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 × 10⁶ 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 × 10⁵ hMSCs). The effects of the following growth factors were examined: human transforming growth factor-β₃ (TGF-β₃) ± 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 mm²). 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-β₃, 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-β₃ 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.