The cell sheets that were maintained in blebbistatin-dosed media (bleb+) contracted to 18% of the original area, but were still significantly greater than the time-matched control (p 0.05). no significant difference in nuclear density was observed among all treated samples. (B) After dispase lifting the siRNA transfected samples, all sheets contracted, but K5 and K14 siRNA treated contracted significantly less than the sample treated with negative control siRNA. *p 0.05, **p 0.01. 1472-6750-13-17-S2.docx (270K) GUID:?8919A015-A60C-48E3-B52D-B9FA671F60F5 Abstract Background There is an increasing need to understand cell-cell interactions for cell and tissue engineering purposes, such as optimizing cell sheet constructs, as well as for examining adhesion defect diseases. For cell-sheet engineering, one major obstacle to sheet function is that cell sheets in suspension are fragile and, over time, will contract. While the role of the cytoskeleton in maintaining the structure and adhesion of cells cultured on a rigid substrate is well-characterized, a systematic examination of the role played by different components of the cytoskeleton in regulating cell sheet contraction and cohesion in the absence of a substrate has been lacking. Results In this study, keratinocytes were cultured until confluent and cell sheets were generated using dispase to remove the influence of the substrate. The effects of disrupting actin, microtubules or intermediate filaments on cell-cell interactions were assessed by measuring cell sheet cohesion and contraction. Keratin intermediate filament disruption caused comparable effects on cell sheet cohesion and contraction, when compared to actin or microtubule disruption. Interfering with actomyosin contraction demonstrated that interfering with cell contraction can also diminish cell cohesion. Ethynylcytidine Conclusions All components of the cytoskeleton are involved Ethynylcytidine in maintaining cell sheet cohesion and contraction, although not to the same extent. These findings demonstrate that substrate-free cell sheet biomechanical properties are dependent on the integrity of the cytoskeleton network. strong class=”kwd-title” Keywords: Cell sheet, Cytoskeleton, Adhesion, Contraction Background The development of cell-sheet tissue engineering, where cells are plated and allowed to form confluent layers which are then dissociated from the plate to form intact, functional sheets, has generated a need for a systematic characterization of cell-cell interactions to better condition constructs for in vivo use [1-3]. Such cell sheets have been generated for a wide variety of tissues, such as skin, heart, corneal and renal components [4-6]. Cell sheets generated for tissue engineering purposes are fragile and are typically handled by using external supports, such as chitin membranes [7]. Methods for improving the Ethynylcytidine strength and other mechanical properties of such sheets is essential for further development of these constructs. However, to be effective, such methods must rely on information regarding the mechanism by which sheet properties are regulated. For example, of interest would be mechanisms by which cell sheet contraction is limited by targeting select aspects of the cell cytoskeleton. To uncover such mechanisms, there needs to be a systematic examination of the role of the cytoskeleton in regulating cell sheet properties. Further, there is a significant amount of recent interest in the relationship between the cytoskeleton and cell-cell interactions to model physiology or disease processes [8-10]. The cellular cytoskeleton primarily consists of three main components in mammalian cells C actin, microtubules and intermediate filaments. For cells that remained attached Ethynylcytidine to a substrate, the contribution of the cytoskeleton to cell-substrate adhesion, spreading, and signaling have been extensively studied [11-21]. Actin is a well-examined cytoskeletal component, since actin Cxcr2 links to the focal adhesion complex and disruption of actin is linked to reduced traction forces and altered mechanotransductive signaling [16,22-26]. Microtubules have a role in supporting the actin framework and destabilizing focal adhesions [27,28], but play more prominent roles in cell division and intracellular transport. Intermediate filaments are much less frequently examined, but are thought to be involved in tissue strength [29-32]. Much less is known about the roles of these components Ethynylcytidine in determining the properties of suspended.