Volkan Mujdat Tiryaki

Nanofibrillar scaffolds induce preferential activation of Rho GTPases in cerebral cortical astrocytes

Cerebral cortical astrocyte responses to polyamide nanofibrillar scaffolds versus poly-L-lysine (PLL)-functionalized planar glass, unfunctionalized planar Aclar coverslips, and PLL-functionalized planar Aclar surfaces were investigated by atomic force microscopy and immunocytochemistry. The physical properties of the cell culture environments were evaluated using contact angle and surface roughness measurements and compared. Astrocyte morphological responses, including filopodia, lamellipodia, and stress fiber formation, and stellation were imaged using atomic force microscopy and phalloidin staining for F-actin. Activation of the corresponding Rho GTPase regulators was investigated using immunolabeling with Cdc42, Rac1, and RhoA. Astrocytes cultured on the nanofibrillar scaffolds showed a unique response that included stellation, cell–cell interactions by stellate processes, and evidence of depression of RhoA. The results support the hypothesis that the extracellular environment can trigger preferential activation of members of the Rho GTPase family, with demonstrable morphological consequences for cerebral cortical astrocytes.

Differentiation of reactive-like astrocytes cultured on nanofibrillar and comparative culture surfaces

AIM To investigate the directive importance of nanophysical properties on the morphological and protein expression responses of dibutyryladenosine cyclic monophosphate (dBcAMP)-treated cerebral cortical astrocytes in vitro. MATERIALS & METHODS Elasticity and work of adhesion characterizations of culture surfaces were performed using atomic force microscopy and combined with previous surface roughness and polarity results. The morphological and biochemical differentiation of dBcAMP-treated astrocytes cultured on promising nanofibrillar scaffolds and comparative culture surfaces were investigated by immunocytochemistry, colocalization, super resolution microscopy and atomic force microscopy. The dBcAMP-treated astrocyte responses were further compared with untreated astrocyte responses. RESULTS & CONCLUSION Nanofibrillar scaffold properties were shown to reduce immunoreactivity responses while poly-L-lysine-functionalized Aclar® (Ted Pella Inc., CA, USA) properties were shown to induce responses reminiscent of glial scar formation. The comparison study indicated that directive cues may differ in wound-healing versus quiescent situations.

AFM Feature Definition for Neural Cells on Nanofibrillar Tissue Scaffolds

A diagnostic approach is developed and implemented that provides clear feature definition in atomic force microscopy (AFM) images of neural cells on nanofibrillar tissue scaffolds. Because the cellular edges and processes are on the same order as the background nanofibers, this imaging situation presents a feature definition problem. The diagnostic approach is based on analysis of discrete Fourier transforms of standard AFM section measurements. The diagnostic conclusion that the combination of dynamic range enhancement with low-frequency component suppression enhances feature definition is shown to be correct and to lead to clear-featured images that could change previously held assumptions about the cell-cell interactions present. Clear feature definition of cells on scaffolds extends the usefulness of AFM imaging for use in regenerative medicine.

Texture-Based Segmentation and a New Cell Shape Index for Quantitative Analysis of Cell Spreading in AFM Images

A new cell shape index is defined for use with atomic force microscopy height images of cell cultures. The new cell shape index reveals quantitative cell spreading information not included in a conventional cell shape index. A supervised learning‐based cell segmentation algorithm was implemented by texture feature extraction and a multi‐layer neural network classifier. The texture feature sets for four different culture surfaces were determined from the gray level co‐occurrence matrix and local statistics texture models using two feature selection algorithms and by considering computational cost. The quantitative morphometry of quiescent‐like and reactive‐like cerebral cortical astrocytes cultured on four different culture environments was investigated using the new and conventional cell shape index. Inclusion of cell spreading with stellation information through use of the new cell shape index was shown to change biomedical conclusions derived from conventional cell shape analysis based on stellation alone. The new CSI results showed that the quantitative astrocyte spreading and stellation behavior was induced by both the underlying substrate and the immunoreactivity of the astrocytes.

Quantitative Investigations of Nanoscale Elasticity of Nanofibrillar Matrices

Recent research indicates that nanophysical properties as well as biochemical cues can influence cellular re-colonization of a tissue scaffold. It has also been shown nanoscale elasticity can strongly influence cellular responses. In the present work, quantitative investigations of the elasticity of a nanofibrillar matrix scaffold that has demonstrated promise for spinal cord injury repair are compared with complementary transmission electron microscopy investigations, performed to assess nanofiber internal structures. Interpretive model improvements are identified and discussed.

A novel quantitative volumetric spreading index definition and assessment of astrocyte spreading in vitro

A novel quantitative volumetric spreading index (VSI) is defined that depends on the total distance between object voxels and the contact surface plane in three‐dimensional (3D) space. The VSI, which ranges from 0 to 1, is rotationally invariant around the z‐axis. VSI can be used to quantify the degree of individual cell spreading, which is important for analysis of cell interactions with their environment. The VSIs of astrocytes cultured on a nanofibrillar surface and three different comparative planar surfaces have been calculated from confocal laser scanning microscope z‐series images, and the effects of both culture surface and immunoreactivity on the degree of cell spreading were investigated. VSI calculations indicated a statistical correlation between increased reactivity, based on immunolabeling for glial fibrillary acidic protein, and decreased cell spreading. Further results provided a quantitative measure for the increased spreading of quiescent‐like and reactive‐like astrocytes on planar substrates functionalized with poly‐l‐lysine.

Astrocyte Cell-Cell Interactions via Long-Range Connective Bridges on Directive Surfaces

In previously reported work [1], we demonstrated that astrocytes cultured on synthetic polyamide nanofibrillar surfaces that mimic the architecture of the capillary basement membrane assumed morphological forms that recapitulated their physiology within the developing central nervous system. In the present work, atomic force microscopy was used to investigate astrocyte cell-cell interactions at 24 h, for cells cultured on nanofibrillar versus planar surfaces. For the nanofibrillar surfaces, high pass spatial filtering was required to distinguish the nanofibrillar background from the nanoscale astrocyte features. Using this approach, details of the physical interactions between astrocytes on nanofibrillar surfaces via connective extensions across ∼50 μm distances were identified, which were not observable in epi-fluorescent microscopy, or in tapping or deflection mode atomic force microscopy. Astrocyte cell-cell interactions were shown to differ in connective extension type, cell body type, and number of interactions. The connective bridges took the form of a filopodia network for planar surfaces but a single extension lamellipodia bridge for the nanofibrillar surfaces. Structures suggestive of adherens versus gap junctions that were part of the connective extensions were also identified. Cell-cell interactions via connective bridges (filopodia bridges, or tunneling nanotubes) over distances much larger than adjacent cell wall-cell wall contact distances have been previously reported for planar substrates. The present research supports this work and adds the dimension that nanofibrillar versus planar surface architectures can also be directive for specific implementations of such long-distance interactions.[1] Delgado-Rivera, R, Harris, SL, Ahmed, I, Babu, AN, Patel, R, Kamal, J, Ayres, V, Flowers, D, Meiners, S, 2009. Increased FGF-2 secretion and ability to support neurite outgrowth by astrocytes cultured on polyamide nanofibrillar matrices. Matrix Bio. 28: 137-147.