Substrate Adhesion Regulates Sealing Zone Architecture and Dynamics in Cultured Osteoclasts

The bone-degrading activity of osteoclasts depends on the formation of a cytoskeletal-adhesive super-structure known as the sealing zone (SZ). The SZ is a dynamic structure, consisting of a condensed array of podosomes, the elementary adhesion-mediating structures of osteoclasts, interconnected by F-actin filaments. The molecular composition and structure of the SZ were extensively investigated, yet despite its major importance for bone formation and remodelling, the mechanisms underlying its assembly and dynamics are still poorly understood. Here we determine the relations between matrix adhesiveness and the formation, stability and expansion of the SZ. By growing differentiated osteoclasts on micro-patterned glass substrates, where adhesive areas are separated by non-adhesive PLL-g-PEG barriers, we show that SZ growth and fusion strictly depend on the continuity of substrate adhesiveness, at the micrometer scale. We present a possible model for the role of mechanical forces in SZ formation and reorganization, inspired by the current data

Evaluation of Ant-Inflammatory and Antimicrobial Resorcinarene-Peptides for biomaterial modification

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Designed polymer structures with antifouling-antimicrobial properties

Designing surfaces with antifouling and antimicrobial properties has an important route to solve problems, such as infections and fouling, in healthcare and industrial applications. Recently, there has been considerable interest in developing surfaces with polymers because of their distinct properties, such as their length scale, their processability, low cost, tunable properties, and diverse functionalities. This article reviews the polymer systems developed as antifouling-antimicrobial surface coatings, termed as biopassive and bioactive polymers respectively. Many different types of bioactive and biopassive polymeric systems and their limitations are presented

The study of polarisation in single cells using model cell membranes

The apicobasal polarisation of epithelial cells within an epithelium is critical for its function as a selective barrier. Microenvironmental parameters, including cell-matrix and cell-cell interactions, contribute to the initiation and orientation of this polarity. However, it is often non-trivial to decipher the differential effects of these parameters in a controlled manner using traditional in vitro platforms. A reductionist platform, consisting of E-cadherin coupled onto laterally mobile supported lipid bilayers, was utilised to mimic E-cadherin presentation in the cell membrane. These functionalised bilayers were generated either on flat 2D surfaces or the interior surfaces of round microwells. This platform enabled the study of E-cadherin adhesion and the initiation of polarisation in a controlled environment, where the dimensionality of the microenvironment, type of protein coating and cell shape could be independently studied. A high proportion of single epithelial cells interacted with and clustered cellular E-cadherin in the presence of E-cadherin functionalised bilayers, which was reduced in the presence of integrin-mediated adhesion. The differential response in E-cadherin clustering correlated with the polarisation of E-cadherin and Na,K-ATPase, a reporter for the induction of basolateral polarity. Neither the three-dimensional presentation of E-cadherin nor the cell shape affected E-cadherin clustering or polarisation in single cells. Thus, the mobile presentation of E-cadherin was sufficient to mimic a cell-cell contact and induce basolateral polarisation in single cells

Engineered 3D environments to elucidate the effect of environmental parameters on drug response in cancer

Traditional in vitro models used for the development of anti-cancer drugs are based on the monolayer culture of cells, which has a limited predictivity of in vivo efficacy. A number of cell culture platforms have been developed in recent years to improve predictivity and further to elucidate the mechanisms governing the differing responses observed in vitro versus in vivo. One detrimental aspect of current in vitro models is their inability to decouple the effect of different extrinsic factors on the responsiveness of the cells to drug treatment. Here, we have used an engineered poly(dimethylsiloxane) (PDMS) microwell array as a reductionist approach to study the effect of environmental parameters, independently of each other. It is observed for MCF-7 breast cancer cells, that culture within the three-dimensional (3D) environment of the microwells alone had an effect on the response to Taxol and results in a reduction of cell death in comparison to cells cultured on flat substrates. Additionally the microwells allowed the response of single versus multicell clusters to be differentiated. It was found that the formation of cell-cell contacts alters the drug response, depending on the type of adhesive protein present. Thus, with this microwell platform it is revealed that the presence of cell-cell contacts in addition to the dimensionality and the matrix composition of the environment are important mediators of altered drug responses. In conclusion the microwell array can not only serve as a platform to reveal which parameters of the extracellular environment affect drug response but further the interdependence of these parameters

Miniaturized pre-clinical cancer models as research and diagnostic tools

Cancer is one of the most common causes of death worldwide. Consequently, important resources are directed towards bettering treatments and outcomes. Cancer is difficult to treat due to its heterogeneity, plasticity and frequent drug resistance. New treatment strategies should strive for personalized approaches. These should target neoplastic and/or activated microenvironmental heterogeneity and plasticity without triggering resistance and spare host cells. In this review, the putative use of increasingly physiologically relevant microfabricated cell-culturing systems intended for drug development is discussed. There are two main reasons for the use of miniaturized systems. First, scaling down model size allows for high control of microenvironmental cues enabling more predictive outcomes. Second, miniaturization reduces reagent consumption, thus facilitating combinatorial approaches with little effort and enables the application of scarce materials, such as patient-derived samples. This review aims to give an overview of the state-of-the-art of such systems while predicting their application in cancer drug development

Brain research : an international multidisciplinary journal devoted to fundamental research in the brain sciences

Microwell arrays have emerged as robust and versatile alternatives to conventional mammalian cell culture substrates. Using standard microfabrication processes, biomaterials surfaces can be topographically patterned to comprise high-density arrays of micron-sized cavities with desirable geometry. Hundreds to thousands of individual cells or cell colonies with controlled size and shape can be trapped in these cavities by simple gravitational sedimentation. Efficient long-term cell confinement allows for parallel analyses and manipulation of cell fate during in vitro culture. These live-cell arrays have already found applications in cell biology, for example to probe the effect of cell colony size on embryonic stem cell differentiation, to dissect the heterogeneity in single cell proliferation kinetics of neural or hematopoietic stem/progenitor cell populations, or to elucidate the role of cell shape on cell function. Here, we highlight the key applications of these platforms, hopefully inspiring biologists to apply these systems for their own studies

Cell shape-dependent early responses of fibroblasts to cyclic strain

Randomly spread fibroblasts on fibronectin-coated elastomeric membranes respond to cyclic strain by a varying degree of focal adhesion assembly and actin reorganization. We speculated that the individual shape of the cells, which is linked to cytoskeletal structure and pre-stress, might tune these integrin-dependent mechanotransduction events. To this aim, fibronectin circles, squares and rectangles of identical surface area (2000μm(2)) were micro-contact printed onto elastomeric substrates. Fibroblasts plated on these patterns occupied the corresponding shapes. Cyclic 10% equibiaxial strain was applied to patterned cells for 30min, and changes in cytoskeleton and cell-matrix adhesions were quantified after fluorescence staining. After strain, megakaryocytic leukemia-1 protein translocated to the nucleus in most cells, indicating efficient RhoA activation independently of cell shape. However, circular and square cells (with radial symmetry) showed a significantly greater increase in the number of actin stress fibers and vinculin-positive focal adhesions after cyclic strain than rectangular (bipolar) cells of identical size. Conversely, cyclic strain induced larger changes in pY397-FAK positive focal complexes and zyxin relocation from focal adhesions to stress fibers in bipolar compared to symmetric cells. Thus, radially symmetric cells responded to cyclic strain with a larger increase in assembly, whereas bipolar cells reacted with more pronounced reorganization of actin stress fibers and matrix contacts. We conclude that integrin-mediated responses to external mechanical strain are differentially modulated in cells that have the same spreading area but different geometries, and do not only depend on mere cell size

Engineered lysozyme amyloid fibril networks support cellular growth and spreading

Fibrous networks assembled from synthetic peptides are promising candidates for biomimetic cell culture platforms and implantable biomaterials. The ability of the materials to reproduce physiological cell-matrix interactions is essential. However, the synthetic complexity of such systems limits their applications, thus alternative materials are desirable. Here, we design lysozyme derived amyloid fibril networks with controllable topographies, and perform a comprehensive study of the response of cultured fibroblast and epithelial cells. At high surface coverage a favorable increase in spreading and the generation of focal adhesions was observed, due to a combination of biomimetic chemistry and morphology. Their ease of synthesis, makes the nanoscale fibrils presented here ideal materials for future clinical applications whereby large volumes of biomimetic biomaterials are required. Furthermore, the surface chemistry of the fibrils is sufficient for the promotion of focal adhesions with cultured cells, eliminating the need for complex protocols for fibril decoration with bioactive moieties