Expansion of autologous chondrocytes in vitro is used to generate adequate populations for cell-based therapies. However, standard (SD) culture methods cause loss of chondrocyte phenotype and dedifferentiation to fibroblast-like cells. Here, we use a novel surface expansion culture system in an effort to inhibit chondrocyte dedifferentiation. A highly elastic silicone rubber culture surface was continuously stretched over a 13-day period to 600% of its initial surface area. This maintained cells at a high density while limiting contact inhibition and reducing the need for passaging. Gene expression analysis, biochemical assays, and immunofluorescence microscopy of follow-on pellet cultures were used to characterize the results of continuous expansion (CE) culture versus SD cultures on rigid polystyrene. CE culture yielded cells with a more chondrocyte-like morphology and higher RNA-level expression of the chondrogenic markers collagen type II, aggrecan, and cartilage oligomeric matrix protein. Furthermore, the expression of collagen type I RNA and a-smooth muscle actin protein were significantly reduced, indicating suppression of fibroblastic features. Pellet cultures from CE chondrocytes contained more sulphated glycosaminoglycan and collagen type II than pellets from SD culture. Additional control cultures on static (unexpanded) silicone (SS culture) indicated that benefits of CE culture were partially due to features of the culture surface itself and partially due to the reduced passaging which that surface enabled through CE. Chondrocytes grown in CE culture may, therefore, be a superior source for cellbased therapies.
Mechanical stimulation influences stem cell differentiation and may therefore provide improved lineage specification control for clinical applications. Low-frequency oscillatory mechanical stimulation (0.01 Hz) has recently been shown to suppress adipogenic differentiation of mesenchymal stem cells, indicating that the range of effective stimulation frequencies is not limited to those associated with locomotion, circulation, and respiration. We hypothesized that low-frequency mechanical stimulation (0.01 Hz) can also promote osteogenic cell differentiation of myoblastic C2C12 cells in combination with BMP-2. Results indicate that lowfrequency mechanical stimulation can significantly enhance osteogenic gene expression, provided that differentiation is initiated by a priming period involving BMP-2 alone. Subsequent application of low-frequency mechanical stimulation appears to act synergistically with continued BMP-2 exposure to promote osteogenic differentiation of C2C12 cells and can even partially compensate for the removal of BMP-2. These effects may be mediated by the ERK and Wnt signalling pathways. Osteogenic induction of C2C12 cells by low-frequency mechanical stimulation is therefore critically dependent upon previous exposure to growth factors, and the timing of superimposed BMP-2 and mechanical stimuli can sensitively influence osteogenesis. These insights may provide a technically simple means for control of stem cell differentiation in cell-based therapies, particularly for the enhancement of differentiation toward desired lineages.
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