Effects Of Micropatterned Curvature On The Physiological And Mechanical Properties Of Airway Smooth Muscle Cells

Cells in vivo live in a dynamic,complex microenvironment which contains a variety of physical factors such as geometry, spatial dimensions and mechanical stimuli. These factors play an important role in regulating cell behavior and function. Large numbers of studies indicate that many topology structures such as lines, ridge, column, and pit can influence cell adhesion and migration. Moreover, recent researches confirm that many of the physiological behavior of cells, such as cell proliferation, differentiation and apoptosis are more or less affected by these physical factors of microenvironment.Controlling physical factors of cellular surrounding micro- environment to research and analyze the interaction between cell function and physical factors of its surrounding microenviroment becomes an important proposition.In fact, many types of cells reside in curved space of tubular organs, such as the blood vessels and bronchial airways. Take the latter as an example, along the bronchial airway tree, the caliber of the airway changes with the periodic contraction and expansion of airway. Dynamical curvature change of the airway walls is likely to affect airway smooth muscle cell physiology and physical behavior. However, most studies on asthma and airway disease ignored the role of curvature of the airway may play, so it is very necessary and important to explore the effect of curvature on cell behaviours. To fill this gap, we explore the effect of curvature on airway smooth muscle cell behaviours and mechanic properties by using micro-contact printing technology to build an array of toroidal strips to simulate the airway curvature in vitro.According to anatomy data of the airway, we designed an array of toroidal strips with curvature varying from 0 to 1/50μm-1, this scope of curvature fully coveris the airway wall curvature range. In this array, the width of rings is 20μm, which only allows a single airway smooth muscle cell to grow and adhere. This ensures the airway smooth muscle cell arrange in the bending curvature perfectly, compeletely feel the existence of curvature and respond to it. In addition, we have also designed the micropattern of a square/circular island adjacent to the strips as the control. Then, we cultured airway smooth muscle cells (ASMCs) on these strips and investigated the cells'motility and mechanical properties using time-lapse imaging microscopy and optical magnetic twisting cytometry (OMTC).We found:1) The remodeling of cytoskeleton and focal adhesion of airway smooth muscle cells are affected by the curvature and change accordingly. When airway smooth muscle cells adhered to the strips coated with collagen, the shape of the cell match the curvature strip perfectly, meanwhile, the fluorescence intensity and the distribution of both F-actin and vinculin change accordingly. For example, the expression of F-actin is most on line strip, and it decreases while curvature increases; the expression of vinculin is most on strip with intermediate curvature, and it increases first and then deceases while the curvature increases.2) The curvature can influence airway smooth muscle cell migration speed.When the curvature increased from 0 to 1/150μm-1, the velocity of cell migration first decreased (0~1/750μm-1), and then increased (1/750~1/150μm-1). And at the intermediate curvature (1/750μm-1) the ASMCs were the least motile.3) The strategy of airway smooth muscle cell migration is affected by the curvature of substrate. In most cases, the cells moved subdiffusively with slightly different power-law exponents, but the motion of the cells on the strips with either low or high curvature became superdiffusive at large time scales.4) Although the slope of the power-law behaviour was almost independent of the curvature, the magnitude of cell stiffness was significantly influenced by the curvature. As the curvature increased from 0 to 1/150μm-1, the cell stiffness changed in a biphasic fashion, i.e., increased from the low to intermediate curvature but decreased from the intermediate to high curvature. Interestingly, the stiffness of the cells grown on the square/circular micropattern appeared to be very close to that on the straight strip.5) The contractility of the ASMCs decreased consistently as the curvature increased. In addition, the contractility of the cells grown on the square/circular micropattern (the controls) was similar to that with intermediate curvature.And indeed, the change of cell contractility with changing curvature was correlated with that of the expression of F-actin within the cells. From geometry point of view, the actin filaments aligned in parallel to the principal direction of the elongated ASMCs are the primary structures contributing to the generation of contraction. This implies that cells on the straight strip would have maximal portion of actin filaments aligned in the principal direction of the cell, thus exhibit the greatest contractility as compared to those on the strips with increasing curvature.In conclusion, the cytoskeleton, motility and mechanical properties of the ASMCs were all influenced by the curvature. The level of F-actin, and vinculin expression within the ASMCs appeared to correlate with the contractility and motility, respectively, in relation to the curvature. These results may provide valuable insights to understanding the heterogeneity of airway constrictions in asthma as well as the developing and functioning of other tubular organs and tissue engineering.