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

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
• 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
• 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
• 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
• 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
• 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
• 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
• 8 Benya P D, Schaffer J D. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gel.  Cell. 1982;  30 215-224
• 9 Caplan A I. The mesengenic process.  Clin Plast Surg. 1994;  21 429-435
• 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
• 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
• 12 Johnstone B, Yoo J O. Autologous mesenchymal progenitor cells in articular cartilage repair.  Clin Orthop. 1999;  367 156-162
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 24 Caplan A I. Tissue engineering designs for the future: new logics, old molecules.  Tissue Engineering. 2000;  6 1-8
• 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
• 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