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Molekulare Charakterisierung von gezüchteten humanen dreidimensionalen Chondrozytentransplantaten

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Zusammenfassung

Die Transplantation von Chondrozyten in resorbierbaren Polymervliesen ist eine vielversprechende Methode zur Behandlung von Knorpeldefekten. Die Züchtung dieser künstlichen Knorpelgewebe ist durch eine Dedifferenzierung im Monolayer, dem Einsatz von abbaubaren Matrizen und Polymeren und der erneuten Expression chondrozytärer Markergene in dreidimensionaler (3D-)Kultur gekennzeichnet.

Das Ziel der vorliegenden Studie war die molekulare Charakterisierung von phänotypischen Veränderungen während des autologen Knorpel-Tissue-engineering. Humane artikuläre Chondrozyten wurden isoliert, mit humanem Serum bis zur Passage 3 expandiert und in humanem Fibrinogen und Polyglactin/Polydioxanon-Vliese eingebettet, um bis zu 4 Wochen 3D kultiviert zu werden. Die Dedifferenzierung im Monolayer und die Ausbildung von Knorpelgewebe in vitro und nach subkutaner Transplantation in Nacktmäuse wurde mittels Expressionsanalyse chondrozytärer Gene und anhand histologischer und immunhistochemischer Verfahren untersucht. Die Vermehrung der Chondrozyten mit humanem Serum führte zur Induktion von Typ-I- und Typ-III-Kollagen, während die knorpelspezifischen Gene Typ-II-Kollagen, "Cartilage-Oligomeric-Matrix-Protein", "Cartilage-Link-Protein" und Aggrecan reprimiert wurden, um erneut in 3D-Kultur in Polyglactin/Polydioxanon-Vliesen induziert zu werden. Subkutane Transplantationen im Mausmodell dokumentierten die Synthese von Proteoglykan und knorpelspezifischem Typ-II-Kollagen. Das 3D-Anordnen von humanen Gelenkchondrozyten in resorbierbaren Polyglactin/Polydioxanon-Vliesen unterstützt die chondrogene Differenzierung und die Bildung einer hyalinähnlichen Knorpelmatrix in vitro und in vivo.

Abstract

Three-dimensional arrangement and subsequent transplantation of chondrocytic cells in resorbable polymers has been shown to be a promising technique for the treatment of cartilaginous defects. Engineering of artificial cartilage tissue includes dedifferentiation of chondrocytes in monolayer culture, the use of biodegradable matrices and polymer scaffolds, and re-expression of chondrocytic marker genes in three-dimensional culture.

The aim of this study was to characterize molecularly the phenotypic changes occurring with autologous cartilage tissue engineering. Human articular chondrocytes were isolated, cultured in medium containing human serum, and expanded up to passage 3. Chondrocytes were embedded in human fibrinogen and in polyglactin-polydioxanon fleeces and cultured three-dimensionally up to 4 weeks. Dedifferentiation of chondrocytes in monolayers and formation of cartilage tissue in vitro or after subcutaneous transplantation into nude mice was assessed by gene expression analysis of typical chondrocytic genes, histology, and immunohistochemistry. The expansion of chondrocytes with human serum resulted in the induction of type I and type III collagens, whereas cartilage-specific type II collagen, cartilage oligomeric matrix protein, cartilage link protein, and aggrecan were repressed and induced again after three-dimensional arrangement of chondrocytes in polyglactin-polydioxanon. Transplantation experiments documented the synthesis of proteoglycan and cartilage-specific type II collagen in vivo. Three-dimensional arrangement of human articular chondrocytes in resorbable polyglactin-polydioxanon fleeces supports chondrogenic differentiation and the formation of a hyaline-like cartilaginous matrix in vitro and in vivo.

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Danksagung

Diese Studie wurde unterstützt von der Deutschen Forschungsgemeinschaft (DFG BU 445/5–4) und vom Senat von Berlin (B002).

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Authors and Affiliations

  1. Labor für Tissue Engineering, Experimentelle Rheumatologie und klinische Immunologie, Medizinische Fakultät, Humboldt-Universität, Charité, Berlin

    C. Kaps, S. Fuchs, M. Endres & M. Sittinger

  2. TransTissue Technologies GmbH, Berlin

    C. Kaps & S. Vetterlein

  3. AG Experimentelle Infektions- und Immunpathologie, Institut für Pathologie, Medizinische Fakultät, Humboldt-Universität, Charité, Berlin

    V. Krenn

  4. Orthopädische Klinik und Poliklinik, Medizinische Fakultät, Humboldt-Universität, Charité, Berlin

    C. Perka

  5. Labor für Tissue Engineering, Experimentelle Rheumatologie und klinische Immunologie, Medizinische Fakultät, Humboldt-Universität, Charité, Tucholskystraße 2, 10117, Berlin

    C. Kaps

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  1. C. Kaps
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  2. S. Fuchs
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Correspondence to C. Kaps.

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Kaps, C., Fuchs, S., Endres, M. et al. Molekulare Charakterisierung von gezüchteten humanen dreidimensionalen Chondrozytentransplantaten. Orthopäde 33, 76–85 (2004). https://doi.org/10.1007/s00132-003-0505-3

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