17 research outputs found
The Biophysical Properties of Basal Lamina Gels Depend on the Biochemical Composition of the Gel
The migration of cells within a three-dimensional extracellular matrix (ECM) depends sensitively on the biochemical and biophysical properties of the matrix. An example for a biological ECM is given by reconstituted basal lamina gels purified from the Engelbreth-HolmSwarm sarcoma of mice. Here, we compare four different commercial variants of this ECM, which have all been purified according to the same protocol. Nevertheless, in those gels, we detect strong differences in the migration behavior of leukocyte cells as well as in the Brownian motion of nanoparticles. We show that these differences correlate with the mechanical properties and the microarchitecture of the gels which in turn arise from small variations in their biochemical composition
Components of the Plasminogen Activation System Promote Engraftment of Porous Polyethylene Biomaterial via Common and Distinct Effects
Rapid fibrovascularization is a prerequisite for successful biomaterial engraftment. In addition to their well-known roles in fibrinolysis, urokinase-type plasminogen activator (uPA) and tissue plasminogen activator (tPA) or their inhibitor plasminogen activator inhibitor-1 (PAI-1) have recently been implicated as individual mediators in non-fibrinolytic processes, including cell adhesion, migration, and proliferation. Since these events are critical for fibrovascularization of biomaterial, we hypothesized that the components of the plasminogen activation system contribute to biomaterial engraftment. Employing in vivo and ex vivo microscopy techniques, vessel and collagen network formation within porous polyethylene (PPE) implants engrafted into dorsal skinfold chambers were found to be significantly impaired in uPA-, tPA-, or PAI-1-deficient mice. Consequently, the force required for mechanical disintegration of the implants out of the host tissue was significantly lower in the mutant mice than in wild-type controls. Conversely, surface coating with recombinant uPA, tPA, non-catalytic uPA, or PAI-1, but not with non-catalytic tPA, accelerated implant vascularization in wild-type mice. Thus, uPA, tPA, and PAI-1 contribute to the fibrovascularization of PPE implants through common and distinct effects. As clinical perspective, surface coating with recombinant uPA, tPA, or PAI-1 might provide a novel strategy for accelerating the vascularization of this biomaterial
Migration trajectories of dHL-60 cells tracked from hours 4–6 after the cells are embedded into the four different basal lamina matrices.
<p>The starting point of all trajectories is shifted to the origin for clarity, and the end point is marked by a dot. The average start-to-end distance (Euclidean distance) travelled by the cells and the respective standard deviation is denoted by <i>ED</i>. The fraction of cells with an <i>ED</i> greater than <i>ED</i> = 105.5 μm (red circle) is denoted by <i>f</i>. (e) Comparison of the migration velocity of dHL60 cells in different ECM gels. The red line denotes the median of the velocity distribution, the box includes 25% of the observed velocities above and below this median, respectively. The remaining 25% of slower as well as the 25% of faster cells are indicated by the dashed lines. Outliers are denoted by a red cross.</p
Gelation kinetics of the four different gels measured with a macrorheometer.
<p>The temperature is increased from 5°C to 37°C after one minute to induce gelation. The curves shown represent averages of three independent measurements. The inset shows the storage moduli <i>G’</i> of the four gels at 30 min. The error bars denote the error of the mean.</p
Content of selected ECM proteins in the four different ECM gel variants.
<p>(a) A coomassie staining of the four gel variants shows extra bands in ECM1 at low molecular weight. The star denotes the band which is further investigated by mass spectroscopy (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118090#pone.0118090.s010" target="_blank">S2 Table</a> for details). (b) The content of fibronectin, laminin, entactin and collagen type IV in the four different ECM gels is analyzed by western blot (for uncropped blots see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118090#pone.0118090.s007" target="_blank">S7 Fig</a>.). (c) Densiometric analysis of fibronectin, laminin, entactin and collagen IV signals. The error bars denote the standard deviations as obtained from four independent gel runs.</p
Micromorphology of the four gels determined by three different methods.
<p>(a) Exemplary trajectories of PEGylated particles with a diameter of 200 nm in the four different gels. Trajectories are shifted for clarity. (b) Micromorphology of the four different basal lamina gel variants as determined by confocal fluorescence microscopy. Representative staining of the matrix component collagen IV. The scale bar in the upper left image denotes 50 μm and applies to all images. (c) Micromorphology of the whole network of the four gel variants imaged by SEM. The scale bar corresponds to 25 μm and applies to all images.</p
Effect of surface coating of implants with DFP-uPA or NE-tPA on vascularization of PPE biomaterial.
<p>Vascularization of PPE implants, which were coated with DFP-uPA or NE-tPA embedded in Matrigel, was analyzed by <i>in vivo</i> fluorescence microscopy in WT mice. Panels show results for the relative increase in absolute (<b>A</b>) and functional (<b>B</b>) vessel density within the implant as compared to Matrigel-coated control implants (mean ± SEM for n = 5 – 6).</p
Role of endogenous uPA, tPA, and PAI-1 for leukocyte-endothelial cell interactions in PPE biomaterial.
<p>Interactions of fluorescence-labeled leukocytes and endothelial cells in newly formed microvessels within the PPE implants in WT as well as in uPA-, tPA-, or PAI-1-deficient mice were analyzed by <i>in vivo</i> fluorescence microscopy. Representative <i>in vivo</i> microscopy images of these processes in a WT mouse are shown (<b>A</b>; scale bar: 100 μm). Panels show results for the number of intravascularly rolling (<b>B</b>) and adherent (<b>C</b>) leukocytes (mean ± SEM for n = 6; * p < 0.05 vs. WT).</p
Effect of uPA, tPA, and PAI-1 on migration of endothelial cells.
<p>The effect of recombinant uPA, tPA, and PAI-1 on migration of endothelial cells was analyzed <i>in vitro</i>. Representative migration plots are shown (<b>A</b>). Panels show results for the forward migration index (FMI) of migrating endothelial cells upon exposure to recombinant uPA, tPA, or PAI-1 (<b>B</b>; mean ± SEM for n = 3; * p < 0.05 vs. +/-).</p