Biocompatibility of cluster-assembled nanostructured TiO2 with primary and cancer cells
Introduction
Cellular behavior in vivo and in vitro is influenced by the mechanical, biochemical and topographical properties of the extracellular microenvironment where cells grow [1], [2], [3]. In particular, the biochemical composition and the mechanical behavior of the extracellular matrix (ECM) play an important role in many developmental phenomena during embryogenesis [4] or tumor-related conditions [5]. Recently also stem cells were shown to be responsive to the extracellular environment with relevant consequences for their self-renewal or differentiation programs [6], [7].
According to the most recent studies on biomaterials [8], [9], cells can actively ‘sense’ and adapt to the surface of adhesion and activate specific intracellular signals that influence cell survival and behavior. In vivo, cell attachment is the consequence of the binding with specific cell adhesion proteins in the ECM, and it is intrinsically influenced, besides by receptor–ligand specific interactions, by the physical and mechanical signals arising from the topography of the external environment [1], [2], [10]. In vitro, on the other hand, cells set up a complex network of interactions both with the artificial surface and with the secreted and serum ECM proteins. The possibility of optimizing cell-substrate interactions can open up new perspectives in the design of biomimetic supports [11], [12].
The topography of the ECMs is characterized by features over different length scales ranging from the nano- to the mesoscale and it regulates the cellular behavior in a way that it is still far from a complete understanding [13], [14], [15]. The coexistence of ECM features at different length scales is probably one of the key factors, however it is not clear if there is a hierarchical organization of different structures and to what extent the various length scales can influence cellular response [16], [17]
In order to elucidate the role of substrate topography and to fabricate biocompatible interfaces capable of mimicking the physiological conditions of the extracellular environment, a large number of studies have been devoted to the investigation of cell interactions with artificially produced nanostructures such as pits, pillars, grooves, dots or random structures obtained by chemically or physically etching metallic, semiconducting and polymeric surfaces [8], [18], [19], [20], [21].
Particular efforts have been devoted to the topographical modification of titanium and titanium dioxide surfaces since these materials are amongst the most studied and well-characterized biomaterials [22]. Pure titanium and titanium alloys are frequently used as dental and orthopedic implants because of their excellent mechanical strength, chemical stability, and biocompatibility [23], [24], which ultimately arise from the thin oxide layer that spontaneously forms on the titanium surfaces [22].
The fabrication strategies employed to create synthetic substrates with tailored topography at the nano- and microscale are essentially top-down and in particular based on hard and soft lithography for the fabrication of ordered structures [25], [26]. These approaches, when not based on natural matrix-related proteins, despite the great improvements in miniaturization and accuracy, are not able to reproduce the morphology and the hierarchical organization typical of the ECMs [27].
Up to now, very few studies have been devoted to the elucidation of the interaction of cells with nanostructured materials obtained by the bottom-up approach of nanoparticle assembling [28], [29], [30], [31]. This is quite surprising since nanoparticle-assembled materials have an increasing role in the fabrication of biocompatible devices as well as diagnostic and therapeutic platforms; moreover, a bottom-up approach can offer more possibilities to organize the structure on a multi-length scale similar to that observed in ECM.
In this paper we report the characterization of the biocompatibility of nanostructured TiO2 films obtained by the deposition of a supersonic beam of TiOx clusters. The films, resulting from a random stacking of nanoparticles, are characterized, at the nanoscale, by a granularity and porosity mimicking those of recently observed ECM structures [32], [33]. Moreover titanium dioxide nanoparticles can form complexes with a variety of chemical groups and immobilize functional peptides and macromolecules [34], [35], [36], [37], [38], [39] which therefore could be employed to functionalize the surface.
In view of the interesting opportunities of integration with microsystems offered by nanostructured TiO2 films assembled by supersonic cluster beam deposition (SCBD) [40], [41], we sought to investigate the biocompatibility of this new substrate with a wide range of cell lines in comparison with a surface which is commonly employed for culturing cells (i.e., gelatin-coated glass coverslips). To explore the biocompatibility of this novel nanostructured surface, cells were analyzed in terms of morphological appearance and growth properties, with the aim to evaluate its possible use as a substrate for different cell-based and tissue-based applications [42], [43], [44], [45], [46], [47].
We have performed short- and long-term studies with different cellular model systems using bright field microscopy for cell morphology, immunofluorescence for cytoskeletal analysis, BrdU incorporation and DAPI staining for cell cycle and cell growth analysis. Our results indicate that cluster-assembled nanostructured TiO2 is a biocompatible surface for cell culturing directly supporting normal growth and adhesion of different primary and cancer cells.
Section snippets
Cluster-assembled nanostructured titanium substrates
Nanostructured TiO2 films (thickness of 50 nm) were grown on round glass coverslips (15 mm diameter, 0.13–0.16 mm thickness, Electron Microscopy Sciences) by depositing under high vacuum a supersonic seeded beam of TiOx clusters produced by a pulsed microplasma cluster source (PMCS). A detailed description of the PMCS and its principle of operation and can be found in Refs. [40], [48]. Briefly, the PMCS operation principle is based on the ablation of a titanium rod by a helium plasma jet, ignited
Morphological and chemical characterization of the substrates
In Fig. 1 we present two AFM images of a cluster-assembled TiO2 film (A) and a gelatin-coated glass coverslip (B). At the nanoscale, both films expose a granular surface, with a grain diameter ranging from a few nanometers up to 20 nm. The nanoscale texture of the two substrates is the result of the aggregation of nanometer-sized building blocks: TiOx clusters, in the case of cluster-assembled TiO2, and proteins in the case of gelatin. Although similar in the nano-scale surface raster, the two
Conclusions
We have described the biocompatibility of nanostructured TiO2 films produced by supersonic cluster beam deposition. The deposition of TiOx clusters on a substrate produces low-density and highly porous films with grains of few nanometers.
Once deposited, the films were not further functionalized before being employed as culture substrates. Cells plated on this substrate adhered and grew with the same morphology as cells grown on a gelatin-coated coverslip; the same cytoskeletal parameters
Acknowledgments
We thank C. Ducati for TEM characterization and G. Giardina and C. Spinelli for their help in melanocytes characterization. This work has been supported by AIRC under OGCG grant “Development and integration of high-throughput technologies for the functional genomics of cancer” and Fondazione CARIPLO under grant “Sviluppo di sistemi micro— e nanostrutturati per analisi fenotipiche di famiglie di geni”.
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