Analysis of Cell Detachment Mechanisms.

<p>(A) The occurrence of different detachment modes are indicated for cells treated with the indicated matrix ligands, cation concentrations, and shear. Cells were characterized as: 1) “Remodeled”, defined as nucleus present but visible cell deformation with paxillin puncta surrounding the cell, e.g. center and lower right images of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102424#pone-0102424-g003" target="_blank">Figure 3</a> (please note that no differentiation was made between deformed cells and aligned cells as the differences are gradual until full alignment is reached), 2) “Aligned”, defined as cells aligned in the direction of shear determined by a combination of cell aspect ratio and direction of major axis, 3) “Detached”, determined from the density difference between other conditions and unspun cells cells. For each condition the same surface area was scanned and analyzed, where the low shear region, e.g.<100 dynes/cm², contained at least 100 cells. Focal adhesion density, (B) based on number of discrete adhesions or (C) on the area of those adhesions versus cell area, was determined for intact cells or detached cells that left behind paxillin-containing puncta. For detached cells, the area was determined by the maximum extent of the puncta. For each condition, at least 80 intact cells from triplicate experiments were scanned and analyzed. *p<0.05.</p

α₅ Integrins Regulate HT1080 Fibrosarcoma Cell Remodeling Under Shear.

<p>(A) Coverslips were washed at the indicated times to eliminate unbound HT1080 fibrosarcoma cells, and the remaining cell density, normalized to untreated (WT) and unwashed controls at the indicated time points, is plotted for the indicated treatments. (B) Cell attachment strength to fibronectin was tested under the indicated conditions. Note that each representative curve represents thousands of cells grouped at set radial distances with data expressed as mean ± standard deviation. (C) Average attachment strength (T₅₀) for cells in the indicated conditions. (D) Fluorescent images of cells stained after application of the indicated shear for nuclei (DNA; blue) and actin (red). A white arrow indicates shear direction and open arrowheads indicate remodeled cells. Dashed lines indicate the regions of inset images, which are outlined in white. Note that all statistical analyses used non-parametric Kruskal-Wallis analysis of variance from triplicate experiments. ***p<0.001.</p

Fibroblast adhesion strength is ligand- and cation-dependent.

<p>(A) Illustration of the Spinning Disc device with cells attached to an extracellular matrix protein-coated coverslip mounted and rotating on a spinning rod in buffer. The radially-dependent shear profile is highlighted showing that cells at the center only rotate in place while those at the edge move around at a high linear velocity. (B) Plot of cell density versus coverslip position for cells that were exposed to shear (spun, gray circles) or were not (unspun, black squares). Inset images show heat maps of cell density for the indicated conditions. (C) Normalized 3T3 cell density was plotted vs. applied shear for cells with or without 0.5 mM Mg²⁺ and with or without 1 mM Ca²⁺ during the 5 min of application of shear as indicated. Note that each representative curve represents thousands of cells bound at set radial distances with data expressed as mean ± standard deviation. (D) Adhesion strength, T₅₀ (measured in dynes/cm²), shown for 3T3 cells on fibronectin- (blue) and type I collagen-coated substrates (green) in absence of calcium but in the presence of 0.01–1000 µM Mg²⁺. Data are fit by sigmoidal curves. (E) Adhesion strength, T₅₀ (measured in dynes/cm²), shown for 3T3 cells on fibronectin- (blue) and type I collagen-coated substrates (green) in the presence of 1–1000 µM Ca²⁺ without Mg²⁺ present. Data are fit by sigmoidal curves. (F) While keeping Mg²⁺ constant at 0.5 mM, adhesion strength was measured as a function of Ca²⁺ for both fibronectin- (blue) and type I collagen-coated substrates (green). Note that each data point in panels D-F represents triplicate experiments of thousands of cells from a coverslip exposed to a radial shear gradient. Data is expressed as mean ± standard deviation of T₅₀ for each shear test at the indicated cation condition.</p

Shear-induced Cell Remodeling.

<p>3T3 fibroblasts are shown under the indicated cation and shear conditions. The shear direction in each image is indicated by a white arrow. Images show paxillin in green, the actin cytoskeleton in red, and the nucleus (DNA) in blue. The approximate pre-shear cell area is indicated by white dashed lines as determined from the focal adhesions that remained on the substrate, which are indicated by open arrowheads. The bottom left image was contrast-enhanced 2-fold to better visualize the focal adhesions that remained on the substrate. Inset images are shown from regions outlined in white.</p

Cell Instructive Microporous Scaffolds through Interface Engineering

The design of novel biomaterials for regenerative medicine requires incorporation of well-defined physical and chemical properties that mimic the native extracellular matrix (ECM). Here, we report the synthesis and characterization of porous foams prepared by high internal phase emulsion (HIPE) templating using amphiphilic copolymers that act as surfactants during the HIPE process. We combine different copolymers exploiting oil–water interface confined phase separation to engineer the surface topology of foam pores with nanoscopic domains of cell inert and active chemistries mimicking native matrix. We further demonstrate how proteins and hMSCs adhere in a domain specific manner

Cell Instructive Microporous Scaffolds through Interface Engineering

The design of novel biomaterials for regenerative medicine requires incorporation of well-defined physical and chemical properties that mimic the native extracellular matrix (ECM). Here, we report the synthesis and characterization of porous foams prepared by high internal phase emulsion (HIPE) templating using amphiphilic copolymers that act as surfactants during the HIPE process. We combine different copolymers exploiting oil–water interface confined phase separation to engineer the surface topology of foam pores with nanoscopic domains of cell inert and active chemistries mimicking native matrix. We further demonstrate how proteins and hMSCs adhere in a domain specific manner

Heterogeneous muscle gene expression patterns in patients with massive rotator cuff tears

<div><p>Detrimental changes in the composition and function of rotator cuff (RC) muscles are hallmarks of RC disease progression. Previous studies have demonstrated both atrophic and degenerative muscle loss in advanced RC disease. However, the relationship between gene expression and RC muscle pathology remains poorly defined, in large part due to a lack of studies correlating gene expression to tissue composition. Therefore, the purpose of this study was to determine how tissue composition relates to gene expression in muscle biopsies from patients undergoing reverse shoulder arthroplasty (RSA). Gene expression related to myogenesis, atrophy and cell death, adipogenesis and metabolism, inflammation, and fibrosis was measured in 40 RC muscle biopsies, including 31 biopsies from reverse shoulder arthroplasty (RSA) cases that had available histology data and 9 control biopsies from patients with intact RC tendons. After normalization to reference genes, linear regression was used to identify relationships between gene expression and tissue composition. Hierarchical clustering and principal component analysis (PCA) identified unique clusters, and fold-change analysis was used to determine significant differences in expression between clusters. We found that gene expression profiles were largely dependent on muscle presence, with muscle fraction being the only histological parameter that was significantly correlated to gene expression by linear regression. Similarly, samples with histologically-confirmed muscle distinctly segregated from samples without muscle. However, two sub-groups within the muscle-containing RSA biopsies suggest distinct phases of disease, with one group expressing markers of both atrophy and regeneration, and another group not significantly different from either control biopsies or biopsies lacking muscle. In conclusion, this study provides context for the interpretation of gene expression in heterogeneous and degenerating muscle, and provides further evidence for distinct stages of RC disease in humans.</p></div

Fold change in expression between pooled RSA biopsies and controls.

<p>As a single pool, RSA biopsies are not significantly different from controls, though expression of pro-myogenic genes trended down while atrophic, adipogenic, and fibrotic genes trended up.</p

Heterogeneous muscle gene expression patterns in patients with massive rotator cuff tears - Fig 7

<p>Fold changes in expression between (A) HIGH and LOW expression muscle groups, (B) HIGH and NO-MUSCLE groups, (C) HIGH muscle and HI-FAT groups, (D) LOW muscle and HI-FAT groups, (E) LOW muscle and NO-MUSCLE groups, and (F) HI-FAT and NO-MUSCLE groups. Solid bars indicate significant up- or down-regulation (p<0.01 and p<0.05 indicated by ‘ = ‘, and ‘ * ‘, respectively).</p

Fold change in expression between the RSA biopsies that contain muscle compared to those without muscle.

<p>Solid bars indicate significant up- or down-regulation (p<0.01 and p<0.05 indicated by ‘ = ‘, and ‘ * ‘, respectively). With muscle present, nearly all genes of interest are significantly differentially regulated, with increased expression of muscle- and fat-related genes and decreased expression of fibrosis-related genes.</p