Focal Adhesion Area Research Articles

Abstract 5418: Lamellipodial protrusions induced by hypoxia depend upon kindlin-2 in prostate cancer cells

Abstract Hypoxia is a physiologically relevant feature of the prostate microenvironment that promotes migration and invasion resulting in extracapsular extension, the first step toward metastatic progression. Prostate cancer invasion depends on integrins as mechanosensing membrane receptors during creation of membrane lamellipodial protrusions and focal adhesions (FAs). The objective of this study was to determine the molecular events that promote membrane protrusions under hypoxia and whether this was dependent upon kindlin-2, an essential integrin adapter that marks activated β1 integrin heterodimers. DU145 cells depleted of one copy of FERMT2+/- (50% kindlin-2 expression) by CRISPR/Cas9 or DU145 FERMT2wt (normal kindlin-2 expression) were grown under acute exposure to hypoxia (1% O2) and compared to cells grown under normal tissue culture conditions. Immunofluorescence microscopy experiments were performed to analyze the spatial temporal expression of kindlin-2 complexes. Kindlin-2 complexes were confirmed by immunoprecipitation using an anti-kindlin-2 3A3-antibody from Sigma-Aldrich. Colocalization was determined by obtaining 2D immunofluorescence microscopy images analyzed using ImageJ 2.1.0/1.53c and Nikon NIS-Elements 5.30.04. Under hypoxic conditions, analysis over four time points (4h, 8h, 12h & 16h) increased the number and area of FAs (marked by paxillin (PXN)) containing kindlin-2 in a time-dependent manner by 2-fold and 1.5-fold, respectively, but not in DU145 FERMT2+/- cells. Additionally, hypoxia increased membrane area, perimeter, and the plasma membrane intensity of kindlin-2 exclusive of FAs in DU145 FERMT2wt cells which was maximal after 8 hours of hypoxia. Interestingly, limiting the kindlin-2 expression in FERMT2+/- cells resulted in a loss of hypoxia-induced lamellipodial protrusions (marked by lamellipodin (RAPH1)) containing kindlin-2 while hypoxia-induced kindlin-2 FA changes were preserved. The current working hypothesis is that lamellipodial protrusions are dependent upon kindlin-2 expression whereas the established FAs are stable under conditions of reduced kindlin-2 expression in hypoxia. This data suggests that an early stage of migration, lamellipodial extensions are sensitive to kindlin-2 availability. Further studies are required to determine the dynamic interplay between protrusive events and focal adhesions in relation to kindlin-2 for prostate cancer cells to migrate and invade. Citation Format: Colin Nelson, Daniel Hernandez-Cortes, Kendra D. Marr, Jaime MC Gard, Allan I. Paxson, William L. Harryman, Natalya K. Seppanen, John M. Ryniawec, Anne E. Cress. Lamellipodial protrusions induced by hypoxia depend upon kindlin-2 in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5418.

Differential effects of the venoms of Russell’s viper and Indian cobra on human myoblasts

Local tissue damage following snakebite envenoming remains a poorly researched area. To develop better strategies to treat snakebites, it is critical to understand the mechanisms through which venom toxins induce envenomation effects including local tissue damage. Here, we demonstrate how the venoms of two medically important Indian snakes (Russell's viper and cobra) affect human skeletal muscle using a cultured human myoblast cell line. The data suggest that both venoms affect the viability of myoblasts. Russell’s viper venom reduced the total number of cells, their migration, and the area of focal adhesions. It also suppressed myogenic differentiation and induced muscle atrophy. While cobra venom decreased the viability, it did not largely affect cell migration and focal adhesions. Cobra venom affected the formation of myotubes and induced atrophy. Cobra venom-induced atrophy could not be reversed by small molecule inhibitors such as varespladib (a phospholipase A2 inhibitor) and prinomastat (a metalloprotease inhibitor), and soluble activin type IIb receptor (a molecule used to promote regeneration of skeletal muscle), although the antivenom (raised against the Indian ‘Big Four’ snakes) has attenuated the effects. However, all these molecules rescued the myotubes from Russell’s viper venom-induced atrophy. This study demonstrates key steps in the muscle regeneration process that are affected by both Indian Russell’s viper and cobra venoms and offers insights into the potential causes of clinical features displayed in envenomed victims. Further research is required to investigate the molecular mechanisms of venom-induced myotoxicity under in vivo settings and develop better therapies for snakebite-induced muscle damage.

Effects of Various Cell Surface Engineering Reactions on the Biological Behavior of Mammalian Cells.

Cell surface engineering technologies can regulate cell function and behavior by modifying the cell surface. Previous studies have mainly focused on investigating the effects of cell surface engineering reactions and materials on cell activity. However, they do not comprehensively analyze other cellular processes. This study exploits covalent bonding, hydrophobic interactions, and electrostatic interactions to modify the macromolecules succinimide ester-methoxy polyethylene glycol (NHS-mPEG), distearoyl phosphoethanolamine-methoxy polyethylene glycol (DSPE-mPEG), and poly-L-lysine (PLL), respectively, on the cell surface. This work systematically investigates the effects of the three surface engineering reactions on the behavior of human umbilical vein endothelial cells (HUVECs) and human skin fibroblasts, including viability, growth, proliferation, cell cycle, adhesion, and migration. The results reveals that the PLL modification method notably affects cell viability and G2/M arrest and has a short modification duration. However, the DSPE-mPEG and NHS-mPEG modification methods have little effect on cell viability and proliferation but have a prolonged modification duration. Moreover, the DSPE-mPEG modification method highly affects cell adherence. Further, the NHS-mPEG modification method can significantly improve the migration ability of HUVECs by reducing the area of focal adhesions. The findings of this study will contribute to the application of cell surface engineering technology in the biomedical field.

Biochip Surfaces Containing Recombinant Cell-Binding Domains of Fibronectin

Surface immobilization and characterization of the functional activity of fibronectin (Fn) type-III domains are reported. The domains FnIII9-10 or FnIII10 containing the RGD loop and PHSRN synergy site were recombinantly produced and covalently bound to chemically activated PEG methacrylate (MA) hydrogel coatings by microcontact printing. Such fabricated biochip surfaces were 6 mm in diameter and consisted of 190 µm wide protein stripes separated by 200 µm spacing. They were analyzed by imaging null ellipsometry, atomic force microscopy and fluorescence microscopy. Also, the coatings were tested in human foreskin fibroblast and HeLa cultures for at least 96 h, thus evaluating their suitability for controlled cell adhesion and proliferation. However, while HeLa cultures were equally well responsive to the FnIII9-10, FnIII10 and Fn surfaces, the fibroblasts displayed lower cell and lower focal adhesion areas, as well as lower proliferation rates on the Fn fragment surfaces as compared to Fn. Nevertheless, full functional activity of the fibroblasts was confirmed by immunostaining of Fn produced by the cells adherent on the biochip surfaces. The observed interaction differences that were either cell type or surface composition-dependent demonstrate the potential use of specifically engineered Fn and other ECM protein-derived domains in biochip architectures.

Abstract 3452: Morphological phenotyping of organotropic brain- and bone-seeking triple negative metastatic breast tumor cells

Abstract Triple negative breast cancer (TNBC) follows a non-random pattern of metastasis to the bone and brain tissue. Prior work has found that brain-seeking breast tumor cells display altered proteomic profiles, leading to alterations in pathways related to cell signaling, cell cycle, metabolism, and extracellular matrix remodeling. Given the unique microenvironmental characteristics of brain and bone tissue, we hypothesized that brain- or bone-seeking TNBC cells may have altered morphologic or migratory phenotypes from each other, or from the parental TNBC cells, as a function of the biochemical or mechanical microenvironment. In this study, we utilized TNBC cells (MDA-MB-231) that were conditioned to metastasize solely to brain (MDA-BR) or bone (MDA-BO) tissue. We quantified characteristics such as cell morphology, migration, and stiffness in response to cues that partially mimic their final metastatic niche. We have shown that MDA-BO cells have a distinct protrusive morphology not found in MDA-P or MDA-BR. Further, MDA-BO cells migrate over a larger area when on a collagen I (abundant in bone tissue) substrate when compared to fibronectin (abundant in brain tissue). However, migration in highly confined environments was similar across the cell types. Modest differences were found in the stiffness of MDA-BR and MDA-BO cells plated on collagen I vs. fibronectin-coated surfaces. Lastly, MDA-BO cells were found to have larger focal adhesion area and density in comparison with the other two cell types. These results initiate a quantitative profile of mechanobiological phenotypes in TNBC, with future impacts in helping to predict metastatic propensities to organ-specific sites in a clinical setting. Citation Format: Ariana Joy L. DeCastro, Marina Pranda, Kelsey Gray, John Merlo-Coyne, Nathaniel Girma, Madelyn Hurwitz, Yuji Zhang, Kimberly Stroka. Morphological phenotyping of organotropic brain- and bone-seeking triple negative metastatic breast tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3452.

Desmin Modulates Muscle Cell Adhesion and Migration.

Cellular adhesion and migration are key functions that are disrupted in numerous diseases. We report that desmin, a type-III muscle-specific intermediate filament, is a novel cell adhesion regulator. Expression of p.R406W mutant desmin, identified in patients with desmin-related myopathy, modified focal adhesion area and expression of adhesion-signaling genes in myogenic C2C12 cells. Satellite cells extracted from desmin-knock-out (DesKO) and desmin-knock-in-p.R405W (DesKI-R405W) mice were less adhesive and migrated faster than those from wild-type mice. Moreover, we observed mislocalized and aggregated vinculin, a key component of cell adhesion, in DesKO and DesKI-R405W muscles. Vinculin expression was also increased in desmin-related myopathy patient muscles. Together, our results establish a novel role for desmin in cell-matrix adhesion, an essential process for strength transmission, satellite cell migration and muscle regeneration. Our study links the patho-physiological mechanisms of desminopathies to adhesion/migration defects, and may lead to new cellular targets for novel therapeutic approaches.

Morphological Phenotyping of Organotropic Brain- and Bone-Seeking Triple Negative Metastatic Breast Tumor Cells.

Triple negative breast cancer (TNBC) follows a non-random pattern of metastasis to the bone and brain tissue. Prior work has found that brain-seeking breast tumor cells display altered proteomic profiles, leading to alterations in pathways related to cell signaling, cell cycle, metabolism, and extracellular matrix remodeling. Given the unique microenvironmental characteristics of brain and bone tissue, we hypothesized that brain- or bone-seeking TNBC cells may have altered morphologic or migratory phenotypes from each other, or from the parental TNBC cells, as a function of the biochemical or mechanical microenvironment. In this study, we utilized TNBC cells (MDA-MB-231) that were conditioned to metastasize solely to brain (MDA-BR) or bone (MDA-BO) tissue. We quantified characteristics such as cell morphology, migration, and stiffness in response to cues that partially mimic their final metastatic niche. We have shown that MDA-BO cells have a distinct protrusive morphology not found in MDA-P or MDA-BR. Further, MDA-BO cells migrate over a larger area when on a collagen I (abundant in bone tissue) substrate when compared to fibronectin (abundant in brain tissue). However, migration in highly confined environments was similar across the cell types. Modest differences were found in the stiffness of MDA-BR and MDA-BO cells plated on collagen I vs. fibronectin-coated surfaces. Lastly, MDA-BO cells were found to have larger focal adhesion area and density in comparison with the other two cell types. These results initiate a quantitative profile of mechanobiological phenotypes in TNBC, with future impacts aiming to help predict metastatic propensities to organ-specific sites in a clinical setting.

Aligned and Oriented Collagen Nanocomposite Fibers as Substrates to Activate Fibroblasts.

Purified collagen possesses weak mechanical properties, hindering its broad application in tissue engineering. Strategies based on manipulating the hydrogel to induce fiber formation or incorporate nanomaterials have been proposed to overcome this issue. Herein, we use a microfluidic device to fabricate, for the first time, collagen hydrogels with aligned and oriented fibers doped with gold nanoparticles and carbon nanotubes. Results based on rheology, atomic force microscopy, and scanning electron microscopy reveal the formation of aligned and oriented collagen fibers possessing greater rigidity and stiffness on the doped hydrogels in comparison with native collagen. The mechanical properties of the hydrogels increased with the nanomaterial loading percentage and the stiffest formulations were those prepared in the presence of carbon nanotubes. We further evaluate the in vitro response of NIH-3T3 fibroblasts to the change in stiffness. The cells were found to be viable on all substrates with directional cell growth observed for the carbon nanotube-doped collagen fibers. No significant differences in the cell area, aspect ratio, and intensification of focal adhesions driven by the increase in stiffness were noted. Nonetheless, fibroblast proliferation and secretion of TGF-β1 were greater on the hydrogels doped with carbon nanotubes. This nanomaterial-collagen composite provides unique features for cell and tissue substrate applications.

The effect of surface modification of TiMg composite on the in-vitro degradation response, cell survival, adhesion, and proliferation

This study is aimed to evaluate the influence of mechanical surface treatment on the degradation response, cell survival, adhesion, and proliferation of a TiMg composite material. Two sets of the TiMg samples with different surface characteristics were studied: i) as-machined samples (TiMg-T) and ii) samples with a mechanically modified surface (TiMg-P). Surface roughness was determined using a confocal microscope. Degradation rates (DR) were evaluated in artificial Plasma, HBSS, and NaCl 0.9%. The cell viability was evaluated using an MTT assay. The initial cell adhesion and spreading were investigated using the direct contact assay. An xCELLigence system was employed to provide real-time cell proliferation. The focal adhesion and cell morphological changes were also examined. The DR of TiMg-P decreased by ⁓5 times compared with that of TiMg-T. Surface of the TiMg-P specimens after 72 h exposure to either HBSS or Plasma was passivated by a layer enriched with bioactive Ca/P species. The cell viability of L929 and Saos-2 after 72 h incubation for TiMg-P was 94.6% and 94.8% compared with 73.8% and 74.3% obtained for TiMg-T, respectively. The direct contact assay showed that the initial adhesion and spreading of the L929 cells incubated with TiMg-P was more pronounced compared with that of TiMg-T. The proliferation rate of Saos-2 cells incubated with TiMg-P was higher when compared with that of TiMg-T, and was almost comparable to that of the DMEM-blank between the 24 and 72 h interval. TiMg-P had a pronounced difference in the number and area of Focal Adhesions (FA) compared with that of TiMg-T. The morphology of cells incubated with TiMg-P was not altered. The results confirmed that the smooth and less strained surface of the TiMg-P samples effectively improved the in-vitro degradation response, cell survival, adhesion, and proliferation.

Bone Piezoelectricity-Mimicking Nanocomposite Membranes Enhance Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells by Amplifying Cell Adhesion and Actin Cytoskeleton.

Ferroelectric biomaterials have been widely investigated and demonstrated to enhance osteogenesis by simulating the inherent electrical properties of bone tissues. Nevertheless, the underlying biological processes are still not wellunderstood. Hence, this study investigated the underlying biological processes by which bone piezoelectricity-mimicking barium titanate/poly(vinylidene fluoride-trifluoroethylene) nanocomposite membranes (BTO nanocomposite membranes) promote osteogenesis of Bone Marrow Mesenchymal Stem Cells (BMSCs). Ourresults revealed that the piezoelectric coefficient (d33) of nanocomposite membranes aftercontrolled corona poling was similar to that of native bone, and exhibited highly-stable piezoelectrical properties and concentrated surface electrical potential. These nanocomposite membranes significantly enhanced the adhesion and spreading of BMSCs, which was manifested as increased number and area of mature focal adhesions. Furthermore, the nanocomposite membranes significantly promoted the expression of integrin receptors genes (α1, α5 andβ3), which in turn enhanced osteogenesis of BMSCs, as manifested by upregulated Alkaline Phosphatase (ALP) and Bone Morphogenetic Protein 2 (BMP2) expression levels. Further investigations found that the Focal Adhesion Kinase (FAK)-Extracellular Signal-Regulated Kinase1/2 (ERK 1/2) signaling axis may be involved in the biological process of polarized nanocomposite membrane-induced osteogenesis. This study thus provides useful insights for betterunderstanding of the biological processes by which piezoelectric or ferroelectric biomaterials promote osteogenesis.

Fibroblasts treated with macrophage conditioned medium results in phenotypic shifts and changes in collagen organization

In tissue regeneration, the goal is to regenerate tissue similar to what was damaged or missing while preventing fibrotic scarring, which may lead to decreased mechanical strength and dissimilar tissue characteristics compared to native tissue. We believe collagen orientation plays a critical role in wound contraction and scarring and that it is modulated by myofibroblasts. We used macrophage conditioned medium to simulate complex events that can influence the fibroblast phenotype during the wound healing process. In addition to examining the effect of macrophage phenotype on fibroblasts, we inhibited focal adhesion kinase (FAK), Rho-associated protein kinase (ROCK), and myosin II for fibroblasts cultured on both tissue culture plastic and methacrylated gellan gum to understand how different pathways and materials influence fibroblast responses. Collagen orientation, α-SMA expression, focal adhesion area, and cell migration were altered by inhibition of FAK, ROCK, or myosin II and macrophage phenotype, along with the substrate. An increase in either focal adhesion area or α-smooth muscle actin (α-SMA) expression correlated with an aligned collagen orientation. Gellan gum hydrogels upregulated α-SMA expression in ROCK inhibited conditioned media and downregulated the FAK area in FAK and ROCK inhibited conditioned media. Myosin II had no impact on the α-SMA expression on the substrate compared to coverslip except for M2 conditioned medium. Gellan gum hydrogel significantly increased cell migration under FAK and Myosin II mediated conditioned media and unconditioned media. Collectively, our study examined how macrophage phenotype influences fibroblast response, which would be beneficial in controlling scar tissue formation.

Hyperthermia induced disruption of mechanical balance leads to G1 arrest and senescence in cells.

Human body temperature limits below 40°C during heat stroke or fever. The implications of prolonged exposure to the physiologically relevant temperature (40°C) on cellular mechanobiology is poorly understood. Here, we have examined the effects of heat stress (40°C for 72 h incubation) in human lung adenocarcinoma (A549), mouse melanoma (B16F10), and non-cancerous mouse origin adipose tissue cells (L929). Hyperthermia increased the level of ROS, γ-H2AX and HSP70 and decreased mitochondrial membrane potential in the cells. Heat stress impaired cell division, caused G1 arrest, induced cellular senescence, and apoptosis in all the tested cell lines. The cells incubated at 40°C for 72 h displayed a significant decrease in the f-actin level and cellular traction as compared with cells incubated at 37°C. Also, the cells showed a larger focal adhesion area and stronger adhesion at 40°C than at 37°C. The mitotic cells at 40°C were unable to round up properly and displayed retracting actin stress fibers. Hyperthermia down-regulated HDAC6, increased the acetylation level of microtubules, and perturbed the chromosome alignment in the mitotic cells at 40°C. Overexpression of HDAC6 rescued the cells from the G1 arrest and reduced the delay in cell rounding at 40°C suggesting a crucial role of HDAC6 in hyperthermia mediated responses. This study elucidates the significant role of cellular traction, focal adhesions, and cytoskeletal networks in mitotic cell rounding and chromosomal misalignment. It also highlights the significance of HDAC6 in heat-evoked senile cellular responses.

Integrin affinity modulation critically regulates atherogenic endothelial activation in vitro and in vivo

While vital to platelet and leukocyte adhesion, the role of integrin affinity modulation in adherent cells remains controversial. In endothelial cells, atheroprone hemodynamics and oxidized lipoproteins drive an increase in the high affinity conformation of α5β1 integrins in endothelial cells in vitro, and α5β1 integrin inhibitors reduce proinflammatory endothelial activation to these stimuli in vitro and in vivo. However, the importance of α5β1 integrin affinity modulation to endothelial phenotype remains unknown. We now show that endothelial cells (talin1 L325R) unable to induce high affinity integrins initially adhere and spread but show significant defects in nascent adhesion formation. In contrast, overall focal adhesion number, area, and composition in stably adherent cells are similar between talin1 wildtype and talin1 L325R endothelial cells. However, talin1 L325R endothelial cells fail to induce high affinity α5β1 integrins, fibronectin deposition, and proinflammatory responses to atheroprone hemodynamics and oxidized lipoproteins. Inducing the high affinity conformation of α5β1 integrins in talin1 L325R endothelial cells suggest that NF-κB activation and maximal fibronectin deposition require both integrin activation and other integrin-independent signaling. In endothelial-specific talin1 L325R mice, atheroprone hemodynamics fail to promote inflammation and macrophage recruitment, demonstrating a vital role for integrin activation in regulating endothelial phenotype.

K+ Channel Tetramerization Domain 5 (KCTD5) Protein Regulates Cell Migration, Focal Adhesion Dynamics and Spreading through Modulation of Ca2+ Signaling and Rac1 Activity.

Cell migration is critical for several physiological and pathophysiological processes. It depends on the coordinated action of kinases, phosphatases, Rho-GTPases proteins, and Ca2+ signaling. Interestingly, ubiquitination events have emerged as regulatory elements of migration. Thus, the role of proteins involved in ubiquitination processes could be relevant to a complete understanding of pro-migratory mechanisms. KCTD5 is a member of Potassium Channel Tetramerization Domain (KCTD) proteins that have been proposed as a putative adaptor for Cullin3-E3 ubiquitin ligase and a novel regulatory protein of TRPM4 channels. Here, we study whether KCTD5 participates in cell migration-associated mechanisms, such as focal adhesion dynamics and cellular spreading. Our results show that KCTD5 CRISPR/Cas9- and shRNA-based depletion in B16-F10 cells promoted an increase in cell migration and cell spreading, and a decrease in the focal adhesion area, consistent with an increased focal adhesion disassembly rate. The expression of a dominant-negative mutant of Rho-GTPases Rac1 precluded the KCTD5 depletion-induced increase in cell spreading. Additionally, KCTD5 silencing decreased the serum-induced Ca2+ response, and the reversion of this with ionomycin abolished the KCTD5 knockdown-induced decrease in focal adhesion size. Together, these data suggest that KCTD5 acts as a regulator of cell migration by modulating cell spreading and focal adhesion dynamics through Rac1 activity and Ca2+ signaling, respectively.

Fiber Crimp Confers Matrix Mechanical Nonlinearity, Regulates Endothelial Cell Mechanosensing, and Promotes Microvascular Network Formation.

Mechanical interactions between cells and their surrounding extracellular matrix (ECM) guide many fundamental cell behaviors. Native connective tissue consists of highly organized, 3D networks of ECM fibers with complex, nonlinear mechanical properties. The most abundant stromal matrix component is fibrillar type I collagen, which often possesses a wavy, crimped morphology that confers strain- and load-dependent nonlinear mechanical behavior. Here, we established a new and simple method for engineering electrospun fibrous matrices composed of dextran vinyl sulfone (DexVS) with controllable crimped structure. A hydrophilic peptide was functionalized to DexVS matrices to trigger swelling of individual hydrogel fibers, resulting in crimped microstructure due to the fixed anchorage of fibers. Mechanical characterization of these matrices under tension confirmed orthogonal control over nonlinear stress-strain responses and matrix stiffness. We next examined ECM mechanosensing of individual endothelial cells (ECs) and found that fiber crimp promoted physical matrix remodeling alongside decreases in cell spreading, focal adhesion area, and nuclear localization of Yes-associated protein (YAP). These changes corresponded to an increase in migration speed, along with evidence for long-range interactions between neighboring cells in crimped matrices. Interestingly, when ECs were seeded at high density in crimped matrices, capillary-like networks rapidly assembled and contained tube-like cellular structures wrapped around bundles of synthetic matrix fibers due to increased physical reorganization of matrix fibers. Our work provides an additional level of mechanical and architectural tunability to synthetic fibrous matrices and implicates a critical role for mechanical nonlinearity in EC mechanosensing and network formation.

Predictive Modeling of the In Vitro Responses of Preosteoblastic MC3T3-E1 Cells on Polymeric Surfaces Using Fourier Transform Infrared Spectroscopy.

Biomaterials' surface properties elicit diverse cellular responses in biomedical and biotechnological applications. Predicting the cell behavior on a polymeric surface is an ongoing challenge due to its complexity. This work proposes a novel modeling methodology based on attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. Spectra were collected on wetted polymeric surfaces to incorporate both surface chemistry and information on water-polymer interactions. Results showed that predictive models built with spectra from wetted surfaces ("wet spectra") performed much better than models built using spectra acquired from dry surfaces ("dry spectra"), suggesting that the water-polymer interaction is critically important to the prediction of subsequent cell behavior. The best model was seen to predict total area of focal adhesions with coefficient of determination for prediction (R2P) of 0.94 and root-mean-square errors of prediction (RMSEP) of 4.03 μm2 when tested on an independent experimental set. This work offers new insights into our understanding of cell-biomaterial interactions. The presence of carboxyl groups in polymers promoted larger adhesion areas, yet the formation of carbonyl-to-water interaction decreased adhesion areas. Surface wettability, which was related to the water-polymer interaction, was proven to highly influence cell adhesion. The good predictive ability opens new possibilities for high throughput monitoring of cell attachment on polymeric substrates.

Magnetic nanoparticles of Ga‐substituted ε‐Fe2O3 for biomedical applications: Magnetic properties, transverse relaxivity, and effects of silica‐coated particles on cytoskeletal networks

Magnetic nanoparticles of ε-Fe1.76 Ga0.24 O3 with the volume-weighted mean size of 17 nm were prepared by thermal treatment of a mesoporous silica template impregnated with metal nitrates and were coated with silica shell of four different thicknesses in the range 6-24 nm. The bare particles exhibited higher magnetization than the undoped compound, 22.4 Am2 kg-1 at 300 K, and were characterized by blocked state with the coercivity of 1.2 T at 300 K, being thus the very opposite of superparamagnetic iron oxides. The relaxometric study of the silica-coated samples at 0.47 T revealed promising properties for MRI, specifically, transverse relaxivity of 89-168 s-1 mmol(f.u.)-1 L depending on the shell thickness was observed. We investigated the effects of the silica-coated nanoparticles on human A549 and MCF-7 cells. Cell viability, proliferation, cell cycle distribution, and the arrangement of actin cytoskeleton were assessed, as well as formation and maturation of focal adhesions. Our study revealed that high concentrations of silica-coated particles with larger shell thicknesses of 16-24 nm interfere with the actin cytoskeletal networks, inducing thus morphological changes. Consequently, the focal adhesion areas were significantly decreased, resulting in impaired cell adhesion.

In Vitro Culture of Human Corneal Endothelium on Non-Mulberry Silk Fibroin Films for Tissue Regeneration.

PurposeThe purpose of this study was to determine if non-mulberry varieties of silk are suitable for the culture of corneal endothelium (CE).MethodsAqueous silk fibroin derived from Philosamia ricini (PR), Antheraea assamensis (AA), and Bombyx mori (BM) were cast as approximately 15 µm films with and without pores on which human CE cells were cultured. Tensile strength, elasticity, transmittance in visible range, and degradation properties of the films were characterised. Adhesion of CE to the silk films was quantified using MTT assay in addition to quantifying the number and area of focal adhesions using paxillin. Expression of CE markers was determined at the gene and protein levels using PCR and immunostaining, respectively. Barrier integrity of the cultured cells was measured as permeability to FITC dextran (10 kDa) in the presence or absence of thrombin.ResultsThe films exhibited robust tensile strength, >95% transmittance and a refractive index comparable to the native cornea. BM degraded significantly faster when compared to PR and AA. A comparison between the three varieties of silk showed that significantly more cells were adhered to PR and AA than to BM. This was also reflected in the expression of stable focal adhesions on PR and AA, thus enabling the formation of intact monolayers of cells on these varieties unlike on BM. Treatment with thrombin significantly increased cellular permeability to dextran.ConclusionsOur data shows that PR and AA varieties sufficiently support the growth and function of CE cells. This could be attributed to the presence of natural cell binding motifs (RGD) in these varieties.Translational RelevanceDevelopment of a suitable carrier for engineering the CE to address a major clinical requirement of healthy donor tissues for transplantation.

Molecular chaperone αB-crystallin regulates the dynamic stability of focal adhesion under mechanical stress conditions

Resilience to stretch stress is an important characteristic that helps maintain cell adhesion and consequently, human health. This study aimed to elucidate the underlying mechanism of adaptation to stretch stress regulated by the molecular chaperone αB-crystallin. Three rat myoblast L6 cell lines, wild type (L6-WT), αB-crystallin knock down (L6-KD), and αB-crystallin overexpressing (L6-OE) cells were used. Muscle cells are less motile because they are specialized for contraction. Forced stretch stress was given to the three cell lines on a soft adhesive sheet, and we found that L6-OE cells showed the highest resilience to stretch stress and the least motility compared to other cell lines. Conversely, L6-KD cells showed the least resilience to stretch stress. Vinculin staining showed that total focal adhesion (FA) size and area of L6-OE cells were significantly larger than those of other cell types. Thus αB-crystallin in myoblast cells contributes the resilience of FA stability during stretch stress.

Directional Topography Influences Adipose Mesenchymal Stromal Cell Plasticity: Prospects for Tissue Engineering and Fibrosis.

Introduction Progenitor cells cultured on biomaterials with optimal physical-topographical properties respond with alignment and differentiation. Stromal cells from connective tissue can adversely differentiate to profibrotic myofibroblasts or favorably to smooth muscle cells (SMC). We hypothesized that myogenic differentiation of adipose tissue-derived stromal cells (ASC) depends on gradient directional topographic features. Methods Polydimethylsiloxane (PDMS) samples with nanometer and micrometer directional topography gradients (wavelength (w) = 464-10, 990 nm; amplitude (a) = 49-3, 425 nm) were fabricated. ASC were cultured on patterned PDMS and stimulated with TGF-β1 to induce myogenic differentiation. Cellular alignment and adhesion were assessed by immunofluorescence microscopy after 24 h. After seven days, myogenic differentiation was examined by immunofluorescence microscopy, gene expression, and immunoblotting. Results Cell alignment occurred on topographies larger than w = 1758 nm/a = 630 nm. The number and total area of focal adhesions per cell were reduced on topographies from w = 562 nm/a = 96 nm to w = 3919 nm/a = 1430 nm. Focal adhesion alignment was increased on topographies larger than w = 731 nm/a = 146 nm. Less myogenic differentiation of ASC occurred on topographies smaller than w = 784 nm/a = 209 nm. Conclusion ASC adherence, alignment, and differentiation are directed by topographical cues. Our evidence highlights a minimal topographic environment required to facilitate the development of aligned and differentiated cell layers from ASC. These data suggest that nanotopography may be a novel tool for inhibiting fibrosis.