Dynamic quantitative visualization of single cell alignment and migration and matrix remodeling in 3-D collagen hydrogels under mechanical force

Abstract

We developed a live imaging system enabling dynamic visualization of single cell alignment induced by external mechanical force in a 3-D collagen matrix. The alignment dynamics and migration of smooth muscle cells (SMCs) were studied by time lapse differential interference contrast and /or phase contrast microscopy. Fluorescent and reflection confocal microcopy were used to study the SMC morphology and the microscale collagen matrix remodeling induced by SMCs. A custom developed program was used to quantify the cell migration and matrix remodeling. Our system enables cell concentration-independent alignment eliminating cell-to-cell interference and enables dynamic cell tracking, high magnification observation and rapid cell alignment accomplished in a few hours compared to days in traditional models. We observed that cells sense and response to the mechanical signal before cell spreading. Under mechanical stretch the migration directionality index of SMCs is 46.3% more than those cells without external stretch; the dynamic direction of cell protrusion is aligned to that of the mechanical force; SMCs showed directional matrix remodeling and the alignment index calculated from the matrix in front of cell protrusions is about 3 fold of that adjacent to cell bodies. Our results indicate that the mechanism of cell alignment is directional cell protrusion. Mechano-sensing, directionality in cell protrusion dynamics, cell migration and matrix remodeling are highly integrated. Our system provides a platform for studying the role of mechanical force on the cell matrix interactions and thus find strategies to optimize selected properties of engineered tissues.

Magnitude-dependent proliferation and contractility modulation of human bladder smooth muscle cells under physiological stretch

“…This finding confirms the observations in the literature showing that smooth muscle cells orient perpendicularly to the direction of maximal stress or stretch [14,15]. However, Pang et al [16] have demonstrated that cells in a 3D environment respond in parallel to the direction of the mechanical force. In addition to the stretch direction, the response of cell reorientation is found to depend on the stretching magnitude as shown in Fig.…”

“…However, most studies in the literature have been mainly focused on growth responses without consideration of contractility, whereas some studies just pay attention to the change of cell contractile phenotype or alteration of contraction marker such as SM-α-actin and myosin heavy chain [12,[15][16][17][18][19][20][21][22]. Although contraction phenotype and marker can be considered as a parameter for the contractility function of cells in a certain extent, the direct effects of stretch on cell contraction have barely been investigated.…”

Biomechanical regulation of vascular smooth muscle cell functions: from in vitro to in vivo understanding

“…Recent reports show that the mechanical properties of the extracellular matrix (ECM) alter VSMC migration and gene expression [43 -45], suggesting that the mechanical properties of the substrate matrix should be taken into account. As reported in a single cell three-dimensional culture under mechanical stimulation, single cell alignment and migration were dynamically and quantitatively visualized in three-dimensional collagen hydrogels under mechanical force [46].…”

Local alignment vectors reveal cancer cell-induced ECM fiber remodeling dynamics

“…Figure 1e appears as only one line because the alignment vector length (green) is exactly equal to the orientational order parameter (blue), meaning that the alignment vector is the accurate measurement of fiber alignment as the orientational order parameter did. Additionally, the alignment vector offers the direction of fiber alignment that other alignment measurements15202122 could not. Figure 1f shows the alignment vector angle for the simulated fibers in Fig.…”

“…Collagen is a major component of the ECM; methods for automated extraction of collagen fiber segments and quantifying fiber alignment have been developed171819. In addition, several measures, including the orientational order parameter15, alignment coefficient20, and alignment index2122 have been developed to quantify fiber alignment. These measures are based on the fiber angular distribution, where the fiber alignment is 0 if all fibers are randomly distributed, and 1 if all fibers are perfectly aligned.…”