Introduction: Recently, mesenchymal stem cells (MSCs) have been known to exhibit substrate stiffness-dependent differentiation, and history of the mechanical dose from culture environment to MSCs sensitively alters its phenotype. A certain level of substrate stiffness and duration period determines the fate of MSCs. In relation to this, we have found before that microelastically- patterned hydrogel with hererogenic distribution of matrix stiffness allow MSCs to suppress fate determination into specific differentiation lineages, and contribute to keep the undifferentiated state. We call such mode of MSCs as “frustrated differentiation”, which serves to construct culture substrate for MSCs to maintain their stemness in high-qualified state. The basis of this phenomenon is in the enforced oscillation of mechanical does from environment to MSCs which eliminate the history of experience on a certain level of stiffness. To make sure the oscillation of the mechanical does and mechanical signal input to MSCs, traction force microscopic analysis has been performed for MSCs culture on the microelastically-striped patterned gelatinous gels.
Methods: Photocurable styrenated gelatin (StG) was used for photolithographic microelasticity patterning of the gel[1],[2]. StG sol solution was spread between vinylated glass and a normal glass substrate, then irradiated with a custom-built, reduction-projection-type photolithographic system. The elastically- alternating 100 μm-wide striped patterns were prepared, and on the top surface of which fluorescence bead were embedded. The surface elasticity of the gels was determined by AFM nanoindentation analysis. The migratory behavior of MSCs and embedded fluorescence beads on the gels were monitored using an automated all-in-one microscope with a temperature- and humidity-controlled cell chamber. Traction force dynamics were analyzed by self-made image processing programs.
Result and Discussion: Bead-embedded elastically striped patterns with 150kPa hard and 10kPa soft regions were successfully prepared.
Fig.1 a,b) Bead-embedded elastically-stripe patterned gel images. c) Distribution of Young's modulus of stripe patterned gel.
MSCs in normadic movement between hard and soft region were confirmed to exhibit characteristic oscillation of local traction forces.
Fig.2 Traction force images on 15/80 kPa stripe patterned gel. MSC on a) hard band and b) soft band.
Figure 2 shows the traction stress mapping. Positive and negative stress values are derived from tensile and compressive 2D local strains in each positions. Tensile stresses was found as mainly distributed outside area of the cell, and compressive stresses were seen inside area of the cell. Inside-cell’s traction stresses tended to be higher on the hard band, while lower on soft band. During this nomadic movement, traction stress distribution on the gel inside and outside cell were confirmed to show dynamic fluctuation.
Besides, RT-PCR analysis showed that osteogenic markers are clearly suppressed on the elastically-striped patterned gels compared with the gels with homogeneous stiffness. Usefulness of mechanobio-materials that modulate and oscillate the mechanical does to stem cells is discussed.
References:
[1] Kidoaki S and Matsuda T., J. Biotechnol. 133:225-230, 2008.
[2] Kawano T. and Kidoaki S., Biomaterials, 32: 2725-2733, 2011.