title: Membrane-based cultures generate scaffold-free neocartilage in vitro: influence of growth factors.
abstract: Scaffold-free cultures provide promising potential in chondrogenic differentiation of human mesenchymal stem cells (hMSCs). In this study, a novel scaffold-free membrane-based culture system, in which hMSCs were cultivated on a cellulose acetate membrane filter at medium-gas interface, was evaluated for chondrogenesis under the addition of growth factors. Chondrogenic differentiation of hMSCs has been described in scaffold-free pellet cultures with good results. In our study membrane-based cultures (1 x 10(6) hMSCs) were produced, maintained at the medium-gas interface and cultured for 21 days. Results were compared with findings from standard pellet cultures (2.5 x 10(5) hMSCs). The effects of the following growth factors were examined: human transforming growth factor-beta(3) (TGF-beta(3)) +/- insulin-like growth factor-1 or +/- human fibroblast growth factor 2. After 3 weeks of culture, chondrogenesis was assessed by Safranin-O staining, immunohistochemistry, a dimethylmethylene blue dye binding assay for glycosaminoglycans, and quantitative real-time polymerase chain reaction for cartilage-specific proteins. Membrane-based cultures containing growth factors formed hemispherical structures with a large surface area (65 mm(2)). When removed from the membrane they showed a histologically smooth cartilage-like surface. Membrane-based cultures stained positive for Safranin-O and collagen type II and contained a high content of glycosaminoglycans. Expression of cartilage-specific markers like collagen type II, aggrecan, and SOX9 was observed under the addition of TGF-beta(3), whereas combinations of growth factors let to a significant increase of collagen type II expression. A markedly reduced expression of collagen type X was found in membrane-based cultures when only TGF-beta(3) was added. Pellet cultures showed similar results besides an increased expression of collagen type X and type II that were observed. Membrane-based cultures provide a differentiation system, comparable in chondrogenesis to pellet cultures, which is able to generate scaffold-free neocartilage. The key benefit factors of membrane-based cultures are a histologically smooth cartilage-like surface and reduced expression of collagen type X, both of which are suitable features for its future application in cartilage regeneration.
authors.affiliation: Department of Orthopaedic Surgery, Klinikum Grosshadern, Ludwig-Maximilians-University, Munich, Germany. susanne.mayer@med.uni-muenchen.de
authors.forename: Susanne
authors.initials: S
authors.lastname: Mayer-Wagner
authors.forename: Tobias S
authors.initials: TS
authors.lastname: Schiergens
authors.forename: Birte
authors.initials: B
authors.lastname: Sievers
authors.forename: Denitsa
authors.initials: D
authors.lastname: Docheva
authors.forename: Matthias
authors.initials: M
authors.lastname: Schieker
authors.forename: Oliver B
authors.initials: OB
authors.lastname: Betz
authors.forename: Volkmar
authors.initials: V
authors.lastname: Jansson
authors.forename: Peter E
authors.initials: PE
authors.lastname: Müller
mesh_terms: D000328:Adult; D002356:Cartilage / Q000187:drug effects / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods*; D024042:Collagen Type I / Q000378:metabolism; D024043:Collagen Type II / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006025:Glycosaminoglycans / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology*; D008567:Membranes, Artificial; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D023822:Tissue Engineering / Q000379:methods*; D054457:Tissue Scaffolds / Q000737:chemistry*; D016212:Transforming Growth Factor beta / Q000494:pharmacology
chemical_list: D024042:Collagen Type I; D024043:Collagen Type II; D006025:Glycosaminoglycans; D036341:Intercellular Signaling Peptides and Proteins; D008567:Membranes, Artificial; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D004247:DNA