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ABSTRACT Aim Materials and Methods Results Conclusion Periodontal disease is driven by oral pathogens, including Porphyromonas gingivalis, and the release of inflammatory cytokines. These cytokines (e.g., TNF) or their receptors (e.g., IL‐1R) are substrates of a disintegrin and metalloproteinases (ADAMs). In this study, we aimed to determine the effects of ADAMs on periodontal disease phenotypes.Western blot and FRET‐based activity measurements of the gingival crevicular fluid (GCF) of patients were compared with those of infected (P. gingivalis) or cytokine‐stimulated oral keratinocytes and primary human neutrophils, respectively. This was accompanied by an analysis of the released extracellular vesicles and MMP9 activity.In the GCF of patients, ADAM8 protein expression and activity were correlated with disease stage, whereas ADAM10 protein expression was inversely correlated with disease stage. Infection and the resulting cytokine release orchestrated the release of soluble ADAM8 by oral keratinocytes and primary neutrophils as soluble ectodomain and on exosomes, respectively. Furthermore, ADAM8 regulated the release of ADAM10 and MMP9.Dysregulation of cell‐associated and extracellular ADAM proteolytic activity may be an essential regulatory element in the progression of periodontal disease driven by ADAM8. The influence of ADAM8 on disease onset and the evaluation of targeting ADAM8 as a potential and novel local treatment option should be addressed in future translational in vivo studies. [ABSTRACT FROM AUTHOR]

The development of self‐healing conductive hydrogels is critical in electroactive nerve tissue engineering. Typical conductive materials such as polypyrrole (PPy) are commonly used to fabricate artificial nerve conduits. Moreover, the field of tissue engineering has advanced toward the use of products such as hyaluronic acid (HA) hydrogels. Although HA‐modified PPy films are prepared for various biological applications, the cell–matrix interaction mechanisms remain poorly understood; furthermore, there are no reports on HA‐modified PPy‐injectable self‐healing hydrogels for peripheral nerve repair. Therefore, in this study, a self‐healing electroconductive hydrogel (HASPy) from HA, cystamine (Cys), and pyrrole‐1‐propionic acid (Py‐COOH), with injectability, biodegradability, biocompatibility, and nerve‐regenerative capacity is constructed. The hydrogel directly targets interleukin 17 receptor A (IL‐17RA) and promotes the expression of genes and proteins relevant to Schwann cell myelination mainly by activating the interleukin 17 (IL‐17) signaling pathway. The hydrogel is injected directly into the rat sciatic nerve‐crush injury sites to investigate its capacity for nerve regeneration in vivo and is found to promote functional recovery and remyelination. This study may help in understanding the mechanism of cell–matrix interactions and provide new insights into the potential use of HASPy hydrogel as an advanced scaffold for neural regeneration. [ABSTRACT FROM AUTHOR]

Transforming growth factor-β (TGF-β) is a multifunctional cytokine that regulates the expression of ECM-associated genes during early injury. Tissue fibrosis development is driven by synergistic cues between the evolving biochemical and mechanical milieu. Few studies have addressed the role of substrate stiffness on TGF-β activity and extracellular matrix (ECM)-associated genes. We used a commercial formulation of polydimethylsiloxane (PDMS) to fabricate substrates of 40 kPa, 300 kPa, and 1.5 MPa stiffness, and cultured the HMF3S fibroblasts on substrates. We quantified TGF-β protein secreted by HMF3S cells on different substrates using a TGF-β responsive promoter reporter assay. We also tested for variations in gene expression levels on the substrates using RT-PCR and Western blotting and determined the MMP-2 and MMP-9 activities with gelatin zymography. The results showed that TGF-β protein activation was significantly compromised at lower stiffnesses. The expression of integrin α5 decreased on lower stiffness substrates and correlated with inefficient TGF-β protein activation. Collagen I, collagen III, and MMP-2 expression levels were lower on softer substrates; there was little MMP-9 activity on all substrates. Cell and nuclear morphologies were more rounded on compliant substrates, correlating with increased tubulin expression. Proliferations were higher on stiffer substrates, whereas cells on softer substrates showed cell cycle arrest. These results demonstrated critical feedback mechanisms between substrate stiffness and ECM regulation by fibroblasts, relevant in fibrosis. [ABSTRACT FROM AUTHOR]

Nonlinear stiffening is a ubiquitous property of major types of biopolymers that make up the extracellular matrices (ECM) including collagen, fibrin, and basement membrane. Within the ECM, many types of cells such as fibroblasts and cancer cells have a spindle-like shape that acts like two equal and opposite force monopoles, which anisotropically stretch their surroundings and locally stiffen the matrix. Here, we first use optical tweezers to study the nonlinear force-displacement response to localized monopole forces. We then propose an effective-probe scaling argument that a local point force application can induce a stiffened region in the matrix, which can be characterized by a nonlinear length scale R* that increases with the increasing force magnitude; the local nonlinear force-displacement response is a result of the nonlinear growth of this effective probe that linearly deforms an increasing portion of the surrounding matrix. Furthermore, we show that this emerging nonlinear length scale R* can be observed around living cells and can be perturbed by varying matrix concentration or inhibiting cell contractility. [ABSTRACT FROM AUTHOR]

Cells continuously sense external forces from their microenvironment, the extracellular matrix (ECM). In turn, they generate contractile forces, which stiffen and remodel this matrix. Although this bidirectional mechanical exchange is crucial for many cell functions, it remains poorly understood. Key challenges are that the majority of available matrices for such studies, either natural or synthetic, are difficult to control or lack biological relevance. Here, we use a synthetic, yet highly biomimetic hydrogel based on polyisocyanide (PIC) polymers to investigate the effects of the fibrous architecture and the nonlinear mechanics on cell-matrix interactions. Live-cell rheology was combined with advanced microscopy-based approaches to understand the mechanisms behind cell-induced matrix stiffening and plastic remodeling. We demonstrate how cell-mediated fiber remodeling and the propagation of fiber displacements are modulated by adjusting the biological and mechanical properties of this material. Moreover, we validate the biological relevance of our results by demonstrating that cellular tractions in PIC gels develop analogously to those in the natural ECM. This study highlights the potential of PIC gels to disentangle complex bidirectional cell-matrix interactions and to improve the design of materials for mechanobiology studies. [ABSTRACT FROM AUTHOR]

Engineered living microtissues such as cellular spheroids and organoids have enormous potential for the study and regeneration of tissues and organs. Microtissues are typically engineered via self‐assembly of adherent cells into cellular spheroids, which are characterized by little to no cell–material interactions. Consequently, 3D microtissue models currently lack structural biomechanical and biochemical control over their internal microenvironment resulting in suboptimal functional performance such as limited stem cell differentiation potential. Here, this work report on stimuli‐responsive cell‐adhesive micromaterials (SCMs) that can self‐assemble with cells into 3D living composite microtissues through integrin binding, even under serum‐free conditions. It is demonstrated that SCMs homogeneously distribute within engineered microtissues and act as biomechanically and biochemically tunable designer materials that can alter the composite tissue microenvironment on demand. Specifically, cell behavior is controlled based on the size, stiffness, number ratio, and biofunctionalization of SCMs in a temporal manner via orthogonal secondary crosslinking strategies. Photo‐based mechanical tuning of SCMs reveals early onset stiffness‐controlled lineage commitment of differentiating stem cell spheroids. In contrast to conventional encapsulation of stem cell spheroids within bulk hydrogel, incorporating cell‐sized SCMs within stem cell spheroids uniquely provides biomechanical cues throughout the composite microtissues' volume, which is demonstrated to be essential for osteogenic differentiation. [ABSTRACT FROM AUTHOR]

Objective: Cancer‐associated fibroblasts (CAFs) are abundantly infiltrated in oral squamous cell carcinoma (OSCC), but the contact‐dependent mechanisms that regulate CAFs phenotype in precursor cells, such as paracancerous fibroblasts (PFs), remain unclear. Here, a fibroblast‐attached organoid (FAO) model was initiated to determine phenotype transition of fibroblasts triggered by contact with OSCC. Material and methods: Organoids and fibroblasts were generated using OSCC and adjacent tissues. Cell‐clusters containing fibroblasts and tumour cells were aggregated to allow for FAOs expansion. Immunoblotting assay was performed to compare expression of Notch intracellular domain (NICD) in CAFs and PFs. Colony formation assay was employed to evaluate morphological activation of fibroblasts. Results: Compared to traditional 3D co‐culture, FAOs better modulated the spatial distribution of fibroblasts with tumour nests. The presence of CAFs with multiple branches was stably observed in FAOs during serial passage. Incorporation with organoids promoted the ability of PFs to form multiple branches. Immunoblotting assay confirmed higher NICD level in CAFs than PFs. Treatment with Notch inhibitor, N‐[N‐(3, 5‐difluorophenacetyl)‐l‐alanyl]‐S‐phenylglycine t‐butyl ester (i.e. DAPT) blocked morphological activation of fibroblasts incorporated into FAO. Conclusion: We developed a robust strategy to study contact‐dependent mechanisms underlying tumour‐stromal interaction, and suggested that Notch activity contributes to biogenesis of OSCC‐associated fibroblasts. [ABSTRACT FROM AUTHOR]

Natural polymer material-based hydrogels normally show inferior mechanical stability and strength to bear large deformation and cyclic loading, therefore their applications in food, biomedical and tissue engineering fields are greatly limited. In this study, gelatin-based hydrogels with remarkable stability, as well as tunable mechanical properties, were prepared via a facile method known as the Hofmeister effect. The higher concentration of potassium sulfatesolution resulted in more dehydration and molecular chain folding, thus the treated hydrogels showed significantly improved tensile and compressive modulus, and decreased equilibrium swelling ratio, as revealed by scanning electron microscopy (SEM), Fourier transform infraredspectroscopy (FTIR), and mechanical tests, etc. Additionally, the reinforced hydrogels were recoverable and biocompatible to modulate the proliferation behavior of human umbilical vein endothelial cells. In conclusion, this paper provides a facile reference for tuning mechanical properties of gelatin-based hydrogels and cell-hydrogel interactions, which show potential capacity in tissue engineering and biomedical fields. [ABSTRACT FROM AUTHOR]

Epithelial tissues mechanically deform the surrounding extracellular matrix during embryonic development, wound repair, and tumor invasion. Ex vivo measurements of such multicellular tractions within three-dimensional (3D) biomaterials could elucidate collective dissemination during disease progression and enable preclinical testing of targeted antimigration therapies. However, past 3D traction measurements have been low throughput due to the challenges of imaging and analyzing information-rich 3D material deformations. Here, we demonstrate a method to profile multicellular clusters in a 96-well-plate format based on spatially heterogeneous contractile, protrusive, and circumferential tractions. As a case study, we profile multicellular clusters across varying states of the epithelial-mesenchymal transition, revealing a successive loss of protrusive and circumferential tractions, as well as the formation of localized contractile tractions with elongated cluster morphologies. These cluster phenotypes were biochemically perturbed by using drugs, biasing toward traction signatures of different epithelial or mesenchymal states. This higher-throughput analysis is promising to systematically interrogate and perturb aberrant mechanobiology, which could be utilized with human-patient samples to guide personalized therapies. [ABSTRACT FROM AUTHOR]

The cornea, as a dynamic and responsive tissue, constantly interacts with mechanical forces in order to maintain its structural integrity, barrier function, transparency and refractive power. Cells within the cornea sense and respond to various mechanical forces that fundamentally regulate their morphology and fate in development, homeostasis and pathophysiology. Corneal cells also dynamically regulate their extracellular matrix (ECM) with ensuing cell-ECM crosstalk as the matrix serves as a dynamic signaling reservoir providing biophysical and biochemical cues to corneal cells. Here we provide an overview of mechanotransduction signaling pathways then delve into the recent advances in corneal mechanobiology, focusing on the interplay between mechanical forces and responses of the corneal epithelial, stromal, and endothelial cells. We also identify species-specific differences in corneal biomechanics and mechanotransduction to facilitate identification of optimal animal models to study corneal wound healing, disease, and novel therapeutic interventions. Finally, we identify key knowledge gaps and therapeutic opportunities in corneal mechanobiology that are pressing for the research community to address especially pertinent within the domains of limbal stem cell deficiency, keratoconus and Fuchs' endothelial corneal dystrophy. By furthering our understanding corneal mechanobiology, we can contextualize discoveries regarding corneal diseases as well as innovative treatments for them. [ABSTRACT FROM AUTHOR]

Dimensionality can have a profound impact on stiffness-mediated differentiation of mesenchymal stem cells (MSCs). However, while we have begun to understand cellular response when encapsulated within 3D substrates, the behavior of cells within macro-porous substrates is relatively underexplored. The goal of this study was to determine the influence of macro-porous topographies on stiffness-mediated differentiation of MSCs. We developed macro-porous recombinant elastin-like protein (ELP) substrates that allow independent control of mechanical properties and ligand chemistry. We then used computational modeling to probe the impact of pore topography on the mechanical stimulus that cells are exposed to within these substrates, and finally we investigated stiffness induced biases towards adipogenic and osteogenic differentiation of MSCs within macro-porous substrates. Computational modeling revealed that there is significant heterogeneity in the mechanical stimuli that cells are exposed to within porous substrates and that this heterogeneity is predominantly due to the wide range of possible cellular orientations within the pores. Surprisingly, MSCs grown within 3D porous substrates respond to increasing substrate stiffness by up-regulating both osteogenesis and adipogenesis. These results demonstrate that within porous substrates the behavior of MSCs diverges from previously observed responses to substrate stiffness, emphasizing the importance of topography as a determinant of cellular behavior. [ABSTRACT FROM AUTHOR]

Animal cells in tissues are supported by biopolymer matrices, which typically exhibit highly nonlinear mechanical properties. While the linear elasticity of the matrix can significantly impact cell mechanics and functionality, it remains largely unknown how cells, in turn, affect the nonlinear mechanics of their surrounding matrix. Here, we show that living contractile cells are able to generate a massive stiffness gradient in three distinct 3D extracellular matrix model systems: collagen, fibrin, and Matrigel. We decipher this remarkable behavior by introducing nonlinear stress inference microscopy (NSIM), a technique to infer stress fields in a 3D matrix from nonlinear microrheology measurements with optical tweezers. Using NSIM and simulations, we reveal large long-ranged cell-generated stresses capable of buckling filaments in the matrix. These stresses give rise to the large spatial extent of the observed cell-induced matrix stiffness gradient, which can provide a mechanism for mechanical communication between cells. [ABSTRACT FROM AUTHOR]

Intestinal epithelial cell differentiation is a highly controlled and orderly process occurring in the crypt so that cells migrating out to cover the villi are already fully functional. Absorptive cell precursors, which originate from the stem cell population located in the lower third of the crypt, are subject to several cycles of amplification in the transit amplifying (TA) zone, before reaching the terminal differentiation compartment located in the upper third. There is a large body of evidence that absorptive cell differentiation is halted in the TA zone through various epigenetic, transcriptional and intracellular signalling events or mechanisms allowing the transient expansion of this cell population but how these mechanisms are themself regulated remains obscure. One clue can be found in the epithelial cell-matrix microenvironment located all along the crypt-villus axis. Indeed, a previous study from our group revealed that α5-subunit containing laminins such as lamimin-511 and 512 inhibit early stages of differentiation in Caco-2/15 cells. Among potential receptors for laminin 511/512 is the integrin α7β1, which has previously been reported to be expressed in the human intestinal crypts and in early stages of Caco-2/15 cell differentiation. In this study, the effects of knocking down ITGA7 in Caco-2/15 cells were studied using shRNA and CRISPR/Cas9 strategies. Abolition of the α7 integrin subunit resulted in a significant increase in the level of differentiation and polarization markers as well as the morphological features of intestinal cells. Activities of focal adhesion kinase and Src kinase were both reduced in α7-knockdown cells while the three major intestinal pro-differentiation factors CDX2, HNFα1 and HNF4α were overexpressed. Two epigenetic events associated with intestinal differentiation, the reduction of tri-methylated lysine 27 on histone H3 and the increase of acetylation of histone H4 were also observed in α7-knockdown cells. On the other hand, the ablation of α7 had no effect on cell proliferation. In conclusion, these data indicate that integrin α7β1 acts as a major repressor of absorptive cell terminal differentiation in the Caco-2/15 cell model and suggest that the laminin-α7β1 integrin interaction occurring in the transit amplifying zone of the adult intestine is involved in the transient halting of absorptive cell terminal differentiation. [Display omitted] • Integrin α7Bβ1 is detected in the transit amplification zone of the intestinal crypt. • α7Bβ1 acts as a major repressor of differentiation in the Caco-2/15 cell model. • ITGA7 abolition reduces focal adhesion kinase and Src phosphorylation. • ITGA7 abolition increases expression of CDX2, HNFα1 and HNF4α. • ITGA7 knockout has no effect on cell proliferation. [ABSTRACT FROM AUTHOR]

During the fabrication of tissue engineering scaffolds and subsequent tissue regeneration, surface bioactivity is vital for cell adhesion, spreading, and proliferation, especially for endothelium dysfunction repair. In this paper, synthetic polymer polycaprolactone (PCL) was blended with natural polymer gelatin at four different weight ratios followed by crosslinking (i.e., 100:0, 70:30, 50:50, 30:70, labeled as PCL-C, P7G3-C, P5G5-C, and P3G7-C) to impart enhanced bioactivity and tunable mechanical properties. The PCL/gelatin blends were first dissolved in 2,2,2-trifluroethanol (TFE) and supplementary acetic acid (1% relative to TFE) solvent, electrospun, and then cross-linked to produce PBS-proof fibrous scaffolds. Scanning electron micrographs (SEM) indicated that fibers of each sample were smooth and homogeneous, with the fiber diameters increasing from 1.01 ± 0.51 μm to 1.61 ± 0.46 μm as the content of gelatin increased. While thermal resistance and crystallization of the blends were affected by the presence of gelatin, as reflected by differential scanning calorimetry (DSC) results, water contact angle (WCA) tests confirmed that the scaffold surfaces became more hydrophilic. Tensile tests showed that PCL-C and P7G3-C scaffolds had mechanical properties comparable to those of human coronary arteries. As for cytocompatibility, skeleton staining images showed that human mesenchymal stem cells (hMSCs) had more favorable binding sites on PCL/gelatin scaffolds than those on PCL scaffolds. Cell proliferation assays revealed that P7G3-C scaffolds could support the most number of hMSCs. The results of this study demonstrated the enhanced cell-matrix interactions and potential use of electrospun PCL/gelatin scaffolds in the tissue engineering field, especially in wound dressings and endothelium regeneration. [ABSTRACT FROM AUTHOR]

The outcome of tissue engineered organ transplants depends on the capacity of the biomaterial to promote a pro-healing response once implanted in vivo. Multiple studies, including ours, have demonstrated the possibility of using the extracellular matrix (ECM) of animal organs as platform for tissue engineering and more recently, discarded human organs have also been proposed as scaffold source. In contrast to artificial biomaterials, natural ECM has the advantage of undergoing continuous remodeling which allows adaptation to diverse conditions. It is known that natural matrices present diverse immune properties when compared to artificial biomaterials. However, how these properties compare between diseased and healthy ECM and artificial scaffolds has not yet been defined. To answer this question, we used decellularized renal ECM derived from WT mice and from mice affected by Alport Syndrome at different time-points of disease progression as a model of renal failure with extensive fibrosis. We characterized the morphology and composition of these ECMs and compared their in vitro effects on macrophage activation with that of synthetic scaffolds commonly used in the clinic (collagen type I and poly-L-(lactic) acid, PLLA). We showed that ECM derived from Alport kidneys differed in fibrous protein deposition and cytokine content when compared to ECM derived from WT kidneys. Yet, both WT and Alport renal ECM induced macrophage differentiation mainly towards a reparative (M2) phenotype, while artificial biomaterials towards an inflammatory (M1) phenotype. Anti-inflammatory properties of natural ECMs were lost when homogenized, hence three-dimensional structure of ECM seems crucial for generating an anti-inflammatory response. Together, these data support the notion that natural ECM, even if derived from diseased kidneys promote a M2 protolerogenic macrophage polarization, thus providing novel insights on the applicability of ECM obtained from discarded organs as ideal scaffold for tissue engineering. [ABSTRACT FROM AUTHOR]

In the past decade, significant advances have been made in the design and optimization of novel biomaterials and microfabrication techniques to generate vascularized tissues. Novel microfluidic systems have facilitated the development and optimization of in vitro models for exploring the complex pathophysiological phenomena that occur inside a microvascular environment. To date, most of these models have focused on engineering of increasingly complex systems, rather than analyzing the molecular and cellular mechanisms that drive microvascular network morphogenesis and remodeling. In fact, mutual interactions among endothelial cells (ECs), supporting mural cells and organ-specific cells, as well as between ECs and the extracellular matrix, are key driving forces for vascularization. This review focuses on the integration of materials science, microengineering and vascular biology for the development of in vitro microvascular systems. Various approaches currently being applied to study cell-cell/cell-matrix interactions, as well as biochemical/biophysical cues promoting vascularization and their impact on microvascular network formation, will be identified and discussed. Finally, this review will explore in vitro applications of microvascular systems, in vivo integration of transplanted vascularized tissues, and the important challenges for vascularization and controlling the microcirculatory system within the engineered tissues, especially for microfabrication approaches. It is likely that existing models and more complex models will further our understanding of the key elements of vascular network growth, stabilization and remodeling to translate basic research principles into functional, vascularized tissue constructs for regenerative medicine applications, drug screening and disease models. [ABSTRACT FROM AUTHOR]

β1,4 Galactosyltransferase-I (GalT-I) is expressed as two nearly identical polypeptides that differ only in the length of their cytoplasmic domains. The longer isoform has been implicated as a cell surface receptor for extracellular glycoside ligands, such as laminin. To more stringently test the function of the long GalT-I isoform during cell interactions with laminin, we created multiple independent fibroblastic cell lines that fail to express the long isoform, but which express the short GalT-I isoform normally and appear to have normal intracellular galactosylation. Cells devoid of the long GalT-I isoform are unable to adhere and spread on laminin substrates as well as control cells, but retain near normal interactions with fibronectin, which do not rely upon surface GalT-I function. The loss of the long GalT-I isoform also leads to a loss of actin stress fibers, focal adhesions and rac GTPase activation. [ABSTRACT FROM AUTHOR]

Lysyl oxidase (LOX) catalyzes crosslink formation between fibrillar collagens and elastins and an increase in LOX activity has been associated with cardiac fibrosis following myocardial infarction (MI). It has been previously reported that LOX expression is regulated by growth factors and cytokines including transforming growth factor (TGF-β1); however, it is unclear how the biophysical and biochemical properties of the cellular microenvironment affect LOX expression. In this study, we isolated rat cardiac fibroblasts (CF) and infarct cardiac fibroblasts (ICF), from healthy and 1-week post-MI left ventricular tissue respectively, and cultured them under varied substrate conditions in vitro to assess their influence on LOX expression. Culture of ICF on collagen I-coated plates increased LOX expression versus uncoated plates with an additional increase observed with the presence of TGF-β1. To further investigate the effect of integrin interactions with collagen I on LOX expression, we inhibited the α2β1 integrin from binding to collagen I and found gene and protein expression of LOX to be downregulated. Together, this demonstrates that the interaction of α2β1 integrin to collagen I in the cellular microenvironment can regulate expression of LOX. Further studies investigating additional integrin interactions may identify therapeutic targets for treating cardiac fibrosis. [ABSTRACT FROM AUTHOR]

Migration of cells is integral in various physiological processes in all facets of life. These range from embryonic development, morphogenesis, and wound healing, to disease pathology such as cancer metastasis. While cell migratory behavior has been traditionally studied using simple assays on culture dishes, in recent years it has been increasingly realized that the physical, mechanical, and chemical aspects of the matrix are key determinants of the migration mechanism. In this paper, we will describe the mechanobiological changes that accompany the dynamic cell–matrix interactions during cell migration. Furthermore, we will review what is to date known about how these changes feed back to the dynamics and biomechanical properties of the cell and the matrix. Elucidating the role of these intimate cell–matrix interactions will provide not only a better multi-scale understanding of cell motility in its physiological context, but also a more holistic perspective for designing approaches to regulate cell behavior. [ABSTRACT FROM AUTHOR]

Reproductive biologists are well-versed in many types of biochemical signaling, and indeed, there are almost innumerable examples in reproduction, including steroid and peptide hormone signaling, receptor-ligand and secondary messenger-mediated signaling, signaling regulated by membrane channels, and many others. Among reproductive scientists, a perhaps lesser-known but comparably important mode of signaling is mechanotransduction: the concept that cells can sense and respond to externally applied or internally generated mechanical forces. Given the cell shape changes and tissue morphogenesis events that are components of many phenomena in reproductive function, it should be no surprise that mechanotransduction has major impacts in reproductive health and pathophysiology. The conference on “Mechanotransduction in the Reproductive Tract” was a valuable launch pad to bring this hot issue in development, cell biology, biophysics, and tissue regeneration to the realm of reproductive biology. The goal of the meeting was to stimulate interest and increased mechanotransduction research in the reproductive field by presenting a broad spectrum of responses impacted by this process. The meeting highlighted the importance of convening expert investigators, students, fellows, and young investigators from a number of research areas resulting in cross-fertilization of ideas and suggested new avenues for study. The conference included talks on tissue engineering, stem cells, and several areas of reproductive biology, from uterus and cervix to the gametes. Specific reproductive health-relevant areas, including uterine fibroids, gestation and parturition, and breast tissue morphogenesis, received particular attention. [ABSTRACT FROM PUBLISHER]

Extracellular matrix is composed of a complex array of molecules that together provide structural and functional support to cells. These properties are mainly mediated by the activity of collagenous and elastic fibers, proteoglycans, and proteins such as fibronectin and laminin. ECM composition is tissue-specific and could include matricellular proteins whose primary role is to modulate cell–matrix interactions. In adults, matricellular proteins are primarily expressed during injury, inflammation and disease. Particularly, they are closely associated with the progression and prognosis of cardiovascular and fibrotic diseases, and cancer. This review aims to provide an overview of the potential use of matricellular proteins in drug delivery including the generation of therapeutic agents based on the properties and structures of these proteins as well as their utility as biomarkers for specific diseases. [ABSTRACT FROM AUTHOR]

Biomaterials-based three dimensional scaffolds with tunable elasticity hold promise in replacing failed organs resulting from injuries, aging, and diseases by providing a suitable cellular microenvironment to facilitate regeneration of damaged tissues. However, controlled presentation of biological signals with tunable tissue mechanics and architecture remain a bottleneck that needs to be addressed to engineer functional artificial tissues. Nanocomposite hydrogels that promote cells adhesion and demonstrate tunable viscoelastic properties could mimic key properties and structures of native tissue. We have developed elastomeric fiber shaped cellular constructs from poly(ethylene glycol) diacrylate, silicate nanoparticles, and gelatin methacrylate via ionic and covalent crosslinking. By controlling the interactions between nanoparticles and polymers, nanocomposite hydrogels with tunable mechanical and degradation properties are fabricated. By encapsulating multiple cell types in these cellular constructs, we demonstrate materials-based control of cell spreading, survival, and proliferation. As a proof-of-concept, we assembled the hydrogel microfibers to obtain multicellular elastomeric tissue constructs. These elastic microfibers may serve as model systems to explore the effect of mechanical stress on cell-matrix interactions. Moreover, such elastomeric hydrogel fibers can be used to engineer scaffold structures, fabric sheets, bundles, or as building blocks for 3D tissue construction. [ABSTRACT FROM AUTHOR]

During angiogenesis, sprouting microvessels interact with the extracellular matrix (ECM) by degrading and reorganizing the matrix, applying traction forces, and producing deformation. Morphometric features of the resulting microvascular network are affected by the interaction between the matrix and angiogenic microvessels. The objective of this study was to develop a continuous-discrete modeling approach to simulate mechanical interactions between growing neovessels and the deformation of the matrix in vitro. This was accomplished by coupling an existing angiogenesis growth model which uses properties of the ECM to regulate angiogenic growth with the nonlinear finite element software FEBio (). FEBio solves for the deformation and remodeling of the matrix caused by active stress generated by neovessel sprouts, and this deformation was used to update the ECM into the current configuration. After mesh resolution and parameter sensitivity studies, the model was used to accurately predict vascular alignment for various matrix boundary conditions. Alignment primarily arises passively as microvessels convect with the deformation of the matrix, but active alignment along collagen fibrils plays a role as well. Predictions of alignment were most sensitive to the range over which active stresses were applied and the viscoelastic time constant in the material model. The computational framework provides a flexible platform for interpreting in vitro investigations of vessel-matrix interactions, predicting new experiments, and simulating conditions that are outside current experimental capabilities. [ABSTRACT FROM AUTHOR]

Background: Claudins are a family of tight junction (TJ) membrane proteins involved in a broad spectrum of human diseases including cancer. Claudin-7 is a unique TJ membrane protein in that it has a strong basolateral membrane distribution in epithelial cells and in tissues. Therefore, this study aims to investigate the functional significance of this non-TJ localization of claudin-7 in human lung cancer cells. Methods: Claudin-7 expression was suppressed or deleted by lentivirus shRNA or by targeted-gene deletion. Cell cycle analysis and antibody blocking methods were employed to assay cell proliferation and cell attachment, respectively. Electron microscopy and transepthelial electrical resistance measurement were performed to examine the TJ ultrastructure and barrier function. Co-immunolocalization and co-immunoprecipitation was used to study claudin-7 interaction with integrin β1. Tumor growth in vivo were analyzed using athymic nude mice. Results: Claudin-7 co-localizes and forms a stable complex with integrin β1. Both suppressing claudin-7 expression by lentivirus shRNA in human lung cancer cells (KD cells) and deletion of claudin-7 in mouse lungs lead to the reduction in integrin β1 and phospho-FAK levels. Suppressing claudin-7 expression increases cell growth and cell cycle progression. More significantly, claudin-7 KD cells have severe defects in cell-matrix interactions and adhere poorly to culture plates with a remarkably reduced integrin β1 expression. When cultured on uncoated glass coverslips, claudin-7 KD cells grow on top of each other and form spheroids while the control cells adhere well and grow as a monolayer. Reintroducing claudin-7 reduces cell proliferation, upregulates integrin β1 expression and increases cell-matrix adhesion. Integrin β1 transfection partially rescues the cell attachment defect. When inoculated into nude mice, claudin-7 KD cells produced significantly larger tumors than control cells. Conclusion: In this study, we identified a previously unrecognized function of claudin-7 in regulating cell proliferation and maintaining epithelial cell attachment through engaging integrin β1. [ABSTRACT FROM AUTHOR]

Background: Claudins are a family of tight junction (TJ) membrane proteins involved in a broad spectrum of human diseases including cancer. Claudin-7 is a unique TJ membrane protein in that it has a strong basolateral membrane distribution in epithelial cells and in tissues. Therefore, this study aims to investigate the functional significance of this non-TJ localization of claudin-7 in human lung cancer cells. Methods: Claudin-7 expression was suppressed or deleted by lentivirus shRNA or by targeted-gene deletion. Cell cycle analysis and antibody blocking methods were employed to assay cell proliferation and cell attachment, respectively. Electron microscopy and transepthelial electrical resistance measurement were performed to examine the TJ ultrastructure and barrier function. Co-immunolocalization and co-immunoprecipitation was used to study claudin-7 interaction with integrin β 1. Tumor growth in vivo were analyzed using athymic nude mice. Results: Claudin-7 co-localizes and forms a stable complex with integrin β 1. Both suppressing claudin-7 expression by lentivirus shRNA in human lung cancer cells (KD cells) and deletion of claudin-7 in mouse lungs lead to the reduction in integrin β 1 and phospho-FAK levels. Suppressing claudin-7 expression increases cell growth and cell cycle progression. More significantly, claudin-7 KD cells have severe defects in cell-matrix interactions and adhere poorly to culture plates with a remarkably reduced integrin β 1 expression. When cultured on uncoated glass coverslips, claudin-7 KD cells grow on top of each other and form spheroids while the control cells adhere well and grow as a monolayer. Reintroducing claudin-7 reduces cell proliferation, upregulates integrin β 1 expression and increases cell-matrix adhesion. Integrin β 1 transfection partially rescues the cell attachment defect. When inoculated into nude mice, claudin-7 KD cells produced significantly larger tumors than control cells. Conclusion: In this study, we identified a previously unrecognized function of claudin-7 in regulating cell proliferation and maintaining epithelial cell attachment through engaging integrin β 1. [ABSTRACT FROM AUTHOR]

Cell-matrix adhesions provide structural stability to the tissue and regulate intracellular signalling pathways that are important for cell fate decisions of the different cell populations within the skin. As a consequence of these central functions, genetic or functional impairment of various key protein components of matrix adhesions plays a causative role in the aetiology or pathophysiology in a large variety of skin disorders. Research towards understanding the molecular composition of these adhesions as well as the mechanisms by which they transmit signals is therefore of obvious importance. In this essay, we discuss the roles of integrin-linked kinase, a key component of cell-matrix adhesions, in the (patho)physiology of skin and in particular highlight its role in regulating mechanical tension and matrix remodelling both in the epidermis and in the dermis. [ABSTRACT FROM AUTHOR]

Objectives We examined whether mouse embryonic stem ( ES) cells can differentiate into odontoblast-like cells without epithelial-mesenchymal interaction. Materials and methods Cells were cultured by the 'hanging drop' method using a collagen type-I scaffold (CS) combined with bone morphogenetic protein (BMP)-4 (CS/BMP-4). Expression of odontoblast-related mRNA and protein, and cell proliferation were performed by reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence staining and WST-1 assay, respectively. Results Cells potently expressed odontoblast-related cell marker mRNAs following induction of odontoblastic differentiation. Dentin sialophosphoprotein, a marker of mature odontoblasts, was strongly expressed in differentiated ES cells. The cells also acquired an odontoblast-like functional phenotype, as evidenced by the appearance of alkaline phosphatase activity and calcification. The cell-surface expression of α2, α6, αV and αVβ3 integrin proteins was rapidly upregulated in differentiated cells. Finally, anti-α2 integrin antibody suppressed the expression of odontoblastic markers in cells grown using this culture system, suggesting that α2 integrin expression in ES cells triggers their differentiation into odontoblast-like cells. Conclusions Mouse ES cells cultured by the 'hanging drop' method are able to differentiate into cells with odontoblast-specific physiological functions and cell-surface integrin protein expression. [ABSTRACT FROM AUTHOR]

Cell sheet engineering has enabled the production of confluent cell sheets stacked together for use as a cardiac patch to increase cell survival rate and engraftment after transplantation, thereby providing a promising strategy for high density stem cell delivery for cardiac repair. One key challenge in using cell sheet technology is the difficulty of cell sheet handling due to its weak mechanical properties. A single-layer cell sheet is generally very fragile and tends to break or clump during harvest. Effective transfer and stacking methods are needed to move cell sheet technology into widespread clinical applications. In this study, we developed a simple and effective micropipette based method to aid cell sheet transfer and stacking. The cell viability after transfer was tested and multi-layer stem cell sheets were fabricated using the developed method. Furthermore, we examined the interactions between stacked stem cell sheets and fibrin matrix. Our results have shown that the preserved ECM associated with the detached cell sheet greatly facilitates its adherence to fibrin matrix and enhances the cell sheet-matrix interactions. Accelerated fibrin degradation caused by attached cell sheets was also observed. [ABSTRACT FROM AUTHOR]

Abstract: Capillary morphogenesis is a multistage, multicellular activity that plays a pivotal role in various developmental and pathological situations. In-depth understanding of the regulatory mechanism along with the capability of controlling the morphogenic process will have direct implications on tissue engineering and therapeutic angiogenesis. Extensive research has been devoted to elucidate the biochemical factors that regulate capillary morphogenesis. The roles of geometric confinement and cell-matrix mechanical interactions on the capillary architecture, nevertheless, remain largely unknown. Here, we show geometric control of endothelial network topology by creating physical confinements with microfabricated fences and wells. Decreasing the thickness of the matrix also results in comparable modulation of the network architecture, supporting the boundary effect is mediated mechanically. The regulatory role of cell-matrix mechanical interaction on the network topology is further supported by alternating the matrix stiffness by a cell-inert PEG-dextran hydrogel. Furthermore, reducing the cell traction force with a Rho-associated protein kinase inhibitor diminishes the boundary effect. Computational biomechanical analysis delineates the relationship between geometric confinement and cell-matrix mechanical interaction. Collectively, these results reveal a mechanoregulation scheme of endothelial cells to regulate the capillary network architecture via cell-matrix mechanical interactions. [Copyright &y& Elsevier]

Abstract: Cell–cell and cell–matrix interactions are important events in directing stem cell chondrogenesis, which can be promoted in matrix microenvironments presenting appropriate ligands. In this study, interfacial polyelectrolyte complexation (IPC) based hydrogels were employed, wherein the unique formation of submicron size fibers facilitated spatial orientation of ligands within such hydrogels. The influence of aligned, collagen type I (Col I) presentation in IPC hydrogel on chondrogenic differentiation of human mesenchymal stem cells (MSC) was investigated. Early morphological dynamics, onset of N-cadherin/β-catenin mediated chondrogenic induction and differentiation were compared between MSCs encapsulated in IPC-Col I and IPC-control (without Col I) hydrogels, and a conventional Col I hydrogel. MSCs in IPC-Col I hydrogel aligned and packed uniformly, resulting in enhanced cell–cell interactions and cellular condensation, facilitating superior chondrogenesis and the generation of mature hyaline neocartilage, with notable downregulation of fibrocartilaginous marker. Inhibition study using function blocking β1-integrin antibodies reversed the aforementioned outcomes, indicating the importance of coupling integrin mediated cell–matrix interactions and N-cadherin/β-catenin mediated downstream signaling events. This study demonstrated the significance of oriented ligand presentation for MSC chondrogenesis, and the importance of facilitating an orderly sequence of differentiation events, initiated by proximal interactions between MSCs and the extracellular matrix for robust neocartilage formation. [Copyright &y& Elsevier]

Although the link between altered stem cell properties and tissue aging has been recognized, the molecular and cellular processes of tendon aging have not been elucidated. As tendons contain stem/progenitor cells ( TSPC), we investigated whether the molecular and cellular attributes of TSPC alter during tendon aging and degeneration. Comparing TSPC derived from young/healthy (Y- TSPC) and aged/degenerated human Achilles tendon biopsies (A- TSPC), we observed that A- TSPC exhibit a profound self-renewal and clonogenic deficits, while their multipotency was still retained. Senescence analysis showed a premature entry into senescence of the A- TSPC, a finding accompanied by an upregulation of p16INK4A. To identify age-related molecular factors, we performed microarray and gene ontology analyses. These analyses revealed an intriguing transcriptomal shift in A- TSPC, where the most differentially expressed probesets encode for genes regulating cell adhesion, migration, and actin cytoskeleton. Time-lapse analysis showed that A- TSPC exhibit decelerated motion and delayed wound closure concomitant to a higher actin stress fiber content and a slower turnover of actin filaments. Lastly, based on the expression analyses of microarray candidates, we suggest that dysregulated cell-matrix interactions and the ROCK kinase pathway might be key players in TSPC aging. Taken together, we propose that during tendon aging and degeneration, the TSPC pool is becoming exhausted in terms of size and functional fitness. Thus, our study provides the first fundamental basis for further exploration into the molecular mechanisms behind tendon aging and degeneration as well as for the selection of novel tendon-specific therapeutical targets. [ABSTRACT FROM AUTHOR]

Abstract: Background and purpose: Extracellular matrix (ECM) reorganization critically contributes to breast cancer (BC) progression and radiotherapy response. We investigated the molecular background and functional consequences of collagen type I (col-I) reorganization by irradiated breast cancer cells (BCC). Materials and methods: Radiation-induced (RI) col-I reorganization was evaluated for MCF-7/6, MCF-7/AZ, T47D and SK-BR-3 BCC. Phase-contrast microscopy and a stressed matrix contraction assay were used for visualization and quantification of col-I reorganization. Cell–matrix interactions were assessed by the inhibition of β1 integrin (neutralizing antibody ‘P5D2’) or focal adhesion kinase (FAK; GSK22560098 small molecule kinase inhibitor). The role of the actomyosin cytoskeleton was explored by western blotting analysis of myosin II expression and activity; and by gene silencing of myosin IIA and pharmacological inhibition of the actomyosin system (blebbistatin, cytochalasin D). BCC death was evaluated by propidium iodide staining. Results: We observed a radiation dose-dependent increase of col-I reorganization by BCC. β1 Integrin/FAK-mediated cell–matrix interactions are essential for RI col-I reorganization. Irradiated BCC are characterized by increased myosin IIA expression and myosin IIA-dependent col-I reorganization. Moreover, RI col-I reorganization by BCC is associated with decreased BCC death, as suggested by pharmacological targeting of the β1 integrin/FAK/myosin IIA pathway. Conclusions: Our data indicate the role of myosin IIA in col-I reorganization by irradiated BCC and reciprocal BCC death. [Copyright &y& Elsevier]

In addition to their obvious biological roles in tissue function, cells often play a significant mechanical role through a combination of passive and active behaviors. This study focused on the passive mechanical contribution of cells in tissues by improving our multiscale model via the addition of cells, which were treated as dilute spherical inclusions. The first set of simulations considered a rigid cell, with the surrounding ECM modeled as (1) linear elastic, (2) Neo-Hookean, and (3) a fiber network. Comparison with the classical composite theory for rigid inclusions showed close agreement at low cell volume fraction. The fiber network case exhibited nonlinear stress-strain behavior and Poisson's ratios larger than the elastic limit of O.5, characteristics similar to those of biological tissues. The second set of simulations used a fiber network for both the cell (simulating cytoskeletal filaments) and matrix, and investigated the effect of varying relative stiffness between the cell and matrix, as well as the effect of a cytoplasmic pressure to enforce incompressibility of the cell. Results showed that the ECM network exerted negligible compression on the cell, even when the stiffness of fibers in the network was increased relative to the cell. Introduction of a cytoplasmic pressure significantly increased the stresses in the cell filament network, and altered how the cell changed its shape under tension. Findings from this study have implications on understanding how cells interact with their surrounding ECM, as well as in the context of mechanosensation. [ABSTRACT FROM AUTHOR]

Small leucine-rich proteoglycans ( SLRPs) are involved in a variety of biological and pathological processes. This review focuses on their regulatory roles in matrix assembly. SLRPs have protein cores and hypervariable glycosylation with multivalent binding abilities. During development, differential interactions of SLRPs with other molecules result in tissue-specific spatial and temporal distributions. The changing expression patterns play a critical role in the regulation of tissue-specific matrix assembly and therefore tissue function. SLRPs play significant structural roles within extracellular matrices. In addition, they play regulatory roles in collagen fibril growth, fibril organization and extracellular matrix assembly. Moreover, they are involved in mediating cell-matrix interactions. Abnormal SLRP expression and/or structures result in dysfunctional extracellular matrices and pathophysiology. Altered expression of SLRPs has been found in many disease models, and structural deficiency also causes altered matrix assembly. SLRPs regulate assembly of the extracellular matrix, which defines the microenvironment, modulating both the extracellular matrix and cellular functions, with an impact on tissue function. [ABSTRACT FROM AUTHOR]

Laminins are a large family of conserved, multidomain trimeric basement membrane proteins that contribute to the structure of extracellular matrix and influence the behavior of associated cells, such as adhesion, differentiation, migration, phenotype stability, and resistance to anoikis. In lower organisms such as Hydra there is only one isoform of laminin, but higher organisms have at least 16 trimeric isoforms with varying degrees of cell/tissue specificity. In vitro protein and cell culture studies, gene manipulation in animals, and laminin gene mutations in human diseases have provided insight into the specific functions of some laminins, but the biological roles of many isoforms are still largely unexplored, mainly owing to difficulties in isolating them in pure form from tissues or cells. In this review, we elucidate the evolution of laminins, describe their molecular complexity, and explore the current knowledge of their diversity and functional aspects, including laminin-mediated signaling via membrane receptors, in vitro cell biology, and involvement in various tissues gained from animal model and human disease studies. The potential use of laminins in cell biology research and biotechnology is discussed. [ABSTRACT FROM AUTHOR]

Abstract: During decades preceding and following the last war, a favourite subject of biochemists was to study glycoproteins. One class of these substances, found in connective tissues were characterised as polysaccharides, most of them found to be linked to proteins, designated later as glycosaminoglycans and proteoglycans. Another family of glycoconjugates represented epithelial mucins as found in the gastro-intestinal and respiratory tracts and conduits. A third family of glycoconjugates is represented by circulating glycoproteins isolated from the blood plasma, mostly studied by medical biochemists in relation to pathological conditions comprising those increasing during the inflammatory reaction: acute phase glycoproteins. Their study suggested that they might be derived from connective tissues. Although inflammatory glycoproteins derive mostly from the liver, the possibility of connective tissue origin of glycoproteins remained open. Using cornea, an avascular tissue, we could show that connective tissues also synthesize glycoproteins. We proposed to designate them “structural glycoproteins” (SGP-s) to distinguish them from circulating, blood-born glycoproteins coming from the liver. They play locally “structural” roles in connective tissues where they are synthesized. Soon after fibronectin was identified and shown to mediate cell-matrix interactions. A large family of glycoproteins were then isolated from a variety of sources, cells, tissues others than liver, confirming our original hypothesis. The first experiments on these glycoproteins were published from 1961/1962 giving the opportunity to recapitulate this biochemical adventure 50 years later, together with the celebration of the foundation of the first connective tissue society in Europe, as described in the first article in this issue. [Copyright &y& Elsevier]

Abstract: The discovery that adherent tissue cells actively sustain internal tension and exert mechanical forces on their surroundings has opened new vistas in the field of cell and tissue mechanics. Cellular forces, generated by acto-myosin contractility, play a central role in numerous aspects of cell behavior and function. Apart from the various specific functions that cells perform by applying forces (e.g., wound healing, remodeling of the extracellular matrix and muscle contraction), cells also apply stresses as a generic means for sensing and responding to the mechanical nature of their environment. In addition, the internal tension plays a role in actively controlling the elastic moduli and shape stability of the cell. In this review, we survey recent theoretical and experimental studies of the physical consequences of cell mechanical activity including its role in cell morphology, adhesion strength, stress-fiber polarization, and the elastic properties of cells. We also discuss the role of cell mechanics in orienting cellular assemblies and in the response of cells to external loads. [Copyright &y& Elsevier]

The anti-cancer drug, emodin, can cause significant mechanobiological changes in human cancer cells, due to the interplay of anti-cell adhesion, actin-disrupting and apoptosis-causing effects of the drug. Here, we used micropillars to monitor the changes in traction forces induced by breast cancer cells in response to this drug stimulus. Our results showed that the variations in the cell traction forces reflected changes in the cytoskeleton integrity and cell behavior. Cell traction forces were found to increase during cell rounding and decrease during the onset of apoptosis and cytoskeleton disruption. These results can provide useful insights into the chemo-mechanical relationships arising from the cell-matrix interactions and can potentially help in the quantitative assessment of drug efficacy. [ABSTRACT FROM AUTHOR]

Podocytes adhere to the glomerular basement membrane by cell surface receptors. Since in other cells these adhesions are enhanced by cell surface proteoglycans, we examined the contribution of these molecules and their glycosaminoglycan side chains to podocyte adhesion by developing immortalized podocyte cell lines with (control) or without (mutant) heparan sulfate glycosaminoglycan chains. In adhesion assays control podocytes attached, spread, and migrated more efficiently compared with mutants, indicating a requirement for heparan sulfate chains in these processes. The proteoglycan syndecan-4 is known to have direct effects on cell attachment, spreading, and cytoskeletal organization. We found it localized to focal adhesions in control podocytes coincident with stress fiber formation. In mutant cells, syndecan-4 was associated with smaller focal contacts and cortical actin organization. Analysis by flow cytometry showed that mutant cells had twice the amount of surface syndecan-4 of control cells. Protein kinase Cα, a signaling molecule bound to and activated by syndecan-4, showed a fourfold increase in membrane localization-activation than that seen in control cells. In vivo, the loss of heparan sulfate glycosaminoglycans in PEXTKO mice led to a loss of glomerular syndecan-4. Overall, our study provides further evidence for a dynamic role of cell surface heparan sulfate glycosaminoglycans in podocyte activity. [ABSTRACT FROM AUTHOR]

Abstract: The effect of different collagen and cell concentrations on the mechanical and remodeling behaviors of corneal stroma wound healing models consisting of collagen hydrogels seeded with human corneal fibroblasts during a 25 day culture period were examined. Human corneal fibroblasts were seeded at 1 × 105, 3 × 105 or 5 × 105 cells per hydrogel, and collagen concentrations of 2.5 mg/ml, 3.5 mg/ml or 4.5 mg/ml were examined. Two non-destructive techniques, spherical indentation and optical coherence tomography, were used to measure the elastic modulus and dimensional changes respectively at several time-points over the culture period. The elastic modulus of the hydrogels increased continuously over 25 days. Hydrogels with higher initial cell seeding densities and lower initial collagen concentrations were found to increase in elastic modulus faster and possessed a higher elastic modulus by the end of the culture period when compared to the other hydrogels. A mathematical equation was applied to accurately fit the change in elastic modulus over time. This study demonstrates a robust in vitro technique able to monitor the effect of different parameters on the cell–matrix mechanical relationship in a corneal stroma model during prolonged culture periods and enhances our understanding on corneal wound healing processes. [ABSTRACT FROM AUTHOR]

Adhesion of mammalian cells to in vitro surfaces is an area of active research and it attracts considerable interest from various scientific disciplines, most notably from medical technology and biotechnology. One important issue in the context of cell-surface adhesion is the time course of attachment and spreading upon surfaces that are decorated with proteins to make them cytocompatible. This article reviews two emerging non-microscopic techniques capable of monitoring the adhesion process label-free and in real-time. Both approaches, electric cell-substrate impedance sensing (ECIS) and the quartz crystal microbalance (QCM), are based on substrate-integrated transducers that transduce cellular adhesion into an electrical signal. A short introduction of both techniques is followed by a set of examples that illustrate the performance of these sensors, their individual merits and limitations. In order to analyze the integral and complex signals of both sensors in contact with mammalian cells in more detail, we also studied their individual readouts during the adsorption of liposomes with well-defined structure and chemical composition. [ABSTRACT FROM AUTHOR]

Glomerulosclerosis is characterized by the loss of glomerular cells by apoptosis and deposition of collagen type I into the normal collagen IV-containing mesangial matrix. We sought to determine the alterations that might contribute to these changes by performing proteomic analysis of rat mesangial cell lysates comparing cells cultured on normal collagen type IV to those grown on abnormal collagen type I surfaces. Subculture on collagen type I was associated with changed expression of several proteins, including a significant upregulation of the paxillin-like LIM protein, hydrogen-peroxide-induced clone 5 (Hic-5), and increased the susceptibility of the cells to apoptosis in response to physiological triggers. When we knocked down Hic-5 (using siRNA), we found mesangial cells grown on collagen type I were protected from apoptosis to the same degree as untreated cells grown on collagen type IV. Further we found that the level of Hic-5 in vivo was almost undetectable in control rats but increased dramatically in the glomerular mesangium of remnant kidneys 90 and 120 days after subtotal nephrectomy. This induction of Hic-5 paralleled the upregulation of mesangial collagen type I expression and glomerular cell apoptosis. Our results suggest that Hic-5 is pivotal in mediating the response of mesangial cells to attachment on abnormal extracellular matrix during glomerular scarring. [ABSTRACT FROM AUTHOR]

Abstract: Prenatal inflammation prevents normal lung morphogenesis and leads to bronchopulmonary dysplasia (BPD), a common complication of preterm birth. We previously demonstrated in a bacterial endotoxin mouse model of BPD that disrupting fibronectin localization in the fetal lung mesenchyme causes arrested saccular airway branching. In this study we show that expression of the fibronectin receptor, integrin α8β1 is decreased in the lung mesenchyme in the same inflammation model suggesting it is required for normal lung development. We verified a role for integrin α8β1 in lung development using integrin α8-null mice, which develop fusion of the medial and caudal lobes as well as abnormalities in airway division. We further show in vivo and in vitro that α8-null fetal lung mesenchymal cells fail to form stable adhesions and have increased migration. Thus we propose that integrin α8β1 plays a critical role in lung morphogenesis by regulating mesenchymal cell adhesion and migration. Furthermore, our data suggest that disruption of the interactions between extracellular matrix and integrin α8β1 may contribute to the pathogenesis of BPD. [Copyright &y& Elsevier]

Abstract: The individual and synergistic effects of extracellular matrix interactions on isolated islet function in culture were investigated within a three-dimensional poly(ethylene glycol) (PEG) hydrogel encapsulation environment. First, we observed similar glucose-stimulated insulin secretion from unencapsulated murine islets and islets photoencapsulated in PEG gels. Then islets were encapsulated in gels containing the basement membrane proteins collagen type IV and laminin, individually and in combination, at a total protein concentration of 100μg/ml, and islet insulin secretion in response to high glucose was measured over time. Specific laminin interactions were investigated via islet encapsulation with adhesive peptide sequences found in laminin as well as via functional blocking of cell surface receptors known to bind laminin. Over 32days, islet interactions with collagen type IV and laminin localized within the three-dimensional extracellular environment contributed to two-fold and four-fold increases in insulin secretion, respectively, relative to islets encapsulated without matrix proteins. Hydrogel compositions containing both matrix proteins and > 75% laminin further increased islet insulin secretion to approximately six-fold that of islets encapsulated in the absence of matrix proteins. Encapsulation with the peptide sequence IKVAV resulted in increased islet insulin secretion, but not to the extent observed in the presence of whole laminin. Increased insulin secretion in the presence of laminin was eliminated when islets were exposed to functionally blocking anti-α6 integrin antibody prior to islet encapsulation with laminin. Our results demonstrate the potential of specific matrix interactions within an islet encapsulation microenvironment to promote encapsulated islet function. [Copyright &y& Elsevier]

Cell-cell and cell-matrix interactions are of utmost importance in the pathogenesis of inflammatory diseases. For example, cell-cell and cell-matrix interactions are crucial for leukocyte homing and recruitment to inflammatory sites. The discovery of the disintegrin and metalloprotease (ADAM) proteins, which have both adhesive and proteolytic activities, raised the question of their involvement in inflammatory processes. More interestingly, the presence of the RGD integrin-binding sequence in the disintegrin domain of ADAM-15 (MDC-15; metar-Odin) highlighted ADAM-15 as a protein particularly involved in cell-cell interactions. These findings therefore prompted authors to investigate the roles of ADAM-15 in inflammatory diseases. Because of the early description of ADAM-15 expression in endothelial cells, work first focused on the roles of ADAM-15 in vascular diseases, and ADAM-15 was found to be associated with atherosclerosis. Other studies also pointed at ADAM-15 as a mediator of rheumatoid arthritis and intestinal inflammation as well as inherent angiogenesis. The roles of ADAM-15 in these diseases appear to involve mechanisms as different as cell-cell interactions, cell-extracellular matrix (ECM) interactions, and shedding activity. Here we review and discuss these recent discoveries pointing to ADAM-15 as a mediator of mechanisms underlying inflammation and as a possible therapeutic target for prevention of inflammatory diseases. [ABSTRACT FROM AUTHOR]

Background/Aims: Because various regions of the mitral valve contain distinctive extracellular matrix enabling the tissues to withstand diverse mechanical environments, we investigated phenotype and matrix production of porcine valvular interstitial cells (VICs) from different regions. Methods: VICswere isolated from the chordae (MCh), the center of the anterior leaflet (AlCtr), and the posterior leaflet free edge (PlFree), then assayed for metabolic, growth, and adhesion rates; collagen and glycosaminoglycan (GAG) production, and phenotype using biochemical assays, flow cytometry, and immunocytochemistry. Results: The AlCtr VICs exhibited the fastest metabolism but slowest growth. PlFree cells grew the fastest, but demonstrated the least smooth muscle α-actin, vimentin, and internal complexity. AlCtr VICs secreted less collagen into the culture medium but more 4-sulfated GAGs than other cells. Adhesion-based separation resulted in altered secretion of sulfated GAGs by MCh and AlCtr cells but not by the PlFree cells. Conclusions: VICs isolated from various regions of the mitral valve demonstrate phenotypic differences in culture, corresponding to the ability of the mitral valve to accommodate the physical stresses or altered hemodynamics that occur with injury or disease. Further understanding of VIC and valve mechanobiology could lead to novel medical or tissue engineering approaches to treat valve diseases. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Cell migration is a process that plays an important role throughout the entire life span. It starts early on during embryogenesis and contributes to shaping our body. Migrating cells are involved in maintaining the integrity of our body, for instance, by defending it against invading pathogens. On the other side, migration of tumor cells may have lethal consequences when tumors spread metastatically. Thus, there is a strong interest in unraveling the cellular mechanisms underlying cell migration. The purpose of this review is to illustrate the functional importance of ion and water channels as part of the cellular migration machinery. Ion and water flow is required for optimal migration, and the inhibition or genetic ablation of channels leads to a marked impairment of migration. We briefly touch cytoskeletal mechanisms of migration as well as cell–matrix interactions. We then present some general principles by which channels can affect cell migration before we discuss each channel group separately. [ABSTRACT FROM AUTHOR]

The development and complete differentiation of salivary glands is a complex process that involves a large number of co-ordinated events. Little is known about the molecular basis for salivary gland development. However, we have reported previously that integrins appear to play a role. Integrins are heterodimeric transmembrane receptors consisting of one α and one β subunit that play a pivotal role in the interaction of cells with the extracellular matrix. Such interactions regulate the organisation of cells of tissues and organs during development as well as cell proliferation and differentiation. Using immunohistochemistry and Western and Northern blot analysis, we mapped the localisation and expression of integrins β1, β3 and β4 in human salivary glands obtained from foetuses ranging from weeks 4–24 of gestation and compared it with adult salivary glands. Integrin β1 first appeared during the canalisation stage and during the differentiation stage. A message first appeared at week 6 of development. The expression of β4 integrin protein and message was observed only in the late stage of differentiation. Integrin β3 was not detected in the developing glands; however, integrins β1, β3 and β4 were all expressed in adult salivary gland tissues. The data suggest that integrins, particularly β1, have a role to play in salivary gland development and differentiation. [ABSTRACT FROM AUTHOR]

Although best known for its role in cholinergic signalling, a substantial body of evidence suggests that acetylcholinesterase (AChE) has multiple biological functions. Previously, we and others identified AChE expression in areas of bone that lacked expression of other neuronal proteins. More specifically, we identified AChE expression at sites of new bone formation suggesting a role for AChE as a bone matrix protein. We have now characterised AChE expression, secretion and adhesive function in osteoblasts. Using Western blot analysis, we identified expression of two AChE species in osteoblastic cells, a major species of 68 kDa and less abundant species of approximately 55 kDa. AChE colocalised with the Golgi apparatus in osteoblastic cells and was identified in osteoblast-conditioned medium. Further analyses revealed differentiation-dependent secretion by osteoblasts, with AChE secretion levels corresponding with alkaline phosphatase activity. AChE expression by osteoblastic cells was also found to be regulated by mechanical strain both in vitro and in vivo. Finally, we investigated the possibility of a functional role for AChE in osteoblast adhesion. Using specific inhibitors, blockade of sites thought to be responsible for AChE adhesive properties caused a concentration-dependent decrease in osteoblastic cell adhesion, suggesting that AChE is involved in regulating cell–matrix interactions in bone. [Copyright &y& Elsevier]

In this study the anti-angiogenic action of a novel non-peptide RGDS-analog named RAM was tested in vitro and in vivo. RAM inhibited FGF-2-induced chemotaxis by 80% in an adhesion-independent way. Further, it induced HUVEC-apoptosis in collagen-seeded HUVEC, indicating that such pro-apoptotic effect was adhesion-independent. In vivo studies revealed that RAM inhibited FGF-2 induced angiogenesis by 60% in the mouse Matrigel-assay and in the chicken-egg chorion-allantoic membrane assay. Finally, RAM was markedly more stable in serum as compared to the template RGDS and after 24h incubation in 100% serum was significantly more active than RGDS. Taken together these results show that RAM exerts anti-chemotactic and pro-apoptotic effects, by an unexpected adhesion-independent mechanism, as we have recently shown for the template RGDS molecule [Blood 103 (2004) 4180], and has in vivo relevant anti-angiogenic properties, with marked stability in serum; therefore, RAM represents a novel promising anti-angiogenic molecule. [Copyright &y& Elsevier]