Chondrogenic Differentiation of Human Bone Marrow Stem Cells in Transwell Cultures: Generation of Scaffold-free Cartilage

¹ UK Centre for Tissue Engineering and Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Faculty of Life Sciences, Michael Smith Building, Oxford Road, Manchester, M13 9PT, U.K.

^* To whom correspondence should be addressed. E-mail: timothy.e.hardingham{at}manchester.ac.uk.

	Abstract

Human bone marrow stem cells (hMSCs) have been shown to differentiate in vitro into a number of cell lineages and are a potential autologous cell source for the repair and replacement of damaged and diseased musculoskeletal tissues. hMSC differentiation into chondrocytes has been described in high-density cell pellets cultured with specific growth and differentiation factors. We now describe how culture of hMSC as a shallow multi-cellular layer on a permeable membrane over 2-4 weeks resulted in a much more efficient formation of cartilaginous tissue than in established chondrogenic assays. In this format, the hMSCs differentiated in 14 days to produce translucent, flexible discs, 6mm diameter by 0.8-1mm thick from 0.5 x 10⁶ cells. The discs contained an extensive cartilage-like extracellular matrix (ECM), with more than 50% greater proteoglycan content per cell than control hMSC differentiated in standard cell pellet cultures. The disc constructs were also enriched in the cartilage-specific collagen II and this was more homogeneously distributed than in cell pellet cultures. The expression of cartilage matrix genes for collagen type II and aggrecan was enhanced in disc cultures, but improved matrix production was not accompanied by increased expression of the transcription factors, SOX9, L-SOX5 andSOX6. The fast continuous growth of cartilage ECM in these cultures up to 4 weeks appeared to result from the geometry of the construct and the efficient delivery of nutrients to the cells. Scaffold free growth of cartilage in this format will provide a valuable experimental system for both experimental and potential clinical studies.

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