The temporal and spatial dynamics of microscale collagen scaffold remodeling by smooth muscle cells

Abstract

Smooth muscle cells (SMCs) and collagen scaffolds are widely used in vascular tissue engineering but their interactions in remodeling at the microscale level remained unclear. We characterized microscale morphologic alterations of collagen remodeled by SMCs in six dimensions: three spatial, time, multi-channel and multi-position dimensions. In live imaging assays, computer-assisted cell tracking showed locomotion characteristics of SMCs; reflection and fluorescent confocal microscopy and spatial reconstruction images of each time point showed detailed morphologic changes of collagen fibers and spatial collagen-SMC interactions. The density of the collagen around the SMCs was changed dynamically by the leading edges of the cells. The density of the collagen following 24 h of cell-induced remodeling increased 51.61 ± 9.73% compared to unremodeled collagen containing cells for 1 h (P < 0.0001, n = 40) (NS vs. collagen without cells). Fast Fourier transform analysis showed that the collagen fibers' orientation changed from random (alignment index = 0.047 ± 0.029, n = 40) after 1 h into concordant with that of the SMCs (alignment index = 0.379 ± 0.098, P < 0.0001, n = 40) after 24 h. Mosaic imaging extended the visual field from a single cell to a group of cells in one image without loss of optical resolution. Direct visualization of alignment of actin fibers and collagen fibers showed the molecular machinery of the process of scaffold remodeling. This is a new approach to better understanding the mechanism of scaffold remodeling and our techniques represent effective tools to investigate the interactions between cells and scaffold in detail at the microscale level.

Analyzing Structure and Function of Vascularization in Engineered Bone Tissue by Video-Rate Intravital Microscopy and 3D Image Processing

“…Three-dimensional volume and orthogonal image reconstruction was performed as documented previously. 19 To visualize both the morphology of the vessel and the blood cells, as well as evaluate the location of the blood cells, vessels were presented in the orthogonal view and cells in the volume view. The reconstructed images were overlaid in 3D.…”

Mechanobiology of tumor invasion: Engineering meets oncology

“…While this method represents the most rigorous numerical description of 3D traction forces to date, it does not account for the fibrillar structure and bioactivity of native ECM, which is known to exhibit complex mechanics and control cell behavior [156]. Thus, our lab and a number of others have measured extracellular matrix deformation and reorganization in natural biopolymer matrices to describe cell contractility in three-dimensional environments [134,157–159]. Such matrices allow in vivo -like interactions between cultured cells and the environment and enable ECM deposition, remodeling, path-making, and path-finding, all of which occur frequently in vivo [156,160].…”

“…Early work with confocal reflectance [145,161,162] and differential interference contrast (DIC) [163] microscopy established the utility of ECM fiber visualization for probing how cells physically interact with their environment, and recently, mechanobiology models have provided better understanding of the regulation and outcome of single-cell traction forces. For example, several groups have studied how cytoskeletal dynamics in cell pseudopodia bring about traction forces and induce ECM fiber alignment [158,159]. Local matrix deformations have been used to describe the temporal and spatial evolution of ECM remodeling by cells embedded within 3D hydrogel scaffolds [139,164].…”

Using a Type 1 Collagen-Based System to Understand Cell-Scaffold Interactions and to Deliver Chimeric Collagen-Binding Growth Factors for Vascular Tissue Engineering

“…We have developed a new microscopic approach to better understand their temporal and spatial interaction dynamics in 6 dimensions: 3 spatial dimensions ( x , y , and z ), a time dimension, a spectral dimension (multichannel of fluorophores), and a multiposition dimension (mosaic imaging). 9 First live time-lapse phase imaging enables a long-term dynamic study of collagen remodeling, and the locomotion characteristics of the cells were studied with computer-assisted cell tracking. Fluorescent and reflection confocal microscopic techniques were used to acquire images and for quantification of microscale changes of both SMCs and collagen fibers during remodeling.…”