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Laryngorhinootologie 2002; 81(7): 521-527
DOI: 10.1055/s-2002-33287
Plastische Chirurgie
© Georg Thieme Verlag Stuttgart · New York

Adulte mesenchymale Stammzellen - neue Möglichkeiten der Gewebezüchtung für die plastisch-rekonstruktive Chirurgie

Mesenchymal Stem Cells - A New Pathway for Tissue Engineering in Reconstructive SurgeryA.  Naumann 1 , J.  Dennis 2 , R.  Staudenmaier 1 , N.  Rotter 1 , J.  Aigner 3 , B.  Ziegelaar 1 , T.  Happ 1 , G.  Rasp 1 , A.  I.  Caplan 2
  • 1 Klinik und Poliklinik für Hals-, Nasen- und Ohrenkranke (Direktor: Prof. Dr. E. Kastenbauer), Ludwig-Maximilians-Universität München
  • 2 Skeletal Research Center (Direktor: Prof. A. I. Caplan), Case Western Reserve University, Cleveland-Ohio, USA
  • 3 Zentralinstitut für Medizintechnik (Direktor: Prof. Dr. E. Wintermantel), Technische Universität München
Herrn Prof. Dr. med. Ernst Kastenbauer zu seinem 65. Geburtstag gewidmet.
Further Information

Publication History

Eingegangen: 18. Februar 2002

Angenommen: 24. April 2002

Publication Date:
12 August 2002 (online)

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Zusammenfassung

Hintergrund: Mesenchymale Stammzellen besitzen die Fähigkeit, unter spezifischen Bedingungen in Knorpelzellen zu differenzieren. Adulte mesenchymale Stammzellen stellen ein beinahe unbegrenztes Zellreservoir für das Tissue Engineering autologer Knorpeltransplantate für Patienten mit Defekten im Kopf-Halsbereich dar. Methode: Humane adulte mesenchymale Stammzellen (hMSZ) wurden mit Hilfe eines Percollgradienten aus Knochenmarksproben isoliert. Die isolierten hMSZ wurden insgesamt 21 Tage in vitro unter konventionellen Zellkulturbedingungen amplifiziert und anschließend mit Hilfe von Transforming Growth Factor-beta 1 (TGF-beta 1) zu Knorpelgewebe in einem dreidimensionalen Aggregatkultursystem differenziert. Die Expression knorpelspezifischer Matrixbestandteile wurde histologisch (Toluidinblau, Safranin-O-Färbung) sowie immunhistochemisch (Kollagen-II- und X-Expression) nach 3 Wochen in dreidimensionaler In-vitro-Kultur untersucht. Ergebnisse: Die hMSZ (12,4 Mio ± 2,6 Zellen nach 1. Passage) konnten innerhalb von 21 Tagen in vitro auf bis zu 80,2 Mio ± 15,3 Zellen/Knochenmarksprobe mit einer über 90 %igen Zellvitalität amplifiziert werden. Die isolierten hMSZ haben unter Zugabe von TGF-beta 1 sowohl knorpelspezifisches Kollagen Typ II und X als auch Glykosaminoglykane in einem dreidimensionalen Zellkultursystem synthetisiert. Schlussfolgerung: Isolierte mesenchymale Stammzellen aus adultem Knochenmark differenzieren in einem dreidimensionalen In-vitro-Kultursystem zu Knorpelgewebe und stellen somit eine neue Möglichkeit zur Herstellung autologer Knorpeltransplantate für die plastisch-rekonstruktive Chirurgie der Zukunft dar.

Abstract

Introduction: Mesenchymal stem cells (MSC) have the capacity to differentiate into chondrocytes with the synthesis of cartilage. This report presents the use of human adult bone marrow derived mesenchymal stem cells for tissue engineering of autologous cartilage grafts. Methods: Human bone marrow aspirates were obtained from the iliac crest and fractionated on a Percoll gradient. The isolated hMSC were plated at 20 × 106 cells per 100 mm2 culture dish. After 21 days in culture at 37 °C with 5 % CO2, the adherent multiplied MSC were trypsinized, counted, and tested for viability by trypan blue assay. The hMSCs were loaded into a sterile 15 ml polypropylene tube (0.5 Mio cells/ml) and centrifuged on the bottom of the tube at 500 g for 5 minutes. The MSC were cultivated for 3 weeks in vitro in a specific chondrogenetic medium composed of Dulbecco's Modified Eagles Medium-High Glucose supplemented with 10 ng/ml transforming growth factor-beta 1, 1 % ITS-Premix medium, 80 µM ascorbic acid, and 100 nM dexamethasone. Results: Histological and immunohistochemical studies performed after 3 weeks in threedimensional culture demonstrated the expression of cartilage specific collagen type II and X as well as proteoglycans. Conclusion: Human adult mesenchymal stem cells derived from bone marrow aspirates have the ability to differentiate into chondrocytes under specific culture conditions by growth factors. The use of adult mesenchymal stem cells may be a promising tool for tissue engineering of autologous cartilage grafts in reconstructive surgery in the future.

Schlüsselwörter

Adulte mesenchymale Stammzellen - Transforming growth factor - Knorpeltransplantate

Key words

Adult mesenchymal stem cells - Collagen expression - Autologous cartilage grafts

Literatur

  • 1 Cao Y L, Vacanti J P, Paige K. Transplantation of chondrocytes utilizing a polymer-cell construct to product tissue engineered cartilage in the shape of a human ear.  Plast Reconstr Surg. 1997;  100 297-302
    CrossrefPubMedGoogle Scholar
  • 2 Rodriguez A, Cao Y L, Ibarra C, Pap S, Vacanti M, Eavey R D, Vacanti C A. Characteristics of cartilage engineered from human pediatric auricular cartilage.  Plast Reconstr Surg. 1999;  103 1111-1119
    CrossrefPubMedGoogle Scholar
  • 3 Hunziker E B, Schenk R K. A differential treatment protocol for inducing cartilage and bone repair in full-thickness articular cartilage defects.  Trans Orthop Res Soc. 1995;  20 170-174
    PubMedGoogle Scholar
  • 4 Naumann A J, Rotter N, Bujía J M, Aigner J. Tissue engineering of autologous cartilage transplants for rhinology.  Am J Rhin. 1998;  12 59-63
    PubMedGoogle Scholar
  • 5 Arévalo-Silva C A, Eavey R D, Cao Y L, Vacanti M, Weng Y, Vacanti C A. Internal support of tissue-engineered cartilage.  Arch Otolaryngol Head Neck Surg. 2000;  126 1448-1452
    PubMedGoogle Scholar
  • 6 Aigner J, Tegeler J, Hutzler P, Campoccia D, Pavesio A, Hammer C, Kastenbauer E, Naumann A. Cartilage tissue engineering with novel nonwoven structured biomaterial based on hyaluronic acid benzyl ester.  J Biomed Mater Res. 1998;  42 172-181
    PubMedGoogle Scholar
  • 7 Ten Koppel P G, van Osch G J, Verwoerd C D, Verwoerd-Verhoef H L. Efficiacy of perichondrium and a trabecular demineralized bone matrix for generating cartilage.  Plast Reconstr Surg. 1998;  102 2012-2020
    PubMedGoogle Scholar
  • 8 Benya P D, Schaffer J D. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gel.  Cell. 1982;  30 215-224
    CrossrefPubMedGoogle Scholar
  • 9 Caplan A I. The mesengenic process.  Clin Plast Surg. 1994;  21 429-435
    PubMedGoogle Scholar
  • 10 Pittenger M F, Mackay A M, Beck S C, Jaiswal R K, Douglas R, Mosca J D, Moorman M A, Simonetti D W, Craig S, Marshak D R. Multilineage potential of adult human mesenchymal stem cells.  Science. 1999;  284 143-147
    CrossrefPubMedGoogle Scholar
  • 11 Dennis J E, Haynesworth S E, Young R G, Caplan A I. Osteogenesis in marrow-derived mesenchymal cell porous ceramic composites transplanted subcutaneously: Effect of fibronectin and laminin on cell retention and rate of osteogenic expression.  Cell Transplant. 1992;  1 23-32
    PubMedGoogle Scholar
  • 12 Johnstone B, Yoo J O. Autologous mesenchymal progenitor cells in articular cartilage repair.  Clin Orthop. 1999;  367 156-162
    PubMedGoogle Scholar
  • 13 O'Keefe R J, Crabb I D, Puzas J E, Rosier R N. Effects of transforming growth factors-beta1 and fibroblast growth factor on DNA synthesis in growth plate chondrocytes are enhanced by insulin-like growth factor-1.  J Bone Miner Res. 1994;  9 1713-1722
    PubMedGoogle Scholar
  • 14 Crabb I D, O'Keefe R J, Puzas R J, Rosier R N. Differential effects of parathyroid hormone on chick growth plate and articular chondrocytes.  Calcif Tissue Int. 1992;  50 61-66
    CrossrefPubMedGoogle Scholar
  • 15 Vortkamp A, Lee K, Lanske B, Segre G V, Kronenberg H M, Tabin C J. Regulation of rate of cartilage differentiation by indian hedgehog and PTH-related protein.  Science. 1996;  273 613-622
    PubMedGoogle Scholar
  • 16 Grimsrud C R, Romano P R, D'Souza M, Puzas J E, Reynolds P R, Rosier R N, O'Keefe R J. BMP-6 is an autocrine stimulator of chondrocyte differentiation.  J Bone Miner Res. 1999;  14 475-482
    PubMedGoogle Scholar
  • 17 Leboy P S, Sullivan T A, Nooreyazdan M, Venezian R A. Rapid chondrocyte maturation in serum-free culture with BMP-2 and ascorbic acid.  J Cell Biochem. 1997;  66 394-403
    CrossrefPubMedGoogle Scholar
  • 18 Campbell I K, Piccoli D S, Roberts M J, Muirden K D, Hamilton J A. Effects of tumor necrosis factor alpha and beta on resorption of human articular cartilage and production of plasminogen activator by human articular chondrocytes.  Arthritis Rheum. 1990;  33 542-552
    CrossrefPubMedGoogle Scholar
  • 19 Moos V, Fickert S, Mueller B, Weber U, Sieper J. Immunohistological analysis of cytokine expression in human osteoarthritic and healthy cartilage.  J Rheumatol. 1999;  26 870-879
    PubMedGoogle Scholar
  • 20 Yoo J U, Barthel T S, Nishimura K, Solchaga L, Caplan A I, Goldberg V M, Johnstone B. The chondrogenic potential of human bone-marrow-derived mesenchymal progenitor cells.  J Bone Joint Surg. 1998;  80 1745-1757
    PubMedGoogle Scholar
  • 21 Cao Y, Rodriguez A, Vacanti M, Ibarra C, Arévalo C A, Vacanti C A. Comparative study of the use of poly(glycolic acid), calcium alginate and pluronic in the engineering of autologous porcine cartilage.  J Biomater Sci Polym Ed. 1998;  9 475-487
    PubMedGoogle Scholar
  • 22 Behrens P, Ehlers E M, Köchermann K U, Rohwedel J, Russlies M, Plötz W. Neues Therapieverfahren für lokalisierte Knorpeldefekte - ermutigende Resultate mit der autologen Chondrozytenimplantation.  MMW-Fortschr Med. 1999;  141 793-795
    PubMedGoogle Scholar
  • 23 Caplan A I, Elyaderani M, Mochizuki Y, Wakitani S, Goldberg V M. Principles of cartilage repair and regeneration.  Clin Orthop Rel Res. 1997;  342 254-269
    PubMedGoogle Scholar
  • 24 Caplan A I. Tissue engineering designs for the future: new logics, old molecules.  Tissue Engineering. 2000;  6 1-8
    CrossrefPubMedGoogle Scholar
  • 25 Solchaga L A, Dennis J E, Goldberg V M, Caplan A I. Hyaluronic acid-based polymers as cell carriers for tissue-engineered repair of bone and cartilage.  J Orthop Res. 1999;  17 205-213
    CrossrefPubMedGoogle Scholar
  • 26 Lennon D P, Haynesworth S E, Bruder S P, Jaiswal N, Caplan A I. Human and animal MPC's from bone marrow: identification of serum for optimal selection and proliferation.  In vitro Cell Dev Biol. 1996;  32 602-611
    PubMedGoogle Scholar

Dr. med. Andreas Naumann

Klinik und Poliklinik für Hals-, Nasen- und Ohrenkranke · Ludwig-Maximilians-Universität München

Marchioninistraße 15 · 81377 München ·

Email: andreas.naumann@hno.med.uni-muenchen.de

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