by Keyword: Topography

Martens KJA, Gobes M, Archontakis E, Brillas RR, Zijlstra N, Albertazzi L, Hohlbein J, (2022). Enabling Spectrally Resolved Single-Molecule Localization Microscopy at High Emitter Densities Nano Letters 22, 8618-8625

Single-molecule localization microscopy (SMLM) is a powerful super-resolution technique for elucidating structure and dynamics in the life- and material sciences. Simultaneously acquiring spectral information (spectrally resolved SMLM, sSMLM) has been hampered by several challenges: an increased complexity of the optical detection pathway, lower accessible emitter densities, and compromised spatio-spectral resolution. Here we present a single-component, low-cost implementation of sSMLM that addresses these challenges. Using a low-dispersion transmission grating positioned close to the image plane, the +1stdiffraction order is minimally elongated and is analyzed using existing single-molecule localization algorithms. The distance between the 0th and 1st order provides accurate information on the spectral properties of individual emitters. This method enables a 5-fold higher emitter density while discriminating between fluorophores whose peak emissions are less than 15 nm apart. Our approach can find widespread use in single-molecule applications that rely on distinguishing spectrally different fluorophores under low photon conditions.

JTD Keywords: cells, multicolor imaging, nanoscopy, particle tracking, point accumulation for imaging in nanoscale topography (paint), precision, single-molecule fo?rster resonance energy transfer (smfret), stochastic optical reconstruction microscopy (storm), Diffraction-limit, Multicolor imaging, Point accumulation for imaging in nanoscale topography (paint), Single-molecule förster resonance energy transfer (smfret), Single-molecule spectroscopy, Stochastic optical reconstruction microscopy (storm)

Casanellas I, Samitier J, Lagunas A, (2022). Recent advances in engineering nanotopographic substrates for cell studies Frontiers In Bioengineering And Biotechnology 10, 1002967

Cells sense their environment through the cell membrane receptors. Interaction with extracellular ligands induces receptor clustering at the nanoscale, assembly of the signaling complexes in the cytosol and activation of downstream signaling pathways, regulating cell response. Nanoclusters of receptors can be further organized hierarchically in the cell membrane at the meso- and micro-levels to exert different biological functions. To study and guide cell response, cell culture substrates have been engineered with features that can interact with the cells at different scales, eliciting controlled cell responses. In particular, nanoscale features of 1-100 nm in size allow direct interaction between the material and single cell receptors and their nanoclusters. Since the first "contact guidance" experiments on parallel microstructures, many other studies followed with increasing feature resolution and biological complexity. Here we present an overview of the advances in the field summarizing the biological scenario, substrate fabrication techniques and applications, highlighting the most recent developments.Copyright © 2022 Casanellas, Samitier and Lagunas.

JTD Keywords: cell response, density, differentiation, lithography, micro, nanofabrication, nanopatterning, nanopatterns, nanoscale, nanotopography, organization, photolithography, Cell response, Nanofabrication, Nanopatterning, Nanotopography, Plasma-membrane, Receptor nanoclustering

Iglesias-Fernandez, M, Buxadera-Palomero, J, Sadowska, JM, Espanol, M, Ginebra, MP, (2022). Implementation of bactericidal topographies on biomimetic calcium phosphates and the potential effect of its reactivity Biomaterials Advances 136, 212797

Since the discovery that nanostructured surfaces were able to kill bacteria, many works have been published focusing on the design of nanopatterned surfaces with antimicrobial properties. Synthetic bone grafts, based on calcium phosphate (CaP) formulations, can greatly benefit from this discovery if adequate nanotopographies can be developed. However, CaP are reactive materials and experience ionic exchanges when placed into aqueous solutions which may in turn affect cell behaviour and complicate the interpretation of the bactericidal results. The present study explores the bactericidal potential of two nanopillared CaP prepared by hydrolysis of two different sizes of alpha-tricalcium phosphate (alpha-TCP) powders under biomimetic or hydrothermal conditions. A more lethal bactericidal response toward Pseudomonas aeruginosa (similar to 75% killing efficiency of adhered bacteria) was obtained from the hydrothermally treated CaP which consisted in a more irregular topography in terms of pillar size (radius: 20-60 nm), interpillar distances (100-1500 nm) and pillar distribution (pillar groups forming bouquets) than the biomimetically treated one (radius: 20-40 nm and interpillar distances: 50-200 nm with a homogeneous pillar distribution). The material reactivity was greatly influenced by the type of medium (nutrient-rich versus nutrient-free) and the presence or not of bacteria. A lower reactivity and superior bacterial attachment were observed in the nutrient-free medium while a lower attachment was observed for the nutrient rich medium which was explained by a superior reactivity of the material paired with the lower tendency of planktonic bacteria to adhere on surfaces in the presence of nutrients. Importantly, the ionic exchanges produced by the presence of materials were not toxic to planktonic cells. Thus, we can conclude that topography was the main contributor to mortality in the bacterial adhesion tests.

JTD Keywords: Adhesion, Antibacterial, Bactericidal, Biomaterials, Calcium deficient hydroxyapatite, Calcium phosphates, Hydroxyapatite, In-vitro, Infections, Nanopillars, Pseudomonas aeruginosa, Pseudomonas-aeruginosa, Reactivity, Recent progress, Silver, Topography, Transmission

Andrian, T, Pujals, S, Albertazzi, L, (2021). Quantifying the effect of PEG architecture on nanoparticle ligand availability using DNA-PAINT Nanoscale Advances 3, 6876-6881

The importance of PEG architecture on nanoparticle (NP) functionality is known but still difficult to investigate, especially at a single particle level. Here, we apply DNA Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT), a super-resolution microscopy (SRM) technique, to study the surface functionality in poly(lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG) NPs with different PEG structures. We demonstrated how the length of the PEG spacer can influence the accessibility of surface chemical functionality, highlighting the importance of SRM techniques to support the rational design of functionalized NPs.

JTD Keywords: chain-length, density, plga, surface, systems, Chain-length, Density, Dna, Microscopy technique, Nanoparticles, Nanoscale topography, Paint, Peg spacers, Plga, Poly lactide-co-glycolide, Poly-lactide-co-glycolide, Polyethylene glycols, Polylactide-co-glycolide, Single-particle, Super-resolution microscopy, Superresolution microscopy, Surface, Surface chemicals, Surface functionalities, Systems

Nashimoto Y, Abe M, Fujii R, Taira N, Ida H, Takahashi Y, Ino K, Ramon-Azcon J, Shiku H, (2021). Topography and Permeability Analyses of Vasculature-on-a-Chip Using Scanning Probe Microscopies Advanced Healthcare Materials 10

Microphysiological systems (MPS) or organs-on-chips (OoC) can emulate the physiological functions of organs in vitro and are effective tools for determining human drug responses in preclinical studies. However, the analysis of MPS has relied heavily on optical tools, resulting in difficulties in real-time and high spatial resolution imaging of the target cell functions. In this study, the role of scanning probe microscopy (SPM) as an analytical tool for MPS is evaluated. An access hole is made in a typical MPS system with stacked microchannels to insert SPM probes into the system. For the first study, a simple vascular model composed of only endothelial cells is prepared for SPM analysis. Changes in permeability and local chemical flux are quantitatively evaluated during the construction of the vascular system. The morphological changes in the endothelial cells after flow stimulation are imaged at the single-cell level for topographical analysis. Finally, the possibility of adapting the permeability and topographical analysis using SPM for the intestinal vascular system is further evaluated. It is believed that this study will pave the way for an in situ permeability assay and structural analysis of MPS using SPM.

JTD Keywords: cell, electrochemical microscopy, membrane-permeability, microphysiological systems, organs-chips, platform, scanning electrochemical microscopy, scanning ion conductance microscopy, scanning probe microscopy, shear-stress, surface-topography, Ion conductance microscopy, Microphysiological systems, Organs-chips, Scanning electrochemical microscopy, Scanning ion conductance microscopy, Scanning probe microscopy

Brennan M, Monahan DS, Brulin B, Gallinetti S, Humbert P, Tringides C, Canal C, Ginebra MP, Layrolle P, (2021). Biomimetic versus sintered macroporous calcium phosphate scaffolds enhanced bone regeneration and human mesenchymal stromal cell engraftment in calvarial defects Acta Biomaterialia 135, 689-704

In contrast to sintered calcium phosphates (CaPs) commonly employed as scaffolds to deliver mesenchymal stromal cells (MSCs) targeting bone repair, low temperature setting conditions of calcium deficient hydroxyapatite (CDHA) yield biomimetic topology with high specific surface area. In this study, the healing capacity of CDHA administering MSCs to bone defects is evaluated for the first time and compared with sintered beta-tricalcium phosphate (β-TCP) constructs sharing the same interconnected macroporosity. Xeno-free expanded human bone marrow MSCs attached to the surface of the hydrophobic β-TCP constructs, while infiltrating the pores of the hydrophilic CDHA. Implantation of MSCs on CaPs for 8 weeks in calvaria defects of nude mice exhibited complete healing, with bone formation aligned along the periphery of β-TCP, and conversely distributed within the pores of CDHA. Human monocyte-osteoclast differentiation was inhibited in vitro by direct culture on CDHA compared to β-TCP biomaterials and indirectly by administration of MSC-conditioned media generated on CDHA, while MSCs increased osteoclastogenesis in both CaPs in vivo. MSC engraftment was significantly higher in CDHA constructs, and also correlated positively with bone in-growth in scaffolds. These findings demonstrate that biomimetic CDHA are favorable carriers for MSC therapies and should be explored further towards clinical bone regeneration strategies. Statement of significance: Delivery of mesenchymal stromal cells (MSCs) on calcium phosphate (CaP) biomaterials enhances reconstruction of bone defects. Traditional CaPs are produced at high temperature, but calcium deficient hydroxyapatite (CDHA) prepared at room temperature yields a surface structure more similar to native bone mineral. The objective of this study was to compare the capacity of biomimetic CDHA scaffolds with sintered β-TCP scaffolds for bone repair mediated by MSCs for the first time. In vitro, greater cell infiltration occurred in CDHA scaffolds and following 8 weeks in vivo, MSC engraftment was higher in CDHA compared to β-TCP, as was bone in-growth. These findings demonstrate the impact of material features such as surface structure, and highlight that CDHA should be explored towards clinical bone regeneration strategies.

JTD Keywords: beta-tricalcium phosphate, bone regeneration, calcium deficient hydroxyapatite, differentiation, engraftment, human bone marrow mesenchymal stromal cells, hydroxyapatite scaffolds, in-vitro, inhibition, osteogenesis, osteoinduction, stem-cells, surface-topography, tissue, Beta-tricalcium phosphate, Bone regeneration, Calcium deficient hydroxyapatite, Engraftment, Human bone marrow mesenchymal stromal cells

Minguela J, Müller DW, Mücklich F, Llanes L, Ginebra MP, Roa JJ, Mas-Moruno C, (2021). Peptidic biofunctionalization of laser patterned dental zirconia: A biochemical-topographical approach Materials Science & Engineering C-Materials For Biological Applications 125,

A dual approach employing peptidic biofunctionalization and laser micro-patterns on dental zirconia was explored, with the aim of providing a flexible tool to improve tissue integration of restorations. Direct laser interference patterning with a femtosecond Ti:Sapphire laser was employed, and two periodic grooved patterns were produced with a periodicity of 3 and 10 μm. A platform containing the cell-adhesive RGD and the osteogenic DWIVA peptides was used to functionalize the grooved surfaces. Topography and surface damage were characterized by confocal laser scanning (CLSM), scanning electron and scanning transmission electron microscopy techniques. The surface patterns exhibited a high homogeneity and subsurface damage was found in the form of nano-cracks and nano-pores, at the bottom of the valleys. Accelerated tests in water steam were carried out to assess hydrothermal degradation resistance, which slightly decreased after the laser treatment. Interestingly, the detrimental effects of the laser modification were reverted by a post-laser thermal treatment. The attachment of the molecule was verified trough fluorescence CLSM and X-ray photoelectron spectroscopy. Finally, the biological properties of the surfaces were studied in human mesenchymal stem cells. Cell adhesion, morphology, migration and differentiation were investigated. Cells on grooved surfaces displayed an elongated morphology and aligned along the patterns. On these surfaces, migration was greatly enhanced along the grooves, but also highly restricted in the perpendicular direction as compared to flat specimens. After biofunctionalization, cell number and cell area increased and well-developed cell cytoskeletons were observed. However, no effects on cell migration were found for the peptidic platform. Although some osteogenic potential was found in specimens grooved with a periodicity of 10 μm, the largest effects were observed from the biomolecule, which favored upregulation of several genes related to osteoblastic differentiation in all the surfaces.

JTD Keywords: alumina toughened zirconia, cell alignment, grain-size, implants, interference, laser patterning, osteogenic differentiation, osteointegration, peptides, surface functionalization, surface-topography, tissue, titanium surface, Laser patterning, Low-temperature degradation, Osteointegration, Peptides, Surface functionalization, Zirconia

Steeves, A.J., Ho, W., Munisso, M.C., Lomboni, D.J., Larrañaga, E., Omelon, S., Martínez, Elena, Spinello, D., Variola, F., (2020). The implication of spatial statistics in human mesenchymal stem cell response to nanotubular architectures International Journal of Nanomedicine 15, 2151-2169

Introduction: In recent years there has been ample interest in nanoscale modifications of synthetic biomaterials to understand fundamental aspects of cell-surface interactions towards improved biological outcomes. In this study, we aimed at closing in on the effects of nanotubular TiO2 surfaces with variable nanotopography on the response on human mesenchymal stem cells (hMSCs). Although the influence of TiO2 nanotubes on the cellular response, and in particular on hMSC activity, has already been addressed in the past, previous studies overlooked critical morphological, structural and physical aspects that go beyond the simple nanotube diameter, such as spatial statistics. Methods: To bridge this gap, we implemented an extensive characterization of nanotubular surfaces generated by anodization of titanium with a focus on spatial structural variables including eccentricity, nearest neighbour distance (NND) and Voronoi entropy, and associated them to the hMSC response. In addition, we assessed the biological potential of a two-tiered honeycomb nanoarchitecture, which allowed the detection of combinatory effects that this hierarchical structure has on stem cells with respect to conventional nanotubular designs. We have combined experimental techniques, ranging from Scanning Electron (SEM) and Atomic Force (AFM) microscopy to Raman spectroscopy, with computational simulations to characterize and model nanotubular surfaces. We evaluated the cell response at 6 hrs, 1 and 2 days by fluorescence microscopy, as well as bone mineral deposition by Raman spectroscopy, demonstrating substrate-induced differential biological cueing at both the short- and long-term. Results: Our work demonstrates that the nanotube diameter is not sufficient to comprehensively characterize nanotubular surfaces and equally important parameters, such as eccentricity and wall thickness, ought to be included since they all contribute to the overall spatial disorder which, in turn, dictates the overall bioactive potential. We have also demonstrated that nanotubular surfaces affect the quality of bone mineral deposited by differentiated stem cells. Lastly, we closed in on the integrated effects exerted by the superimposition of two dissimilar nanotubular arrays in the honeycomb architecture. Discussion: This work delineates a novel approach for the characterization of TiO2 nanotubes which supports the incorporation of critical spatial structural aspects that have been overlooked in previous research. This is a crucial aspect to interpret cellular behaviour on nanotubular substrates. Consequently, we anticipate that this strategy will contribute to the unification of studies focused on the use of such powerful nanostructured surfaces not only for biomedical applications but also in other technology fields, such as catalysis.

JTD Keywords: Nanotubes, Nanotopography, Spatial statistics, Stem cells, Bone quality

Ciapetti, G., Di Pompo, G., Avnet, S., Martini, D., Diez-Escudero, A., Montufar, E. B., Ginebra, M. P., Baldini, N., (2017). Osteoclast differentiation from human blood precursors on biomimetic calcium-phosphate substrates Acta Biomaterialia 50, 102-113

The design of synthetic bone grafts to foster bone formation is a challenge in regenerative medicine. Understanding the interaction of bone substitutes with osteoclasts is essential, since osteoclasts not only drive a timely resorption of the biomaterial, but also trigger osteoblast activity. In this study, the adhesion and differentiation of human blood-derived osteoclast precursors (OCP) on two different micro-nanostructured biomimetic hydroxyapatite materials consisting in coarse (HA-C) and fine HA (HA-F) crystals, in comparison with sintered stoichiometric HA (sin-HA, reference material), were investigated. Osteoclasts were induced to differentiate by RANKL-containing supernatant using cell/substrate direct and indirect contact systems, and calcium (Ca++) and phosphorus (P5+) in culture medium were measured. We observed that OCP adhered to the experimental surfaces, and that osteoclast-like cells formed at a rate influenced by the micro- and nano-structure of HA, which also modulate extracellular Ca++. Qualitative differences were found between OCP on biomimetic HA-C and HA-F and their counterparts on plastic and sin-HA. On HA-C and HA-F cells shared typical features of mature osteoclasts, i.e. podosomes, multinuclearity, tartrate acid phosphatase (TRAP)-positive staining, and TRAP5b-enzyme release. However, cells were less in number compared to those on plastic or on sin-HA, and they did not express some specific osteoclast markers. In conclusion, blood-derived OCP are able to attach to biomimetic and sintered HA substrates, but their subsequent fusion and resorptive activity are hampered by surface micro-nano-structure. Indirect cultures suggest that fusion of OCP is sensitive to topography and to extracellular calcium. Statement of Significance: The novelty of the paper is the differentiation of human blood-derived osteoclast precursors, instead of mouse-derived macrophages as used in most studies, directly on biomimetic micro-nano structured HA-based surfaces, as triggered by osteoblast-produced factors (RANKL/OPG), and influenced by chemistry and topography of the substrate(s). Biomimetic HA-surfaces, like those obtained in calcium phosphate cements, are very different from the conventional calcium phosphate ceramics, both in terms of topography and ion exchange. The role of these factors in modulating precursors’ differentiation and activity is analysed. The system is closely reproducing the physiological process of attachment of host cells and further maturation to osteoclasts toward resorption of the substrate, which occurs in vivo after filling bone defects with the calcium phosphate grafts.

JTD Keywords: Bone resorption, Differentiation, Hydroxyapatite, Ionic exchange, Osteoclasts, Topography

Abadías, Clara, Serés, Carme, Torrent-Burgués, J., (2015). AFM in peak force mode applied to worn siloxane-hydrogel contact lenses Colloids and Surfaces B: Biointerfaces 128, 61-66

The objective of this work is to apply Atomic Force Microscopy in Peak Force mode to obtain topographic characteristics (mean roughness, root-mean-square roughness, skewness and kurtosis) and mechanical characteristics (adhesion, elastic modulus) of Siloxane-Hydrogel Soft Contact Lenses (CLs) of two different materials, Lotrafilcon B of Air Optix (AO) and Asmofilcon A of PremiO (P), after use (worn CLs). Thus, the results obtained with both materials will be compared, as well as the changes produced by the wear at a nanoscopic level. The results show significant changes in the topographic and mechanical characteristics of the CLs, at a nanoscopic level, due to wear. The AO CL show values of the topographic parameters lower than those of the P CL after wear, which correlates with a better comfort qualification given to the former by the wearers. A significant correlation has also been obtained between the adhesion values found after the use of the CLs with tear quality tests, both break-up-time and Schirmer.

JTD Keywords: Adhesion, Atomic force microscopy-peak force mode, Surface topography, Worn siloxane-hydrogel contact lenses, Young modulus

Álvarez, Z., Sena, E., Mattotti, M., Engel, E., Alcántara, S., (2014). An efficient and reproducible method to culture Bergmann and cortical radial glia using textured PMMA Journal of Neuroscience Methods , 232, 93-101

Background: Radial glia cells comprise the principal population of neural stem cells (NSC) during development. Attempts to develop reproducible radial glia and NSC culture methods have met with variable results, yielding non-adherent cultures or requiring the addition of growth factors. Recent studies demonstrated that a 2-μm patterned poly-methyl methacrylate (ln2 PMMA) grooved scaffold, by mimicking the biophysical and microtopographic properties of the embryonic NSC niche, induces the de-differentiation of glial cells into functional radial glia cells. New method: Here we describe a method for obtaining cultures of adherent Bergmann radial glia (BRG) and cortical radial glia (CRG). The growth substrate is ln2 PMMA and the addition of growth factors is not required. Results: Postnatal glia obtained from mouse cerebellum or cerebral cortex and grown on ln2 PMMA adopted a BRG/CRG phenotype characterized by a bipolar shape, the up-regulation of progenitor markers such as nestin and Sox2, and the down-regulation of vimentin and GFAP. Neurons cultured over the BRG/CRG aligned their processes with those of the glial shafts, thus mimicking the behavior of migrating neuronal cells. Comparison with existing methods: The ln2 PMMA culture method offers an ideal system for analyzing both the biochemical factors controlling the neurogenic potential of BRG/CRG and neuronal migration. Conclusions: The ln2 PMMA method is a reproducible system to obtain immature BRG/CRG preparations in vitro. It can be used to study the properties of CNS progenitor cells as well as the interactions between radial glia and neurons, and supports cultured progenitors for use in different applications. © 2014 Elsevier B.V.

JTD Keywords: Astrocytes, Bergmann glia, Micro-patterning, Poly-methyl methacrylate (PMMA), Progenitors, Radial glia, Surface topography

Mendes, A. C., Smith, K. H., Tejeda-Montes, E., Engel, E., Reis, R. L., Azevedo, H. S., Mata, Alvaro, (2013). Co-assembled and microfabricated bioactive membranes Advanced Functional Materials 23, (4), 430-438

The fabrication of hierarchical and bioactive self-supporting membranes, which integrate physical and biomolecular elements, using a single-step process that combines molecular self-assembly with soft lithography is reported. A positively charged multidomain peptide (with or without the cell-adhesive sequence arginine-glycine-aspartic acid-serine (RGDS)) self-assembles with hyaluronic acid (HA), an anionic biopolymer. Optimization of the assembling conditions enables the realization of membranes with well-controlled and easily tunable features at multiple size scales including peptide sequence, building-block co-assembly, membrane thickness, bioactive epitope availability, and topographical pattern morphology. Membrane structure, morphology, and bioactivity are investigated according to temperature, assembly time, and variations in the experimental setup. Furthermore, to evaluate the physical and biomolecular signaling of the self-assembled microfabricated membranes, rat mesenchymal stem cells are cultured on membranes exhibiting various densities of RGDS and different topographical patterns. Cell adhesion, spreading, and morphology are significantly affected by the surface topographical patterns and the different concentrations of RGDS. The versatility of the combined bottom-up and top-down fabrication processes described may permit the development of hierarchical macrostructures with precise biomolecular and physical properties and the opportunity to fine tune them with spatiotemporal control.

JTD Keywords: Membrane scaffolds, Mesenchymal stem cells, Microfabrication, Self-assembly, Topography

Mattotti, Marta, Alvarez, Zaida, Ortega, Juan A., Planell, Josep A., Engel, Elisabeth, Alcántara, Soledad, (2012). Inducing functional radial glia-like progenitors from cortical astrocyte cultures using micropatterned PMMA Biomaterials 33, (6), 1759-1770

Radial glia cells (RGC) are multipotent progenitors that generate neurons and glia during CNS development, and which also served as substrate for neuronal migration. After a lesion, reactive glia are the main contributor to CNS regenerative blockage, although some reactive astrocytes are also able to de-differentiate in situ into radial glia-like cells (RGLC), providing beneficial effects in terms of CNS recovery. Thus, the identification of substrate properties that potentiate the ability of astrocytes to transform into RGLC in response to a lesion might help in the development of implantable devices that improve endogenous CNS regeneration. Here we demonstrate that functional RGLC can be induced from in vitro matured astrocytes by using a precisely-sized micropatterned PMMA grooved scaffold, without added soluble or substrate adsorbed biochemical factors. RGLC were extremely organized and aligned on 2 μm line patterned PMMA and, like their embryonic counterparts, express nestin, the neuron-glial progenitor marker Pax6, and also proliferate, generate different intermediate progenitors and support and direct axonal growth and neuronal migration. Our results suggest that the introduction of line patterns in the size range of the RGC processes in implantable scaffolds might mimic the topography of the embryonic neural stem cell niche, driving endogenous astrocytes into an RGLC phenotype, and thus favoring the regenerative response in situ.

JTD Keywords: Polymethylmethacrylate, Micropatterning, Surface topography, Astrocyte, Nerve guide, Co-culture

Comelles, J., Hortigüela, V., Samitier, J., Martinez, E., (2012). Versatile gradients of covalently bound proteins on microstructured substrates Langmuir 28, (38), 13688-13697

In this work, we propose an easy method to produce highly tunable gradients of covalently bound proteins on topographically modified poly(methyl methacrylate). We used a rnicrofluidic approach to obtain linear gradients with high slope (0.5, relevant at the single-cell level. These protein gradients were characterized using fluorescence microscopy and surface plasmon resonance. Both experimental results and theoretical modeling on the protein gradients generated have proved them to be highly reproducible, stable up to 7 days, and easily tunable. This method enables formation of versatile cell culture platforms combining both complex biochemical and physical cues in an attempt to approach in vitro cell culture methods to in vivo cellular microenvironments.

JTD Keywords: Cell-migration, Microfluidic channel, Surface, Streptavidin, Molecules, Topography, Mechanisms, Generation, Responses, Guidance

Gustavsson, J., Ginebra, M. P., Planell, J., Engel, E., (2012). Osteoblast-like cellular response to dynamic changes in the ionic extracellular environment produced by calcium-deficient hydroxyapatite Journal of Materials Science-Materials in Medicine , 23, (10), 2509-2520

Solution-mediated reactions due to ionic substitutions are increasingly explored as a strategy to improve the biological performance of calcium phosphate-based materials. Yet, cellular response to well-defined dynamic changes of the ionic extracellular environment has so far not been carefully studied in a biomaterials context. In this work, we present kinetic data on how osteoblast-like SAOS-2 cellular activity and calcium-deficient hydroxyapatite (CDHA) influenced extracellular pH as well as extracellular concentrations of calcium and phosphate in standard in vitro conditions. Since cells were grown on membranes permeable to ions and proteins, they could share the same aqueous environment with CDHA, but still be physically separated from the material. In such culture conditions, it was observed that gradual material-induced adsorption of calcium and phosphate from the medium had only minor influence on cellular proliferation and alkaline phosphatase activity, but that competition for calcium and phosphate between cells and the biomaterial delayed and reduced significantly the cellular capacity to deposit calcium in the extracellular matrix. The presented work thus gives insights into how and to what extent solution-mediated reactions can influence cellular response, and this will be necessary to take into account when interpreting CDHA performance both in vitro and in vivo.

JTD Keywords: Alkaline-phosphatase activity, Saos-2 cells, In-vitro, bone mineralization, Biological basis, Differentiation, Culture, Matrix, Proliferation, Topography

Pegueroles, M., Aparicio, C., Bosio, M., Engel, E., Gil, F. J., Planell, J. A., Altankov, G., (2010). Spatial organization of osteoblast fibronectin matrix on titanium surfaces: Effects of roughness, chemical heterogeneity and surface energy Acta Biomaterialia 6, (1), 291-301

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-label led 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.

JTD Keywords: Fibronectin, Extracellular matrix organization, Titanium, Surface topography, Surface energy

Martinez, E., Engel, E., Planell, J. A., Samitier, J., (2009). Effects of artificial micro- and nano-structured surfaces on cell behaviour Annals of Anatomy-Anatomischer Anzeiger , 191, (1), 126-135

Substrate topography, independently of substrate chemistry, has been reported to have significant effects on cell behaviour. Based on the use of fabrication techniques developed by the silicon microtechnology industry, numerous studies can now be found in the literature analyzing cell behaviour as to various micro- and nanofeatures such as lines, wells, holes and more. Most of these works have been found to relate the micro- and nano-sized topographical features with cell. orientation, migration, morphology and proliferation. In recent papers, even the influence of substrate nanotopography on cell gene expression and differentiation has been pointed out. However, despite the large number of papers published on this topic, significant general trends in cell behaviour are difficult to establish due to differences in cell type, substrate material, feature aspect-ratio, feature geometry and parameters measured. This paper intends to compile and review the relevant existing information on the behaviour of cells on micro- and nano-structured artificial substrates and analyze possible general behavioural trends.

JTD Keywords: Microstructure, Topography, Cell behaviour, Cell morphology, Cell orientation

Engel, E., Martinez, E., Mills, C. A., Funes, M., Planell, J. A., Samitier, J., (2009). Mesenchymal stem cell differentiation on microstructured poly (methyl methacrylate) substrates Annals of Anatomy-Anatomischer Anzeiger , 191, (1), 136-144

Recent studies on 2D substrates have revealed the importance of surface properties in affecting cell behaviour. In particular, surface topography appears to influence and direct cell migration. The development of new technologies of hot embossing and micro-imprinting has made it possible to study cell interactions with controlled micro features and to determine how these features can affect cell behaviour. Several studies have been carried out on the effect of microstructures on cell adhesion, cell guidance and cell proliferation. However, there is still a lack of knowledge on how these features affect mesenchymal stem cell differentiation. This study was designed to evaluate whether highly controlled microstructures on PMMA could induce rMSC differentiation into an osteogenic lineage. Structured PMMA was seeded with rMSC and cell number; cell morphology and cell differentiation were evaluated. Results confirm that microstructures not only affect cell proliferation and alignment but also have a synergistic effect with osteogenic medium on rMSC differentiation into mature osteoblasts.

JTD Keywords: Mesenchymal stem cells, Osteoblasts, Topography, Microstructures

Fernandez, Javier G., Mills, C. A., Martinez, E., Lopez-Bosque, M. J., Sisquella, X., Errachid, A., Samitier, J., (2008). Micro- and nanostructuring of freestanding, biodegradable, thin sheets of chitosan via soft lithography Journal of Biomedical Materials Research - Part A , 85A, (1), 242-247

A technique for imparting micro- and nano-structured topography into the surface of freestanding thin sheets of chitosan is described. Both micro- and nanometric surface structures have been produced using soft lithography. The soft lithography method, based on solvent evaporation, has allowed structures similar to 60 nm tall and similar to 500 X 500 nm(2) to be produced on freestanding similar to 0.5 mm thick sheets of the polymer when cured at 293 K, and structures similar to 400 nm tall and 5 X 5 mu m(2) to be produced when cured at 283 K. Nonstructured chitosan thin sheets (similar to 200 mu m thick) show excellent optical transmission properties in the visible portion of the electromagnetic spectrum. The structured sheets can be used for applications where optical microscopic analysis is required, such as cell interaction experiments and tissue engineering.

JTD Keywords: Chitin/chitosan, Microstructure, Nanotopography, Polymerization, Soft lithography

Martinez, E., Engel, E., Lopez-Iglesias, C., Mills, C. A., Planell, J. A., Samitier, J., (2008). Focused ion beam/scanning electron microscopy characterization of cell behavior on polymer micro-/nanopatterned substrates: A study of cell-substrate interactions Micron , 39, (2), 111-116

Topographic micro and nanostructures can play an interesting role in cell behaviour when cells are cultured on these kinds of patterned substrates. It is especially relevant to investigate the influence of the nanometric dimensions topographic features on cell morphology, proliferation, migration and differentiation. To this end, some of the most recent fabrication technologies, developed for the microelectronics industry, can be used to produce well-defined micro and nanopatterns on biocompatible polymer substrates. In this work, osteoblast-like cells are grown on poly(methyl methacrylate) substrates patterned by nanoimprint lithography techniques. Examination of the cell-substrate interface can reveal important details about the cell morphology and the distribution of the focal contacts on the substrate surface. For this purpose, a combination of focused ion beam milling and scanning electron microscopy techniques has been used to image the cell-substrate interface. This technique, if applied to samples prepared by freeze-drying methods, allows high-resolution imaging of cross-sections through the cell and the substrate, where the interactions between the nanopatterned substrate, the cell and the extracellular matrix, which are normally hidden by the bulk of the cell, can be studied.

JTD Keywords: Electron microscopy, Interface, Nanotopography, Osteoblast, Adhesion molecule, Cell morphology