intestinal sous-muqueux, ainsi que des cellules adhérant à ladite couche. Selon la présente invention, ces cellules comprennent des cellules chondrocytes.
PCT/US2002/011497 2001-04-12 2002-04-12 Methode de transplantation autologue WO2002083878A1 (fr)

Priority Applications (9)

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EP02726736A EP1390471A4 (fr) 2001-04-12 2002-04-12 Methode de transplantation autologue
IL15837102A IL158371A0 (en) 2001-04-12 2002-04-12 Method for autologous transplantation
HU0401450A HUP0401450A3 (en) 2001-04-12 2002-04-12 Method for autologous transplantation
CA002444004A CA2444004A1 (fr) 2001-04-12 2002-04-12 Methode de transplantation autologue
MXPA03009312A MXPA03009312A (es) 2001-04-12 2002-04-12 Metodo para trasplante autologo.
BR0208879-7A BR0208879A (pt) 2001-04-12 2002-04-12 Artigo
SK1389-2003A SK13892003A3 (sk) 2001-04-12 2002-04-12 Spôsob autológnej transplantácie
JP2002582217A JP2005502390A (ja) 2001-04-12 2002-04-12 自家移植方法
NO20034581A NO20034581L (no) 2001-04-12 2003-10-10 Fremgangsmåte for autolog transplantasjon

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US28325301P 2001-04-12 2001-04-12
US60/283,253 2001-04-12

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007537778A (ja) * 2004-03-09 2007-12-27 オステオバイオロジックス， インコーポレイテッド 自己組織もしくは同種組織と組合せた移植片足場
JP2008519614A (ja) * 2004-11-10 2008-06-12 ファイブローゲン、インコーポレーテッド 移植可能なコラーゲン組成物
EP1937293A2 (fr) * 2005-09-02 2008-07-02 Ed. Geistlich Söhne Ag Für Chemische Industrie Méthode pour réparer des déchirures méniscales
WO2009106642A1 (fr) * 2008-02-29 2009-09-03 Coloplast A/S Matrice cartilagineuse biosynthétique et ses procédés de fabrication
US8877246B2 (en) 2008-02-29 2014-11-04 Coloplast A/S Compositions and methods for augmentation and regeneration of living tissue in a subject
US9125871B2 (en) 2005-06-30 2015-09-08 Biotissue Ag Cell-free graft

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MX2015000460A (es) * 2012-07-11 2015-07-06 Osiris Therapeutics Inc Productos de cartilago alterados.

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5759190A (en) * 1996-08-30 1998-06-02 Vts Holdings Limited Method and kit for autologous transplantation

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5788625A (en) * 1996-04-05 1998-08-04 Depuy Orthopaedics, Inc. Method of making reconstructive SIS structure for cartilaginous elements in situ
US6171340B1 (en) * 1998-02-27 2001-01-09 Mcdowell Charles L. Method and device for regenerating cartilage in articulating joints

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5759190A (en) * 1996-08-30 1998-06-02 Vts Holdings Limited Method and kit for autologous transplantation

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007537778A (ja) * 2004-03-09 2007-12-27 オステオバイオロジックス， インコーポレイテッド 自己組織もしくは同種組織と組合せた移植片足場
JP2008519614A (ja) * 2004-11-10 2008-06-12 ファイブローゲン、インコーポレーテッド 移植可能なコラーゲン組成物
US9125871B2 (en) 2005-06-30 2015-09-08 Biotissue Ag Cell-free graft
EP1937293A2 (fr) * 2005-09-02 2008-07-02 Ed. Geistlich Söhne Ag Für Chemische Industrie Méthode pour réparer des déchirures méniscales
EP1937293A4 (fr) * 2005-09-02 2011-07-20 Geistlich Soehne Ag Méthode pour réparer des déchirures méniscales
US8084428B2 (en) 2005-09-02 2011-12-27 Ed. Geistlich Soehne Ag Fuer Chemische Industrie Method of repairing meniscal tears
AU2006287197B2 (en) * 2005-09-02 2013-02-14 Ed. Geistlich Soehne Ag Fuer Chemische Industrie Method of repairing meniscal tears
WO2009106642A1 (fr) * 2008-02-29 2009-09-03 Coloplast A/S Matrice cartilagineuse biosynthétique et ses procédés de fabrication
US8877246B2 (en) 2008-02-29 2014-11-04 Coloplast A/S Compositions and methods for augmentation and regeneration of living tissue in a subject

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CA2444004A1 (fr) 2002-10-24
EP1390471A4 (fr) 2005-04-13
JP2005502390A (ja) 2005-01-27
NO20034581L (no) 2003-12-09
EP1390471A1 (fr) 2004-02-25
CN1514877A (zh) 2004-07-21
NO20034581D0 (no) 2003-10-10
HUP0401450A3 (en) 2006-11-28
IL158371A0 (en) 2004-05-12
BR0208879A (pt) 2004-06-29
PL367295A1 (en) 2005-02-21
RU2003132877A (ru) 2005-03-10
HUP0401450A2 (hu) 2004-11-29
SK13892003A3 (sk) 2004-07-07
CZ20033051A3 (en) 2004-05-12

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