Modulation of Stem Cell Adipogenic Differentiation in Response to Mechano-Topographical Factors

Both biochemical and mechanical cues have been shown to affect stem cell differentiation. Substrate topography and stiffness have been shown to play a critical role in directing differentiation and lineage commitment. By combining two or more of these cues, synergistic stem cell differentiation is also plausible. For adipogenesis, effects of the combination of mechanical and topographical cues remain elusive. This study was designed to evaluate adipogenic differentiation of human mesenchymal stem cells (hMSCs) in response to microfabricated topographical cues with varied stiffness. hMSCs were cultured on patterned silicone substrates containing 50x50 micron posts and a 200 micron pitch that were fabricated using standard soft lithography methods. The pattern was designed to have parameters in the micron and submicron range. Mechanical stiffness of topographical features was altered by varying the cross linker to the silicone polymer base ratio. Young’s modulus of the substrates was determined using AFM microindentation. Assessment of adipogenic differentiation was performed at days 4, 7, and 14 of differentiation by Oil Red O staining and fluorescent microscopy. Quantitative analysis of differentiation was determined by measuring optical density at 510 nm of Oil Red O stained samples and image analysis for calculating the relative percent lipid area staining. Image analysis revealed more lipid formation on patterned substrates at early time points (Days 4 and 7) compared to plain substrates. Based on calculation of the percent lipid area formation, varied stiffness of the patterned substrates also altered differentiation. This seems to be consistent with qualitative images of Oil Red O staining. Optical density measurements for the most part support the image analysis of the relative percent area lipid formation. Images of cells show proliferation and differentiation in aggregates that were formed mostly on top of patterned areas. Increased cell-cell contact as a result of aggregate formation on the patterned locations may have played a role in the improved adipogenic differentiation. The results indicate that topographical cues could be used to modulate stem cell adipogenic differentiation. Both mechanical and topographical cues may be introduced to manipulate stem cell responses and therefore assist in strategic design of in vitro models for tissue engineering applications.