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Browsing by Author "Sharma, Puja"

Now showing 1 - 3 of 3
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    Aligned fibers direct collective cell migration to engineer closing and nonclosing wound gaps
    Sharma, Puja; Ng, Colin; Jana, Aniket; Padhi, Abinash; Szymanski, Paige; Lee, Jerry S. H.; Behkam, Bahareh; Nain, Amrinder S. (2017-09-15)
    Cell emergence onto damaged or organized fibrous extracellular matrix (ECM) is a crucial precursor to collective cell migration in wound closure and cancer metastasis, respectively. However, there is a fundamental gap in our quantitative understanding of the role of local ECM size and arrangement in cell emergence-based migration and local gap closure. Here, using ECM-mimicking nanofibers bridging cell monolayers, we describe a method to recapitulate and quantitatively describe these in vivo behaviors over multispatial (single cell to cell sheets) and temporal (minutes to weeks) scales. On fiber arrays with large interfiber spacing, cells emerge (invade) either singularly by breaking cell-cell junctions analogous to release of a stretched rubber band (recoil), or in groups of few cells (chains), whereas on closely spaced fibers, multiple chains emerge collectively. Advancing cells on fibers form cell streams, which support suspended cell sheets (SCS) of various sizes and curvatures. SCS converge to form local gaps that close based on both the gap size and shape. We document that cell stream spacing of 375 mu m and larger hinders SCS advancement, thus providing abilities to engineer closing and nonclosing gaps. Altogether we highlight the importance of studying cell-fiber interactions and matrix structural remodeling in fundamental and translational cell biology.
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    The Mechanistic Influence of Aligned Nanofiber Networks on Cell Shape, Migration and Blebbing Dynamics of Glioma Cells
    Sharma, Puja; Sheets, Kevin; Elankumaran, Subbiah; Nain, Amrinder S. (The Royal Society of Chemistry, 2013-06-03)
    Investigating the mechanistic influence of the tumor microenvironment on cancer cell migration and membrane blebbing is crucial in the understanding and eventual arrest of cancer metastasis. In this study, we investigate the effect of suspended and aligned nanofibers on the glioma cytoskeleton, cell shape, migration and plasma membrane blebbing dynamics using a non-electrospinning fiber-manufacturing platform. Cells attached in repeatable shapes of spindle on single fibers, rectangular on two parallel fibers and polygonal on intersecting fibers. Structural stiffness (N m_1) of aligned and suspended nanofibers (average diameter: 400 nm, length: 4, 6, and 10 mm) was found to significantly alter the migration speed with higher migration on lower stiffness fibers. For cells attached to fibers and exhibiting blebbing, an increase in cellular spread area resulted in both reduced bleb count and bleb size with an overall increase in cell migration speed. Blebs no longer appeared past a critical cellular spread area of approximately 1400 _m2. Our results highlighting the influence of the mechanistic environment on the invasion dynamics of glioma cells add to the understanding of how biophysical components influence glioma cell migration and blebbing dynamics.
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    A Suspended Fiber Network Platform for the Investigation of Single and Collective Cell Behavior
    Sharma, Puja (Virginia Tech, 2016-10-04)
    Cells interact with their immediate fibrous extracellular matrix (ECM); alignment of which has been shown influence metastasis. Specifically, intra-vital imaging studies on cell invasion from tumor-matrix interface and wounds along aligned fibers describe invasion to occur as singular leader (tip) cells, or as collective mass of a few chain or multiple tip cells. Recapitulation of these behaviors in vitro promises to provide new insights in how, when and where cells get the stimulus to break cell-cell junctions and ensue invasion by migrating along aligned tracks. Using Spinneret based Tunable Engineered Parameters (STEP) technique, we fabricated precise layout of suspended fibers of varying diameters (300, 500 and 1000 nm) mimicking ECM dimensions, which were interfaced with cell monolayers to study invasion. We demonstrated that nanofiber diameter and their spacing were key determinants in cells to invade either as singularly, chains of few cells or multiple-chains collectively. Through time-lapse microscopy, we reported that singular cells exhibited a peculiar invasive behavior of recoiling analogous to release of a stretched rubber band; detachment speed of which was influenced with fiber diameter (250, 425 and 400 µm/hr on small, medium and large diameter fibers respectively). We found that cells initiated invasion by putting protrusion on fibers; dynamics of which we captured using a contrasting network of mismatched diameters deposited orthogonally. We found that vimentin, a key intermediate filament upregulated in cancer invasion localized within a protrusion only when the protrusion had widened at the base, signifying maturation. To develop a comprehensive picture of invasion, we also developed strategies to quantify migratory speeds and the forces exerted by cells on fibers. Finally, we extended our findings of cell invasion to report a new wound healing assay to examine gap closure. We found that gaps spanned by crosshatch network of fibers closed faster than those on parallel fibers and importantly, we reported that gaps of 375 µm or larger did not close over a 45-day period. In summary, the methods and novel findings detailed from this study can be extended to ask multiple sophisticated hypotheses in physiologically relevant phenomenon like wound healing, morphogenesis, and cancer metastasis.