Abstract
The tissue microenvironment is a major contributor to cellular functions, such as cell adhesion, migration and invasion. A critical physical parameter for determining the effect of the microenvironment on cellular functions is the average pore-size of filamentous scaffolds, such as 3D collagen fiber matrices, which are assembled by the polymerization of biopolymers. The scaffolds of these matrices can be analyzed easily by using state-of-the-art laser scanning confocal imaging. However, the generation of a quantitative estimate of the pore-size in a 3D microenvironment is not trivial. In this study, we present a reliable and fast analytical method, which relies on a two-step 3D pore-size analysis utilizing several state-of-the-art image analysis methods, such as total variation (TV) denoising and adaptive local thresholds, and another crucial parameter, such as pore-coverage. We propose an iterative approach of pore-size analysis to determine even the smallest and obscure pores in a collagen scaffold. Additionally, we propose a novel parameter, the pseudo-pore-size, which describes a virtual scaffold porosity. In order to validate the advanced two-step pore-size analysis different types of artificial collagens, such as a rat and bovine mixture with two different collagen concentrations have been utilized. Additionally, we compare a traditional approach with our method using an artificially generated network with predefined pore-size distributions. Indeed, our analytical method provides a precise, fast and parameter-free, user-independent and automatic analysis of 3D pore topology, such as pore-sizes and pore-coverage. Additionally, we are able to determine non-physiological network topologies by taking the pore-coverage as a goodness-of-fit parameter.
Highlights
Filamentous networks play a role on various length scales that range from the interior cell level based cytoskeletal networks, over extracellular matrix based microenvironmental networks to organ-scale networks, such as vascular networks that are subject to blood coagulation
We recorded a height-map of the same sample using Atomic Force Microscope (AFM) and segmented it into fibril and non-fibril areas
The findings in this publication show that our novel approach of multi-step pore-size analysis is well suited to describe the pore-sizes and structure of collagen scaffolds and biopolymer networks in general
Summary
Filamentous networks play a role on various length scales that range from the interior cell level based cytoskeletal networks, over extracellular matrix based microenvironmental networks to organ-scale networks, such as vascular networks that are subject to blood coagulation. These networks are commonly formed by the polymerization of biomacromolecules, such as monomeric proteins, and assemble to three-dimensional (3D) structures. The physical properties, such as the pore-size, fiber diameter and fiber length determine the matrix mechanical properties and modulate cellular or tissue functions. A morphological closing can be used to remove rarely occurring, small, not connected objects and artifacts, which represent no obstacle for the migration of cells, such as cancer cells or fibroblasts, through connective tissues
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