Fast and reliable advanced two-step pore-size analysis of biomimetic 3D extracellular matrix scaffolds

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.

Inhomogeneities in 3D Collagen Matrices Impact Matrix Mechanics and Cancer Cell Migration

“…an advanced pore size analysis ( Figure 4B), published previously (Fischer et al, 2019). We found that all observed collagen compositions featured networks with significantly decreasing pore sizes by increasing collagen concentrations ( Supplementary Table S2) (p = 0.01, or less for R collagens, p = 0.05 or less for RB and B collagens).…”

“…The pore size is another crucial parameter to determine structural traits (Fischer et al, 2019). Concerning confined scaffolds with spatial hindrances the pore size is a key value for describing a fibrillary network.…”

“…Concerning confined scaffolds with spatial hindrances the pore size is a key value for describing a fibrillary network. Applying advanced 3D pore size analysis represents a solid method to characterize variations of different networks (Molteni et al, 2013;Münster and Fabry, 2013;Fischer et al, 2019;Sauer et al, 2019). The pore size specifies structural differences due to concentration variations within a certain collagen composition.…”

Migration of Cytotoxic T Lymphocytes in 3D Collagen Matrices

“…If the channels are too narrow for the nucleus to pass, the T cells would appear to halt at the position until the matrix breaks loose or until they manage to squeeze themselves out. According to the experimentally determined pore size distribution in collagen matrices with various concentration [40], one can estimate that the average pore diameter is around 7.5 µm for collagen density 2 mg/ml. The diameter distribution is broad enoughto have some pores with diameter around 9 µm, where T cells with diameter 10 µm can squeeze through without deforming the surrounding collagen fibers.…”

“…show a persistent motion for all collagen concentrations, slow T cells perform anti-persistent motion in 2 mg/ml collagen and become persistent in denser ones. A possible explanation is that the average pore size increases with decreasing collagen density [40] which implies that for low collagen densities, T cells can more easily find some pores around them that are large enough to get into them; this might allow slow T cells to change their direction when they face a constriction while creating a channel. This is, however, less probable in denser collagens, as most pores are smaller than T cells and need equal effort to pass.…”