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.

The interferon-stimulated gene 15 (ISG15) pathway is highly elevated in breast cancer; however, very little is known about how the ISG15 pathway contributes to breast tumorigenesis. In the current study, using the gene disruption approach, we demonstrate that both ISG15 and UbcH8 (ISG15-specific conjugating enzyme) disrupt F-actin architecture and formation of focal adhesions in ZR-75-1 breast cancer cells. In addition, ISG15 and UbcH8 promote breast cancer cell migration. We also demonstrate that ISG15 inhibits ubiquitin/26S proteasome-mediated turnover of proteins implicated in tumor cell motility, invasion and metastasis. Together, our results suggest that the aberrant activation of the ISG15 pathway confers a motile phenotype to breast cancer cells by disrupting cell architecture and stabilizing proteins involved in cell motility, invasion and metastasis. Because the cellular architecture is conserved and the ISG15 pathway is constitutively activated in tumor cells of different lineages, it is reasonable to assume that our observations in breast cancer must hold true for many other tumors.

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.

Microscale plasma-initiated patterning (μPIP) is a novel micropatterning technique used to create biomolecular micropatterns on polymer surfaces. The patterning method uses a polydimethylsiloxane (PDMS) stamp to selectively protect regions of an underlying substrate from oxygen plasma treatment resulting in hydrophobic and hydrophilic regions. Preferential adsorption of the biomolecules onto either the plasma-exposed (hydrophilic) or plasma-protected (hydrophobic) regions leads to the biomolecular micropatterns. In the current work, laminin-1 was applied to an electrospun polyamide nanofibrillar matrix following plasma treatment. Radial glial clones (neural precursors) selectively adhered to these patterned matrices following the contours of proteins on the surface. This work demonstrates that textured surfaces, such as nanofibrillar scaffolds, can be micropatterned to provide external chemical cues for cellular organization.

Continuous biomaterial advances and the regenerating potential of the adult human peripheral nervous system offer great promise for restoring full function to innervated tissue following traumatic injury via synthetic nerve guidance conduits (NGCs). To most effectively facilitate nerve regeneration, a tissue engineering scaffold within a conduit must be similar to the linear microenvironment of the healthy nerve. To mimic the native nerve structure, aligned poly(lactic-co-glycolic acid)/bioactive polyanhydride fibrous substrates were fabricated through optimized electrospinning parameters with diameters of 600 ± 200 nm. Scanning electron microscopy images show fibers with a high degree of alignment. Schwann cells and dissociated rat dorsal root ganglia demonstrated elongated and healthy proliferation in a direction parallel to orientated electrospun fibers with significantly longer Schwann cell process length and neurite outgrowth when compared to randomly orientated fibers. Results suggest that an aligned polyanhydride fiber mat holds tremendous promise as a supplement scaffold for the interior of a degradable polymer NGC. Bioactive salicylic acid-based polyanhydride fibers are not limited to nerve regeneration and offer exciting promise for a wide variety of biomedical applications.

Long distance intercellular communication between astrocytes on nanofibrillar and planar surfaces was investigated by epi-fluorescence microscopy and atomic force microscopy. We found that astrocytes on nanofibrillar surfaces and astrocytes on planar surfaces diverged in apparent cell-cell contact structures. Astrocytes on nanofibrillar surfaces exhibited a “single cellular process” response, while astrocytes on planar surfaces exhibited a filopodial network response. The possibility that astrocytes can sense their geometrical environment and form different cell-to-cell contacts on nanofibrillar versus planar surfaces, with activation of different signaling pathways, is discussed.

Tenascin-C (TNC), a major component of the extracellular matrix, is strongly upregulated after injuries of the central nervous system (CNS) but its role in tissue repair is not understood. Both regeneration promoting and inhibiting roles of TNC have been proposed considering its abilities to both support and restrict neurite outgrowth in vitro. Here, we show that spontaneous recovery of locomotor functions after spinal cord injury is impaired in adult TNC-deficient (TNC(-/-)) mice in comparison to wild-type (TNC(+/+)) mice. The impaired recovery was associated with attenuated excitability of the plantar Hoffmann reflex (H-reflex), reduced glutamatergic input, reduced sprouting of monaminergic axons in the lumbar spinal cord and enhanced post-traumatic degeneration of corticospinal axons. The degeneration of corticospinal axons in TNC(-/-) mice was normalized to TNC(+/+) levels by application of the alternatively spliced TNC fibronectin type III homologous domain D (fnD). Finally, overexpression of TNC-fnD via adeno-associated virus in wild-type mice improved locomotor recovery, increased monaminergic axons sprouting, and reduced lesion scar volume after spinal cord injury. The functional efficacy of the viral-mediated TNC indicates a potentially useful approach for treatment of spinal cord injury.

The introduction of scaffolding materials with appropriate biochemical cues and physical properties into damaged sites within the central nervous system can encourage endogenous or exogenous cellular re-colonization. The scaffolding material currently under investigation is a synthetic electrospun polyamide nanofibrillar matrix that has demonstrated promise in tissue engineering approaches for the repair of the injured spinal cord and is architecturally mimetic for the capillary basement membrane at the blood brain barrier. We will present quantitative investigations of the nanofibrillar matrix, which are achieved through use of a dynamic new mode of atomic force microscopy, Scanning Probe Recognition Microscopy (SPRM). SPRM uniquely allows auto-tracking along individual nanofibers, which are then compiled into a statistical representation of the nanofibrillar matrix as a whole. Complementary transmission electron microscopy (TEM) and nuclear magnetic resonance (NMR) investigations are performed to assess nanofiber internal structures that contribute to elasticity, and growth factor binding site information, respectively. Results of investigations of unmodified nanofibers and nanofibers covalently modified with fibroblast growth factor-2 (FGF-2), a prevailing cytokine involved in regulation of the growth of astrocytes, neurons, and other neural cells, will be presented. Astrocytes are neural cells that can be considered to be the cellular bridge between the capillary basement membrane and neurons. They are therefore directly responsive to the biochemical cues and physical properties of the native or prosthetic basement membrane. We will present results achieved through SPRM, immunocytochemistry, and Western blot techniques that indicate that there are significant differences in the astrocyte response to 2D planar substrates versus 3D nanofibrillar substrates versus 3D nanofibrillar substrates that are covalently modified with FGF-2, mimicking the sequestration of growth factors on the basement membrane. Differences in astrocyte physiology, substrate probing through lamellipodia and filopodia extension and FGF-2 up-regulation will be discussed.

Scanning probe recognition microscopy (SPRM) with auto-tracking of individual nanofibres is used for investigation of the key nanoscale properties of polyamide a nanofibrillar matrix that promotes more in vivo-like forms and functions for cultured cells. Both unmodified and fibroblast growth factor-2-modified nanofibres are considered. The contributions of nanofibrillar matrix elasticity and surface roughness to cellular behaviour are examined.

Recent advances in regenerative medicine have improved understanding of the materials properties of nanofibrillar scaffolds used to aid cell system re-establishment. A picture is emerging that a successful scaffold has a defined set of physical properties including mechanical properties, topographical properties, and growth factor inclusion, and that the weighting of each may vary depending on the cell system and its functionality. In the present work, results from investigations of a slowly biodegradable nanofiber prosthetic for neural cell system re-establishment are presented. Preliminary data from in-vivo investigations (rat model) indicate that a nanofiber prosthetic device of FGF-2-modified nanofibers contributes to system re-establishment including aligned guidance for regenerating axons across an injury gap, and angiogenesis. Research by Meiners' group also demonstrated that FGF-2 retains biological activity significantly longer when immobilized on nanofibers than when presented as a soluble molecule [1]. The present investigations use atomic force microscopy operated in a new mode, Scanning Probe Recognition Microscopy (SPRM) [2], developed by Ayres' group, to quantitatively investigate properties of FGF-2-modified nanofibers. The SPRM system is given the ability to auto-track on regions of interest through incorporation of recognition-based tip control realized using algorithms and techniques from computer vision, pattern recognition and signal processing fields. Statistically meaningful numbers of reliable data points are extracted using an automatic procedure that maintains uniformity of experimental conditions. Properties under quantitative SPRM investigation include nanofiber stiffness and surface roughness, nanofiber curvature, nanofiber mesh density and porosity, and growth factor presentation and distribution. Each of these factors has been demonstrated to have global effects on cell morphology, function, proliferation, morphogenesis, migration, and differentiation. [1] A. Nur-E-Kamal et al., Mol Cell Biochem 309 (2008) 157-166. [2] Y. Fan et al., Int. J. Nanomedicine 2 (2007) 651-661.

Implantable biodegradable nerve guidance conduits (NGCs) have the potential to align and support regenerating cells, as well as prevent scar formation. In this study in vitro bioassays and in vivo material evaluations were performed using a nerve guidance conduit material made from a novel polyanhydride blend. In vitro cytotoxicity studies with both fibroblasts and primary chick neurons demonstrated that the proposed polyanhydride blend was non-cytotoxic. Subcutaneous implantation for 7days in rats resulted in an initial fibrin matrix, minimal macrophage presence and angiogenesis in the surrounding tissues. Nerve guidance conduits fabricated from the proposed polyanhydride blend material may serve as favorable biocompatible tissue engineering devices.

An electrospun nonwoven matrix of polyamide nanofibers was employed as a new model for the capillary basement membrane at the blood-brain barrier (BBB). The basement membrane separates astrocytes from endothelial cells and is associated with growth factors, such as fibroblast growth factor-2 (FGF-2). FGF-2 is produced by astrocytes and induces specialized functions in endothelial cells, but also has actions on astrocytes. To investigate potential autocrine actions of FGF-2 at the BBB, astrocytes were cultured on unmodified nanofibers or nanofibers covalently modified with FGF-2. The former assumed an in vivo-like stellate morphology that was enhanced in the presence of cross-linked FGF-2. Furthermore, astrocyte monolayers established on unmodified nanofibers were more permissive for neurite outgrowth when cultured with an overlay of neurons than similar monolayers established on standard tissue culture surfaces, while astrocytes cultured on FGF-2-modifed nanofibers were yet more permissive. The observed differences were due in part to progressively increasing amounts of FGF-2 secreted by the astrocytes into the medium; hence FGF-2 increases its own expression in astrocytes to modulate astrocyte-neuron interactions. Soluble FGF-2 was unable to replicate the effects of cross-linked FGF-2. Nanofibers alone up-regulated FGF-2, albeit to a lesser extent than nanofibers covalently modified with FGF-2. These results underscore the importance of both surface topography and growth factor presentation on cellular function. Moreover, these results indicate that FGF-2-modified nanofibrillar scaffolds may demonstrate utility in tissue engineering applications for replacement and regeneration of lost tissue following central nervous system (CNS) injury or disease.

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.

Activation of fibroblast growth factor receptors (FGFRs) requires the formation of a ternary complex between fibroblast growth factors (FGFs), FGFRs, and heparan sulfate proteoglycans, which are all located on the cell surface and the basement membrane (BM)/extracellular matrix (ECM). Heparan sulfate proteoglycans appear to stabilize FGFs by inhibiting the rapid degradation of FGFs normally observed in solution. Because of the pivotal role of FGFs in proliferative and developmental pathways, a number of recent studies have attempted to engineer microenvironments to stabilize growth factors for use in applications in tissue culture and regenerative medicine. In this communication, we demonstrate that covalent linkage of FGF-2 to nanofibrillar surfaces (defined as covalently bound FGF-2) composed of a network of polyamide nanofibers resulted in the maintenance of the biological efficacy of FGF-2 when stored dry for at least 6 months at 25 degrees C or 4 degrees C. Moreover, covalently bound FGF-2 was more potent than FGF-2 in solution when measured in cellular assays of proliferation and viability using a variety of cell types. Covalently bound FGF-2 also strongly activated FGFR, extracellular signal-regulated kinase (ERK1/2), and c-fos. Hence cell-signaling molecules can be incorporated into a synthetic nanofibrillar surface, providing a novel means to enhance their stability and biological activity.

Preliminary data from in-vivo investigations (rat model) suggest that a nanofiber prosthetic device of FGF-2-modified nanofibers can correctly guide regenerating axons across an injury gap with aligned functional recovery. Scanning Probe Recognition Microscopy with auto-tracking of individual nanofibers is used for investigation of the key nanoscale properties of the nanofiber prosthetic device for spinal cord tissue engineering and repair.

Growth of cells in tissue culture is generally performed on two-dimensional (2D) surfaces composed of polystyrene or glass. Recent work, however, has shown that such 2D cultures are incomplete and do not adequately represent the physical characteristics of native extracellular matrix (ECM)/basement membrane (BM), namely dimensionality, compliance, fibrillarity, and porosity. In the current study, a three-dimensional (3D) nanofibrillar surface composed of electrospun polyamide nanofibers was utilized to mimic the topology and physical structure of ECM/BM. Additional chemical cues were incorporated into the nanofibrillar matrix by coating the surfaces with fibronectin, collagen I, or laminin-1. Results from the current study show an enhanced response of primary mouse embryonic fibroblasts (MEFs) to culture on nanofibrillar surfaces with more dramatic changes in cell spreading and reorganization of the cytoskeleton than previously observed for established cell lines. In addition, the cells cultured on nanofibrillar and 2D surfaces exhibited differential responses to the specific ECM/BM coatings. The localization and activity of myosin II-B for MEFs cultured on nanofibers was also compared. A dynamic redistribution of myosin II-B was observed within membrane protrusions. This was previously described for cells associated with nanofibers composed of collagen I but not for cells attached to 2D surfaces coated with monomeric collagen. These results provide further evidence that nanofibrillar surfaces offer a significantly different environment for cells than 2D substrates.

BACKGROUND: The design of implants comprised of biodegradable electrospun nanofibers for the purpose of bridging injuries in damaged spinal cord is discussed. Electrospun nanofibers structurally mimic the extracellular matrix on which neurons and other cell types grow in vivo. This property has created great interest for their use in tissue engineering applications. However, their employment as biornimetic surfaces for such in vivo applications is still in its infancy.RESULTS: A nonwoven fabric comprised of electrospun polyamide nanofibers supported modest axonal regeneration in injured adult rat spinal cord. Covalent modification of the nanofibers with a bioactive peptide derived from the neuroregulatory extracellular matrix molecule tenascin-C enhanced the ability of the nanofibers to facilitate axonal regrowth. However, the random orientation of the nanofibrillar fabric folds was an impediment to the forward movement of axons.CONCLUSIONS: Polyamide nanofibers covalently modified with neuroactive molecules provide a promising material for grafts to promote spinal cord regeneration. However, for the proper guidance of regrowing axons, attention must be paid to the engineering of ordered nanofibrillar structures. (c) 2007 Society of Chemical Industry.