DE19632404A1 - Artificial tissue comprising three-dimensional extracellular matrix, cell interaction system - Google Patents
Artificial tissue comprising three-dimensional extracellular matrix, cell interaction systemInfo
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- DE19632404A1 DE19632404A1 DE19632404A DE19632404A DE19632404A1 DE 19632404 A1 DE19632404 A1 DE 19632404A1 DE 19632404 A DE19632404 A DE 19632404A DE 19632404 A DE19632404 A DE 19632404A DE 19632404 A1 DE19632404 A1 DE 19632404A1
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- 230000008614 cellular interaction Effects 0.000 title abstract 2
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- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims description 3
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- 101000669513 Homo sapiens Metalloproteinase inhibitor 1 Proteins 0.000 claims description 2
- 102000000589 Interleukin-1 Human genes 0.000 claims description 2
- 108010002352 Interleukin-1 Proteins 0.000 claims description 2
- 102000019223 Interleukin-1 receptor Human genes 0.000 claims description 2
- 108050006617 Interleukin-1 receptor Proteins 0.000 claims description 2
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- 210000001612 chondrocyte Anatomy 0.000 claims 1
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- AEMRFAOFKBGASW-UHFFFAOYSA-N Glycolic acid Polymers OCC(O)=O AEMRFAOFKBGASW-UHFFFAOYSA-N 0.000 description 1
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Classifications
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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
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- 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
- C12N2503/00—Use of cells in diagnostics
- C12N2503/04—Screening or testing on artificial tissues
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- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Rheumatology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Materials For Medical Uses (AREA)
- Prostheses (AREA)
Abstract
Description
Die Erfindung betrifft transplantierbare Knorpelgewebe mit immunsuppressiven Eigenschaften, Verfahren zu ihrer Herstellung und ihre Verwendung, im engeren Sinne die künstliche Herstellung von Knorpelgeweben aus Zellen, einer mechanischen Stützstruktur und einem Gel.The invention relates to transplantable cartilage tissue with immunosuppressive Properties, processes for their production and their use, in more detail Artificial production of cartilage tissue from cells, one mechanical support structure and a gel.
Künstlich hergestelltes Knorpelgewebe ist während und nach einer Transplantation einer Reihe von negativen Einflüssen ausgesetzt, die eine langfristige Stabilität des Gewebes gefährden. (Bujia-J; et al, Ann-Rheum-Dis. 1994 Apr; 53(4): 229-34). Besonders wichtig ist dies z. B. bei Patienten, die unter Arthrose oder rheumatoider Arthritis leiden. Hier muß das implantierte Gewebe den krankhaft degenerativen und entzündlichen Prozessen lokal entgegenwirken.Artificially made cartilage is during and after a transplant exposed to a number of negative influences that affect long term stability of the Endanger tissue. (Bujia-J; et al, Ann-Rheum-Dis. 1994 Apr; 53 (4): 229-34). This is particularly important. B. in patients suffering from osteoarthritis or rheumatoid Suffering from arthritis. Here the implanted tissue must be the pathologically degenerative and counteract inflammatory processes locally.
Es sind mehrere Verfahren zur Herstellung transplantierbarer Knorpelersatzgewebe bekannt (Vacanti CA et al, Plastic and Reconstructive Surgery, 8: 753-759, 1991 und Sittinger M. et al., Biomaterials 1994, 15: 451-456).There are several methods of making transplantable Cartilage replacement tissue known (Vacanti CA et al, Plastic and Reconstructive Surgery, 8: 753-759, 1991 and Sittinger M. et al., Biomaterials 1994, 15: 451-456).
Mit der DE 43 06 661 wird die Herstellung eines Transplantates aus Zellkulturen beansprucht.DE 43 06 661 describes the production of a transplant from cell cultures claimed.
Es sind auch Möglichkeiten beschrieben worden, Zellen der Gelenkinnenhaut ex vivo mit vorwiegend antientzündlichen Genen auszustatten und dann wieder zu implantieren (Evans, C. H. and P. D. Robbins, 1994: Gene therapy for arthritis. In Gene therapeutics: Methods and applications of direct gene transfer. J. A. Wolff, editor. Birkhäuser, Boston, 312-343).Possibilities have also been described for ex to be provided with predominantly anti-inflammatory genes in vivo and then closed again implant (Evans, C.H. and P.D. Robbins, 1994: Gene therapy for arthritis. In Gene therapeutics: Methods and applications of direct gene transfer. J. A. Wolff, editor. Birkhauser, Boston, 312-343).
Der Erfindung liegt die Aufgabe zugrunde, die gewebetypischen Eigenschaften des Transplantats zu stabilisieren und immunologische/entzündliche Prozesse im Bereich des Transplantats zu unterdrücken oder abzugrenzen.The invention has for its object the tissue-typical properties of Stabilize graft and immunological / inflammatory processes in the Suppress or delineate the area of the graft.
Die Aufgabe wurde durch eine genetische Manipulation von Zellen des Transplantats gelöst und speziell dadurch, daß menschliche Zellen mit bestimmten Genen, z. B. bone morphogenic protein, Interleukin-1-Rezeptor-Antagonist oder TGF-β ausgestattet und danach zu einem neuen Knorpelgewebe gezüchtet werden.The task was accomplished through a genetic manipulation of cells of the Transplant solved and specifically in that human cells with certain Genes, e.g. B. bone morphogenic protein, interleukin-1 receptor antagonist or TGF-β equipped and then grown to a new cartilage will.
Erfindungsgemäß wird ein Gerüst aus Polymeren, z. B. resorbierbare Fasern, vorgeformt, mit einer Zellsuspension gefüllt und die Suspension anschließend verfestigt. Die Zellen produzieren in dieser dreidimensionalen Struktur eine neue extrazelluläre Matrix (ECM). Diese Matrix bestimmt im wesentlichen die typischen Eigenschaften eines Bindegewebes.According to the invention, a framework made of polymers, e.g. B. resorbable fibers, preformed, filled with a cell suspension and then the suspension solidified. The cells produce a new one in this three-dimensional structure extracellular matrix (ECM). This matrix essentially determines the typical ones Properties of a connective tissue.
Die erfindungsgemäß hergestellten Knorpelgewebe besitzen insbesondere immunsuppressive Eigenschaften.The cartilage tissues produced according to the invention have in particular immunosuppressive properties.
Es hat sich überraschenderweise herausgestellt, daß die erfindungsgemäße Vorgehensweise zum in vivo Schutz und zur in vivo Erhaltung in vitro hergestellter Knorpelgewebe geeignet ist und daß die erfindungsgemäßen Knorpelgewebe zur Therapie von Knorpelschäden bei Patienten mit rheumatoider Arthritis eingesetzt werden können. Es ist ferner überraschend, daß die erfindungsgemäß eingesetzte semipermeable Membran, mit der die hergestellte Knorpelgewebe vor, während und einige Zeit nach der Implantation umgeben werden, Schutz bieten vor immunologischen und dedifferenzierenden Reaktivitäten.It has surprisingly been found that the invention Procedure for in vivo protection and for in vivo maintenance of in vitro manufactured Cartilage tissue is suitable and that the cartilage tissue according to the invention for Therapy of cartilage damage is used in patients with rheumatoid arthritis can be. It is also surprising that the one used according to the invention semipermeable membrane with which the cartilage tissue prepared before, during and be surrounded some time after the implantation, provide protection from immunological and dedifferentiating reactivities.
Die erfindungsgemäßen transplantierbaren Knorpelgewebe bestehen aus einer neuen extrazellulären Matrix (ECM) in dreidimensionalen Strukturen, aus vermehrten Knorpelzellen, aus vernetzbaren/polymerisierbaren Polypeptiden oder Polysacchariden und aus Zellen, die aufgrund gentechnischer Manipulation Matrixmoleküle, immunsuppressive oder zelldifferenzierende Faktoren freisetzen. Ihre Strukturen bestehen aus dreidimensionalen Faserstrukturen, dreidimensionalen Geweben, aus Fibrin und aus einer das künstliche Knorpelsystem umgebenden semipermeablen Membran.The transplantable cartilage tissues according to the invention consist of a new extracellular matrix (ECM) in three-dimensional structures increased cartilage cells, from crosslinkable / polymerizable polypeptides or Polysaccharides and from cells due to genetic engineering Release matrix molecules, immunosuppressive or cell differentiating factors. Their structures consist of three-dimensional fiber structures, three-dimensional fabrics, made of fibrin and one artificial Cartilage system surrounding semipermeable membrane.
Eine gewünschte Form besteht z. B. aus einem Silicon-Negativ, Faserwolle oder Faserstrukturen aus resorbierbaren Polymeren, wie a-Hydroxysäuren und die semipermeable Membran aus Polyelektrolytkomplexen, wie Polylysin oder Hyaluronsäure.A desired shape is e.g. B. from a silicone negative, fiber wool or Fiber structures made from resorbable polymers, such as a-hydroxy acids and the semipermeable membrane made of polyelectrolyte complexes, such as polylysine or Hyaluronic acid.
Die Verfahren zur Herstellung von transplantierbarem Knorpelgewebe bestehen darin, daß polymerisierbare Lösungen enthaltende Knorpelzellsuspensionen und gentherapeutisch veränderte Zellen in dreidimensionale Strukturen gefüllt, mit Thrombin verfestigt, die Zellen in dieser dreidimensionalen Struktur eine neue extrazelluläre Matrix (ECM) produzieren und das Knorpelsystem mit einer semipermeablen Membran umgeben wird.The procedures for making transplantable cartilage exist in that cartilage cell suspensions containing polymerizable solutions and genetically modified cells filled in three-dimensional structures, with Thrombin solidified, the cells in this three-dimensional structure a new one produce extracellular matrix (ECM) and the cartilage system with a semipermeable membrane is surrounded.
Die polymerisierbaren Lösungen bestehen aus vernetzbaren/polymerisierbaren Polypeptiden oder Polysacchariden und die veränderten Zellen aus Zellen, die aufgrund gentechnischer Manipulation Matrixmoleküle, immunsuppressive oder zelldifferenzierende Faktoren freisetzen.The polymerizable solutions consist of cross-linkable / polymerizable Polypeptides or polysaccharides and the altered cells from cells that due to genetic engineering manipulation matrix molecules, immunosuppressive or release cell-differentiating factors.
Als Polypeptid wird Fibrinogen in der Weise eingesetzt, daß fibrinogenhaltige Zellsuspensionen in Strukturen aus dreidimensionalen Faserstrukturen oder aus dreidimensionalen Geweben gefüllt und mit Thrombin verfestigt werden. Das Fibrinogen wird aus dem Patienten gewonnen, von dem die Zellen stammen und/oder in den das dreidimensionale Knorpelgewebe implantiert werden soll. Erfindungsgemäß werden alle oder ein Teil der Zellen mit einem oder mehreren der folgenden Gene gentherapeutisch verändert: TGF-β, bone morphogenetic proteins, Morphogene, Rezeptoren für Morphogene, antiinflammatorische Zytokine, Zytokinantagonisten, wie IL-1-Antagonisten, IL-1-Rezeptorantagonist, TNF- Antagonisten oder mit Proteaseinhibitoren (TIMP, PAI).Fibrinogen is used as the polypeptide in such a way that fibrinogen-containing Cell suspensions in structures made of three-dimensional fiber structures or out three-dimensional tissues are filled and solidified with thrombin. The fibrinogen is obtained from the patient from whom the cells originate and / or into which the three-dimensional cartilage tissue is to be implanted. According to the invention, all or part of the cells have one or more of the following genes have been genetically modified: TGF-β, bone morphogenetic proteins, morphogens, receptors for morphogens, anti-inflammatory cytokines, Cytokine antagonists, such as IL-1 antagonists, IL-1 receptor antagonists, TNF- Antagonists or with protease inhibitors (TIMP, PAI).
Dabei lassen sich verschiedene Zellen mit jeweils nur einer genetischen Manipulation oder mehrere Zellen mit verschiedenen genetischen Manipulationen mit unveränderten Zellen kombinieren. Die eingebrachten Gene werden vorübergehend oder permanent exprimiert.Different cells can be identified with only one genetic Manipulation or multiple cells with different genetic manipulations combine with unchanged cells. The genes introduced are expressed temporarily or permanently.
Überraschenderweise lassen sich Formgebung und genetische Manipulation einsetzen, um eine Polarisierung, basierend auf einer ungleichen Verteilung von genetisch manipulierten Zellen und unveränderten Zellen, zu erreichen. Ein weiteres Merkmal der Erfindung besteht darin, daß genetisch manipulierte und unveränderte Zellen in getrennte künstliche Gewebe eingebracht und geformt und zu einem Gesamtgewebe kombiniert oder geformte Gewebe mit genetisch manipulierten Zellen und Gewebe mit unveränderten Zellen schichtweise oder durch Umhüllung oder zwei bzw. mehrere Gewebe mit jeweils verschiedenen genetischen Manipulationen schichtweise oder durch Umhüllung kombiniert werden.Surprisingly, shaping and genetic manipulation can be done use polarization based on an uneven distribution of genetically manipulated cells and unchanged cells. Another feature of the invention is that genetically manipulated and unchanged cells introduced and shaped into separate artificial tissues and combined into a total tissue or shaped tissue with genetic manipulated cells and tissue with unchanged cells in layers or by wrapping or two or more tissues, each with different genetic manipulations in layers or combined by wrapping will.
Gewebe, die genetisch manipulierte Zellen enthalten, lassen sich mit Geweben, die genetisch unveränderten Zellen enthalten, kombinieren. Tissues that contain genetically manipulated cells can be identified with tissues that contain genetically unmodified cells, combine.
Transplantation von künstlich hergestelltem Knorpelgewebe zur Therapie von Knorpelschäden bei Patienten mit rheumatoider Arthritis. Mesenchymale Zellen einer Gewebeprobe eines Patienten werden in vitro ausreichend in Monolayerkultur vermehrt und teilweise mit einem Gen für BMP (bone morphogenetic protein) transfiziert. Die Zellen werden in einer fibrinogenhaltigen Lösung suspendiert und dann in eine Trägerstruktur gebracht (z. B. resorbierbare Polymervliese aus Polylaktid und Polyglykolid). Die Suspension wird dann in der Struktur durch Thrombinzugabe verfestigt. Das Gewebe kann sofort oder nach einer in vitro Reifung in das Gelenk des Patienten transplantiert werden.Transplantation of artificially produced cartilage tissue for Treatment of cartilage damage in patients with rheumatoid arthritis. Mesenchymal cells of a patient's tissue sample are grown in vitro sufficiently propagated in monolayer culture and partly with a gene for BMP (bone morphogenetic protein) transfected. The cells are in one fibrinogen-containing solution suspended and then placed in a support structure (e.g. resorbable polymer nonwovens made of polylactide and polyglycolide). The suspension is then solidified in the structure by adding thrombin. The tissue can transplanted into the patient's joint immediately or after in vitro maturation will.
Vergleichbar zu Beispiel 1 wird ein Teil der Zellen mit einem gewünschten Gen manipuliert und diese gentherapeutisch veränderte Population unter Verwendung von Trägerstrukturen und Fibrin in eine bestimmte Form gebracht. Dieser Struktur werden dann genetisch unveränderte Zellen ebenfalls unter Verwendung formgebender Strukturen angelagert, so daß ein Diffusions- und damit Wirkungsgradient für gentechnisch eingebrachte Wirkstoffe auf die unbehandelten Zellen einwirken kann. Dies dient zur Polarisierung der in vitro hergestellten Gewebestrukturen vergleichbar der Polarisierung im nativen Gelenkknorpel.Comparable to Example 1, a part of the cells with a manipulated the desired gene and this gene therapy modified population using support structures and fibrin into a specific shape brought. This structure then also becomes genetically unchanged cells deposited using shaping structures, so that a diffusion and thus gradient of effectiveness for genetically introduced active substances on the untreated cells can act. This serves to polarize the in vitro fabric structures comparable to the polarization in the native Articular cartilage.
Claims (23)
Priority Applications (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19632404A DE19632404A1 (en) | 1996-08-02 | 1996-08-02 | Artificial tissue comprising three-dimensional extracellular matrix, cell interaction system |
| EP96941582A EP0801676A2 (en) | 1995-10-20 | 1996-10-18 | New artificial tissue, method for the production and the use thereof |
| PCT/DE1996/002025 WO1997015655A2 (en) | 1995-10-20 | 1996-10-18 | New artificial tissue, method for the production and the use thereof |
| US08/860,111 US5932459A (en) | 1995-10-20 | 1996-10-18 | Artificial tissues, methods for the production and the use thereof |
| JP51620297A JP3452366B2 (en) | 1995-10-20 | 1996-10-18 | New artificial tissue, its production method and its use |
| US09/268,894 US6143501A (en) | 1995-10-20 | 1999-03-16 | Artificial tissues, methods for the production and the use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19632404A DE19632404A1 (en) | 1996-08-02 | 1996-08-02 | Artificial tissue comprising three-dimensional extracellular matrix, cell interaction system |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| DE19632404A1 true DE19632404A1 (en) | 1998-04-02 |
Family
ID=7802380
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DE19632404A Ceased DE19632404A1 (en) | 1995-10-20 | 1996-08-02 | Artificial tissue comprising three-dimensional extracellular matrix, cell interaction system |
Country Status (1)
| Country | Link |
|---|---|
| DE (1) | DE19632404A1 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001013826A1 (en) * | 1999-08-20 | 2001-03-01 | Metz Stavenhagen Peter | Vertebral column segment |
| DE19944682A1 (en) * | 1999-08-20 | 2001-03-22 | Metz Stavenhagen Peter | Spine segment |
| DE19949290A1 (en) * | 1999-10-12 | 2001-04-26 | Albrecht Bettermann | Particulate construct for use in transplantation medicine |
| DE102011002536A1 (en) * | 2011-01-11 | 2012-07-12 | Aesculap Ag | Packaging containing a medical product for the treatment of human or animal cartilage damage |
-
1996
- 1996-08-02 DE DE19632404A patent/DE19632404A1/en not_active Ceased
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001013826A1 (en) * | 1999-08-20 | 2001-03-01 | Metz Stavenhagen Peter | Vertebral column segment |
| DE19944682A1 (en) * | 1999-08-20 | 2001-03-22 | Metz Stavenhagen Peter | Spine segment |
| DE19944682C2 (en) * | 1999-08-20 | 2001-10-11 | Metz Stavenhagen Peter | Spine segment |
| US6719798B2 (en) | 1999-08-20 | 2004-04-13 | Peter Metz-Stavenhagen | Vertebral column segment |
| DE19949290A1 (en) * | 1999-10-12 | 2001-04-26 | Albrecht Bettermann | Particulate construct for use in transplantation medicine |
| DE102011002536A1 (en) * | 2011-01-11 | 2012-07-12 | Aesculap Ag | Packaging containing a medical product for the treatment of human or animal cartilage damage |
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Owner name: TRANSTISSUE TECHNOLOGIES GMBH, 10117 BERLIN, DE |
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| 8181 | Inventor (new situation) |
Free format text: SITTINGER, MICHAEL, DR.RER.NAT., 12349 BERLIN, DE SCHULTZ, OLAF, DR., 10119 BERLIN, DE HAEUPL, THOMAS, DR., 15537 ERKNER, DE BURMESTER, GERD R., PROF. DR.MED., 12277 BERLIN, DE |
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