Effect of nanoscale topography on fibronectin adsorption, focal adhesion size and matrix organisation (original) (raw)
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International Journal of …, 2008
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Acta Biomaterialia, 2015
Sculptured thin film (STF) substrates consist of nanocolumns with precise orientation, intercolumnar spacing, and optical anisotropy, which can be used as model biomaterial substrates to study the effect of homogenous nanotopogrophies on the three-dimensional distribution of adsorbed proteins. Generalized ellipsometry was used to discriminate between the distributions of adsorbed FN either on top of or within the intercolumnar void spaces of STFs, afforded by the optical properties of these precisely crafted substrates. Generalized ellipsometry indicated that STFs with vertical nanocolumns enhanced total FN adsorption two-fold relative to flat control substrates and the FN adsorption studies demonstrate different STF characteristics influence the degree of FN immobilization both on top and within intercolumnar spaces, with increasing spacing and surface area enhancing total protein adsorption. Mouse fibroblasts or mouse mesenchymal stem cells were subsequently cultured on STFs, to investigate the effect of highly ordered and defined nanotopographies on cell adhesion, spreading, and proliferation. All STF nanotopographies investigated in the absence of adsorbed FN were found to significantly enhance cell adhesion relative to flat substrates; and the addition of FN to STFs was found to have cell-dependent effects on enhancing cell-material interactions. Furthermore, the amount of FN adsorbed to the STFs did not correlate with comparative enhancements of cell-material interactions, suggesting that nanotopography predominantly contributes to the biocompatibility of homogenous nanocolumnar surfaces. This is the first study to correlate precisely defined nanostructured features with protein distribution and cellnanomaterial interactions. STFs demonstrate immense potential as biomaterial surfaces for applications in tissue engineering, drug delivery, and biosensing. (A.K. Pannier). Acta Biomaterialia xxx (2015) xxx-xxx Contents lists available at ScienceDirect Acta Biomaterialia j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l oc a t e / a c t a b i o m a t Please cite this article in press as: Kasputis T et al. Use of precisely sculptured thin film (STF) substrates with generalized ellipsometry to determine spatial distribution of adsorbed fibronectin to nanostructured columnar topographies and effect on cell adhesion. Acta Biomater (2015), http://dx.
Small surface nanotopography encourages fibroblast and osteoblast cell adhesion
Rsc Advances, 2013
In this paper, we report the initial response of 3T3 fibroblast and MG63 osteoblast cells to engineered nanotopography gradients of three nanoparticle diameters (16 nm, 38 nm and 68 nm). These nanoengineered surfaces were designed to provide a range of nanoparticle densities and comparable surface area across the gradients of different nanoparticle sizes. Importantly, we provided a uniform surface chemistry in order to be able to examine the effect of pure surface nanotopography. We found that nanotopography features of 16 nm encourage the adhesion of both cell types and that there is a critical nanoparticle density between 50 and 140 particles per mm 2 where cells adhered in the greatest numbers. When nanotopography features increased to 38 nm the 3T3 cells adhered and spread well, however, the MG63 cells adhered and spread poorly. Both cell types adhered in lower numbers when the nanotopography feature size increased to 68 nm. This work demonstrates that there is a specific nanotopography scale that encourages cell adhesion and spreading, however, the preferential lateral spacing and height of the nanotopography is different for different cell types.
Biomaterials, 2007
An important consideration in developing physical biomimetic cell-stimulating cues is that the in vivo extracellular milieu includes nanoscale topographic interfaces. We investigated nanoscale topography regulation of cell functions using human fetal osteoblastic (hFOB) cell culture on poly(L-lactic acid) and polystyrene (50/50 w/w) demixed nanoscale pit textures (14, 29, and 45 nm deep pits). Secondary ion mass spectroscopy revealed that these nanotopographic surfaces had similar surface chemistries to that of pure PLLA because of PLLA component surface segregation during spin casting. We observed that 14 and 29 nm deep pit surfaces increased hFOB cell attachment, spreading, selective integrin subunit expression (e.g., av relative to a5, b1, or b3), focal adhesive paxillin protein synthesis and paxillin colocalization with cytoskeletal actin stress fibers, and focal adhesion kinase (FAK) and phosphorylated FAK (pY397) expression to a greater degree than did 45 nm deep pits or flat PLLA surfaces. Considering the important role of integrin-mediated focal adhesion and intracellular signaling in anchorage-dependent cell function, our results suggest a mechanism by which nanostructured physical signals regulate cell function. Modulation of integrin-mediated focal adhesion and related cell signaling by altering nanoscale substrate topography will have powerful applications in biomaterials science and tissue engineering.
Cellular Activity and Biomaterial's Surface Topography
Materials Science Forum, 2007
The contact of a cell on the biomaterial's surface is mediated by its adhesion components. The topography of titanium surfaces influences these adhesion components of osteoblasts, e.g. the integrins, the adapter proteins and the actin cytoskeleton. In our current experiments we were interested in why osteoblasts were strongly aligned to the grooves of a structured pure titanium surface (grade 2). The titanium was characterized by EIS to get insights in the electro-chemically active surface. We used MG-63 human bone cells, cultured in DMEM with 10% FCS at 37°C. For protein adsorption the titanium discs were incubated for 24h with complete medium containing soluble fibronectin at 37°C. Interestingly, only in the grooves cells adhered and were aligned and this is not dependent on the gravitation. The cell adhesion seems to depend on the protein adsorption of fibronectin which we could find to be adsorbed exclusively in the valleys. We speculate that there are local differences in electro-chemical characteristics of this structured titanium surface.
Acta Biomaterialia, 2010
We investigated the early events of bone matrix formation, and specifically the role of fibronectin (FN) in the initial osteoblast interaction and the subsequent organization of a provisional FN matrix on different rough titanium (Ti) surfaces. Fluorescein isothiocyanate-labelled FN was preadsorbed on these surfaces and studied for its three-dimensional (3-D) organization by confocal microscopy, while its amount was quantified after NaOH extraction. An irregular pattern of adsorption with a higher amount of protein on topographic peaks than on valleys was observed and attributed to the physicochemical heterogeneity of the rough Ti surfaces. MG63 osteoblast-like cells were further cultured on FN-preadsorbed Ti surfaces and an improved initial cellular interaction was observed with increasing roughness. 3-D reconstruction of the immunofluorescence images after 4 days of incubation revealed that osteoblasts deposit FN fibrils in a specific facet-like pattern that is organized within the secreted total matrix overlying the top of the samples. The thickness of this FN layer increased when the roughness of the underlying topography was increased, but not by more than half of the total maximum peak-to-valley distance, as demonstrated with images showing simultaneous reconstruction of fluorescence and topography after 7 days of cell culture.
The influence of nanoscale topographical cues on initial osteoblast morphology and migration
European cells & materials, 2010
The natural environment of a living cell is not only organized on a micrometer, but also on a nanometer scale. Mimicking such a nanoscale topography in implantable biomaterials is critical to guide cellular behavior. Also, a correct positioning of cells on biomaterials is supposed to be very important for promoting wound healing and tissue regeneration. The exact mechanism by which nanotextures can control cellular behavior are thus far not well understood and it is thus far unknown how cells recognize and respond to certain surface patterns, whereas a directed response appears to be absent on other pattern types. Focal adhesions (FAs) are known to be involved in the process of specific pattern recognition and subsequent response by cells. In this study, we used a high throughput screening "Biochip" containing 40 different nanopatterns to evaluate the influence of several nanotopographical cues like depth, width, (an)isotropy and spacing (ridge-groove ratio) on osteoblast ...
Journal of Orthopaedic Research, 2007
Integration of an orthopedic prosthesis for bone repair must be associated with osseointegration and implant fixation, an ideal that can be approached via topographical modification of the implant/bone interface. It is thought that osteoblasts use cellular extensions to gather spatial information of the topographical surroundings prior to adhesion formation and cellular flattening. Focal adhesions (FAs) are dynamic structures associated with the actin cytoskeleton that form adhesion plaques of clustered integrin receptors that function in coupling the cell cytoskeleton to the extracellular matrix (ECM). FAs contain structural and signalling molecules crucial to cell adhesion and survival. To investigate the effects of ordered nanotopographies on osteoblast adhesion formation, primary human osteoblasts (HOBs) were cultured on experimental substrates possessing a defined array of nanoscale pits. Nickel shims of controlled nanopit dimension and configuration were fabricated by electron beam lithography and transferred to polycarbonate (PC) discs via injection molding. Nanopits measuring 120 nm diameter and 100 nm in depth with 300 nm center-center spacing were fabricated in three unique geometric conformations: square, hexagonal, and near-square (300 nm spaced pits in square pattern, but with AE50 nm disorder). Immunofluorescent labeling of vinculin allowed HOB adhesion complexes to be visualized and quantified by image software. Perhipheral adhesions as well as those within the perinuclear region were observed, and adhesion length and number were seen to vary on nanopit substrates relative to smooth PC. S-phase cells on experimental substrates were identified with bromodeoxyuridine (BrdU) immunofluorescent detection, allowing adhesion quantification to be conducted on a uniform flattened population of cells within the S-phase of the cell cycle. Findings of this study demonstrate the disruptive effects of ordered nanopits on adhesion formation and the role the conformation of nanofeatures plays in modulating these effects. Highly ordered arrays of nanopits resulted in decreased adhesion formation and a reduction in adhesion length, while introducing a degree of controlled disorder present in near-square arrays, was shown to increase focal adhesion formation and size. HOBs were also shown to be affected morphologicaly by the presence and conformation of nanopits. Ordered arrays affected cellular spreading, and induced an elongated cellular phenotype, indicative of increased motility, while near-square nanopit symmetries induced HOB spreading. It is postulated that nanopits affect osteoblast-substrate adhesion by directly or indirectly affecting adhesion complex formation, a phenomenon dependent on nanopit dimension and conformation. ß
The nanomorphology of cell surfaces of adhered osteoblasts
Beilstein Journal of Nanotechnology
The functionality of living cells is inherently linked to subunits with dimensions ranging from several micrometers down to the nanometer scale. The cell surface plays a particularly important role. Electric signaling, including information processing, takes place at the membrane, as well as adhesion and contact. For osteoblasts, adhesion and spreading are crucial processes with regard to bone implants. Here we present a comprehensive characterization of the 3D nanomorphology of living, as well as fixed, osteoblastic cells using scanning ion conductance microscopy (SICM), which is a nanoprobing method that largely avoids mechanical perturbations. Dynamic ruffles are observed, manifesting themselves in characteristic membrane protrusions. They contribute to the overall surface corrugation, which we systematically study by introducing the relative 3D excess area as a function of the projected adhesion area. A clear anticorrelation between the two parameters is found upon analysis of c...
Materials Science Forum, 2014
Cell-biomaterial interactions are strongly affected by topographical and chemical surface characteristics. We found out earlier that geometric titanium (Ti) pillar structures in the micrometer range induce the cells to rearrange their actin cytoskeleton in short fibers solely on the top of the pillars. As a result, cell physiology was hampered concerning collagen I synthesis and spreading capacity. Furthermore, the position-dependent initial cell adhesion strength was declined near the edges. We asked whether these observed cellular effects can be performed only in combination with Ti or occur independently of chemical surface features. In addition, the specific culture conditions, e.g. serum content or influence of gravity, were of interest. Human primary osteoblasts were cultured in Osteoblast Growth Medium with serum containing SupplementMix on pure silicon pillars (5x5x5 µm) or on samples additionally sputtered with Ti (as reference) or gold. To offer the cells ligands for their adhesion receptors, we coated the pillars with collagen I or alternatively with a plasma polymer layer from allylamine. Different from standard culture conditions, the cells were cultured against gravity as well as without serum. The actin cytoskeleton was stained with phalloidin-TRITC after 24 h and analyzed by confocal laser scanning microscopy. Interestingly, on all modifications tested the cell's actin cytoskeleton was distinctly organized in short fibers on the top of the pillars. Thus, we were able to exclude the influence of (i) the material chemistry (gold, silicon, physical plasma vs. Ti), (ii) the protein deposition on the pillar top and edges, and (iii) the impression caused by gravity.