Characterization of Engineered Tissue Development Under Biaxial Stretch Using Nonlinear Optical Microscopy

Abstract

Little is known about the precise mechanical stimuli that cells sense and respond to as they maintain or refashion the extracellular matrix in multiaxially loaded native or bioengineered tissues. Such information would benefit many areas of research involving soft tissues, including tissue morphogenesis, wound healing, and tissue engineering. A custom tissue culture device has been constructed that can impart well defined biaxial stretches on cruciform-shaped, fibroblast-seeded collagen gels and be mounted on the stage of a nonlinear optical microscopy (NLOM) system for microscopic characterization of matrix organization. The cruciform geometry permitted direct comparison of matrix (re-) organization within regions of the collagen gel exposed to either uniaxial or biaxial boundary conditions and examined by NLOM for up to six days. In addition, sequential NLOM measurements of collagen fiber orientations within gels while stretched, unloaded, or decellularized delineated contributions of applied stretches, cell-mediated tractions and matrix remodeling on the measured distributions. The integration of intravital NLOM with novel bioreactors enables visualization of dynamic tissue properties in culture.

Taking a deep look: modern microscopy technologies to optimize the design and functionality of biocompatible scaffolds for tissue engineering in regenerative medicine

“…Mechanical loading induced collagen alignment, which was improved by applying intermittent strain. Comparable studies have been carried out by Hu et al [156], who characterized tissue development under biaxial stretch using fibroblast-seeded or cell-free collagen gels. The collagen I fibre orientations (SHG) revealed contributions of applied stretches, of cell-mediated tractions and matrix remodelling on the measured fibre distributions.…”

Enabling tools for engineering collagenous tissues integrating bioreactors, intravital imaging, and biomechanical modeling

“…Distributions of collagen fiber orientations were calculated from SHG images using Fourier analysis and custom software (Continuity, courtesy of A. D. McCulloch, University of California at San Diego, La Jolla, CA). Quantification included an Alignment Index (AI), which is defined as the fraction of collagen fibers oriented within 20°of a predominant direction and ranges from random (1.0) to strong (4.6) alignment (22). At 2 weeks of culture, collagen was adjacent to seeded cells and PGA fibers, although the sparseness of collagen and noncompacted nature of the PGA scaffold precluded accurate computation of an average fiber alignment (Fig.…”

A Bioreactor to Identify the Driving Mechanical Stimuli of Tissue Growth and Remodeling

“…Initially, bioreactor systems were developed to study the effects of mechanical stimuli under static conditions. 5,[8][9][10] As cardiovascular tissues are mainly exposed to dynamic loading conditions, other bioreactors applied dynamic deformation to the tissue engineered (TE) constructs. [11][12][13][14][15][16] For example, Rubbens et al and Gould et al applied cyclic strain to study the remodeling of 3D TE constructs.…”