Osteoinduction of human adipose stem cells in 3D hydrogel culture using mechanical stimulation

Low-magnitude high frequency vibration has an ability to stimulate bone formation in vivo and in vitro 2D cultured stem cells. Highly absorbent hydrogels made from natural or synthetic polymers have been shown to mimic the structure of extracellular matrix offering cells a niche to undertake their physiological functions. The use of the 3D extracellular matrix analogous biomaterials is an attractive strategy for studying the effects of the mechanical stimulation to the osteogenic differentiation of stem cells. External forces caused by vibration are thought to conduct via hydrogel to the cells and activate osteogenic signaling pathways inside the cell. In this study, the capability of Collagen I hydrogel purified from rat tail to host human adipose stem cells (hASC) cultured in three dimension and induce their osteogenic differentiation in osteogenic conditions under the vibration loading were demonstrated. Both, the role of the culture medium and vibration loading on hASC osteogenic differentiation were evaluated. Cells were cultured in three distinct conditions: in static cultures with the basic medium, static cultures with the osteogenic medium and under the vibration loading in osteogenic medium. hASC used were derived from three different donors. A vertical vibration for cell plates was achieved with high magnitude high frequency vibration loading device, a peak acceleration of 2.5 g, using a sine wave at 100 Hz with an effective vibration period of 30 minutes with 150 min break. Device carried out five vibration periods in 24 hour. Effects of the vibration and biomaterial on hASC viability were evaluated by live/dead staining after 6 7 and 12 13 days of culture. Also the alkaline phosphatase activity (ALP) and mineralization was measured to assess the osteogenic differentiation of the cells. Furthermore adipoand osteogenic differentiation of the cells was estimated with real time PCR. Assays showed sustained cell viability and proliferation throughout the hydrogels over 12 days and moreover. Mineralization occurred by the cells under osteogenic conditions in vibrated cultures. However, there was a difference in mineralization between separate cell lines in the static control cultures. Exposure to the vibration seemed to speed up the proliferation of hASCs. However, mechanically stimulated cells did not show increased expression of key bone forming genes Runx2, Collagen I and ALP as quantified by RT-PCR. Instead, expression of adipogenic marker gene aP2 and osteogenic marker gene Dlx5 were increased in vibrated cells compared to static cultures. These findings imply that vibration loading may enhance osteoblast differentiation in hASCs seeded in collagen I hydrogel, since Dlx5 is a strong marker of osteogenesis. However, simultaneous induction of adipogenic gene expression may indicate that the vibration stimulation is not very specific stimulant of differentiation or that the 3D setting hinders the stimulus. Variable cellular responses elicited could be partly caused by donor variation and partly by collagen I hydrogel shrinkage due to the increased proliferation and pulling forces created by the growing cell population.