Nanotopographical Features Research Articles

Conductive Silk-Polypyrrole Composite Scaffolds with Bioinspired Nanotopographic Cues for Cardiac Tissue Engineering.

We report on the development of bioinspired cardiac scaffolds made from electroconductive acid-modified silk fibroin-poly(pyrrole) (AMSF+PPy) substrates patterned with nanoscale ridges and grooves reminiscent of native myocardial extracellular matrix (ECM) topography to enhance the structural and functional properties of cultured human pluripotent stem cells (hPSC)-derived cardiomyocytes. Nanopattern fidelity was maintained throughout the fabrication and functionalization processes, and no loss in conductive behavior occurred due to the presence of the nanotopographical features. AMSF+PPy substrates were biocompatible and stable, maintaining high cell viability over a 21-day culture period while displaying no signs of PPy delamination. The presence of anisotropic topographical cues led to increased cellular organization and sarcomere development, and electroconductive cues promoted a significant improvement in the expression and polarization of connexin 43 (Cx43), a critical regulator of cell-cell electrical coupling. The combination of biomimetic topography and electroconductivity also increased the expression of genes that encode key proteins involved in regulating the contractile and electrophysiological function of mature human cardiac tissue.

Layered titanates with fibrous nanotopographic features as reservoir for bioactive ions to enhance osteogenesis

In this study, an osteogenic environment was constructed on Ti alloy implants by in-situ formation of nanosized fibrous titanate, Na2Ti6O13, loaded with bioactive ions, i.e. Sr, Mg and Zn, to enhance surface bioactivity. The bioactive ions were loaded by ion exchange with sodium located at inter-layer positions between the TiO6 slabs, and their release was not associated with the degradation of the structural unit of the titanate. In-vitro cell culture experiments using MC3T3-E1 cells proved that both bioactive ions and nanotopographic features are critical in promoting osteogenic differentiation of the cells. It was found that the osteogenic functions of the titanate can be modulated by the type and amount of ions incorporated. This study points out that nanosized fibrous titanate formed on the Ti alloy can be a promising reservoir for bioactive ions. The major advantage of this approach over other alternatives for bioactive ion delivery using degradable bioceramic coatings is its capacity of maintaining the structural integrity of the coating and thus avoiding structural deterioration and potential mechanical failure.

Towards the cell-instructive bactericidal substrate: exploring the combination of nanotopographical features and integrin selective synthetic ligands

Engineering the interface between biomaterials and tissues is important to increase implant lifetime and avoid failures and revision surgeries. Permanent devices should enhance attachment and differentiation of stem cells, responsible for injured tissue repair, and simultaneously discourage bacterial colonization; this represents a major challenge. To take first steps towards such a multifunctional surface we propose merging topographical and biochemical cues on the surface of a clinically relevant material such as titanium. In detail, our strategy combines antibacterial nanotopographical features with integrin selective synthetic ligands that can rescue the adhesive capacity of the surfaces and instruct mesenchymal stem cell (MSC) response. To this end, a smooth substrate and two different high aspect ratio topographies have been produced and coated either with an αvβ3-selective peptidomimetic, an α5β1-selective peptidomimetic, or an RGD/PHSRN peptidic molecule. Results showed that antibacterial effects of the substrates could be maintained when tested on pathogenic Pseudomonas aeruginosa. Further, functionalization increased MSC adhesion to the surfaces and the αvβ3-selective peptidomimetic-coated nanotopographies promoted osteogenesis. Such a dual physicochemical approach to achieve multifunctional surfaces represents a first step in the design of novel cell-instructive biomaterial surfaces.

The Role of Controlled Surface Topography and Chemistry on Mouse Embryonic Stem Cell Attachment, Growth and Self-Renewal.

The success of stem cell therapies relies heavily on our ability to control their fate in vitro during expansion to ensure an appropriate supply. The biophysical properties of the cell culture environment have been recognised as a potent stimuli influencing cellular behaviour. In this work we used advanced plasma-based techniques to generate model culture substrates with controlled nanotopographical features of 16 nm, 38 nm and 68 nm in magnitude, and three differently tailored surface chemical functionalities. The effect of these two surface properties on the adhesion, spreading, and self-renewal of mouse embryonic stem cells (mESCs) were assessed. The results demonstrated that physical and chemical cues influenced the behaviour of these stem cells in in vitro culture in different ways. The size of the nanotopographical features impacted on the cell adhesion, spreading and proliferation, while the chemistry influenced the cell self-renewal and differentiation.

Nanotopographic Influence on the In Vitro Behavior of Induced Pluripotent Stem Cells.

While the influence of nanotopography on stem cell behavior has been extensively investigated on adult stem cells, far fewer studies have investigated the interaction of induced pluripotent stem cells (iPSCs) with various nanotopographical patterns. The purpose of this study was to identify nanopatterns that can influence the stemness and proliferation, as well as the adhesive properties in iPSCs, and thereby explore the feasibility of applying these nano-features for regenerative medicine. Three kinds of nanopatterns were fabricated from polydimethylsiloxane membranes, irregular patterned membrane (IPM), groove patterned membrane (GPM), and postpatterned membrane (PPM), in addition to flat patterned membrane (FPM) which did not have any nanotopographic features and was used as the control pattern. On the surfaces of GPM or PPM, iPSCs showed tendency for aggregation and did not spread out well at passage 1. However, with continued passaging (P6, P10), the tendency to form aggregates was greatly reduced. While iPSCs cultured on GPM and PPM had low population doubling time values compared with FPM and IPM at P1, the differences disappeared in later passages. The expression of the cell proliferation marker Ki67 in iPSCs gradually decreased with continued passaging in cells cultured on FPM and IPM, but not in those cultured on GPM and PPM. The expression of Oct3/4 and Nanog, marker of stemness, was significantly higher on GPM and PPM than on FPM at P6 and P10. At P5, numerous filopodia were demonstrated in the peripheral attachments of iPSC colonies on FPM and IPM, while GPM and PPM generally had globular appearance. The expression of the focal adhesion (FA) molecules α-actinin, vinculin, phalloidin, or FA kinase was significantly greater on GPM and PPM than on FPM and IPM at P6 or P10. In conclusion, continued passaging on regular nanopatterns, including groove- and post-forms, was effective in maintaining an undifferentiated state and proliferation of iPSCs and also in increasing the expression of FA molecules.

Directional Matrix Nanotopography with Varied Sizes for Engineering Wound Healing.

Topographic features play a crucial role in the regulation of physiologically relevant cell and tissue functions. Here, an analysis of feature-size-dependent cell-nanoarchitecture interactions is reported using an array of scaffolds in the form of uniformly spaced ridge/groove structures for engineering wound healing. The ridge and groove widths of nanopatterns are varied from 300 to 800 nm and the nanotopography features are classified into three size ranges: dense (300-400 nm), intermediate (500-600 nm), and sparse (700-800 nm). On these matrices, fibroblasts demonstrate a biphasic trend of cell body and nucleus elongation showing the maximum at intermediate feature density, whereas maximum migration speed is observed at the dense case with monotonic decrease upon increasing feature size. The directional organization of cell-synthesized fibronectin fibers can be regulated differently via the nanotopographical features. In an in vitro wound healing model, the covering rate of cell-free regions is maximized on the dense nanotopography and decreased with increasing feature size, showing direct correlation with the trend of migration speed. It is demonstrated that the properties of repaired tissue matrices in the process of wound healing may be controlled via the feature-size-dependent cell-nanoarchitecture interactions, which can be an important consideration for designing tissue engineering scaffolds.

Neuron–Material Nanointerfaces: Surface Nanotopography Governs Neuronal Differentiation and Development

AbstractThis review summarizes recent experimental results regarding the influences of surface nanotopography on stem cell differentiation into neurons and neuronal development. Collections of observations and their interpretations are provided. We specifically focus on the in vivo implications of the results, by suggesting that: (i) neural cells use their intracellular machineries to actively recognize surface nanotopography; (ii) the preferential differentiation of stem cells into neurons and the developmental acceleration of neurons occur on a range of similar nanotopographical features, implying that they have similar biological backgrounds; and (iii) surface nanotopography, therefore, is deeply involved in brain development. We believe that this review will provide biological insights to the field of nano–neuron interfaces and encourage interdisciplinary efforts capable of addressing unsolved biological problems and increasing engineering needs.

The Role of Titanium Surface Nanostructuring on Preosteoblast Morphology, Adhesion, and Migration.

Surface structuring of titanium-based implants is known to modulate the behavior of adherent cells, but the influence of different nanotopographies is poorly understood. The aim is to investigate preosteoblast proliferation, adhesion, morphology, and migration on surfaces with similar surface chemistry but distinct nanotopographical features. Sonochemical treatment and anodic oxidation are employed to fabricate disordered, mesoporous titania (TMS) and ordered titania nanotubular (TNT) topographies on titanium, respectively. Morphological evaluation reveals that cells are polygonal and well-spread on TMS, but display an elongated, fibroblast-like morphology on TNT surfaces, while they are much flatter on glass. Both nanostructured surfaces impair cell adhesion, but TMS is more favorable for cell growth due to its support of cell attachment and spreading in contrast to TNT. A quantitative wound healing assay in combination with live-cell imaging reveals that cell migration on TMS surfaces has a more collective character than on other surfaces, probably due to a closer proximity between neighboring migrating cells on TMS. The results indicate distinctly different cell adhesion and migration on ordered and disordered titania nanotopographies, providing important information that can be used in optimizing titanium-based scaffold design to foster bone tissue growth and repair while allowing for the encapsulation of drugs into porous titania layer.

PDT light sources and PDT devices: The necessary tools for performing good photodynamic therapy treatments

Nanoscale topography plays a central role in adult-stem-cell-niches. In this study we have demonstrated that by an accurate material design, mimicking the composition and structure of bone extracellular matrix, it is possible to guide human adipose stem cells (hASCs) to attain an osteogenic commitment, in vitro, without the requirement of soluble additives or grow factors. Alkaline Phosphatase (ALP) activity and Alizarin Red S test demonstrated that hASCs cultured onto hydroxyapatite frameworks dry-coating differentiated into mature osteoblasts even in the absence of specific inducing factors. Optical and electron scanning microscopic (SEM) observations revealed a direct cellular-material interaction that indicated an appropriate communication between nanotopographical features and the integrin receptors in the cell´s focal adhesions. Real-time quantitative PCR (RT-qPCR) analysis confirmed the expression of specific markers of pre-osteoblast and mature osteoblast stages as osterix (OSX), osteopontin (OPN), osteocalcin (OC) and; specific markers of extracellular matrix maturation and mineralization stages as alkaline phosphatase (ALPL), collagen type I alpha 1 (COL1A1) and osteonectin (ON). Osteoprotegerin (OPG), ALPL, COL1A1, and sclerostin (SOST) expressions were up regulated compared to osteogenic differentiation media conditions.

Engineered vesiscles from gingival stem cells: a new approach in 3D printed bone tissue regeneration

In the bone regeneration field, properties of 3D scaffold could be improved using cellular and their released products. Even if previously documented, poly-(lactide) (PLA) scaffolds were not thoroughly evaluated for their design-related characteristics. The aim of the study was to investigate the properties of 3D printed PLA scaffolds for bone regeneration obtained through 3D printing, evaluating the differences in terms of structural properties, in vitro and in vivo cellular responses induced by different scaffold structures. Biofabrication is to generate a construct with biological function. In particular in our reserach we describe the fully process, including printing scaffold step, in vitro culture phase and subsequently in vivo transplantation. Five porous scaffold designs (A-B-C-D-E) were fabricated from a poly-(lactide) (PLA) filament. Scaffold structural parameters, such as porosity and pore size, were measured using scanning electron microscopy, and micro-computed tomography. Nano-topographic surface features were investigated by means of atomic force microscopy. Over a 112-day period, scaffolds were hydrolytically degraded and changes in weight, pH and mechanical properties were measured during degradation. Osteogenic differentiation of hPDLSCs on different scaffold designs after 21 days of culture was measured by means of RT-PCR and Western Blot. In vivo study was performed using C57BL/6 mice and was designed in 5 different groups: - Group1: Scaffold loaded with hPDLSCs - Group2: Scaffold loaded with conditioned medium (CM) derived from hPDLSCs - Group3: Scaffold loaded with exosomes (Exo) purified from CM - Group 4: Scaffold loaded with engineered exosomes (e-Exo), exosome treated with PEI (poly ether imide) - Group5: Scaffold, used as control. Histological analysis were performed after 60 days of in vivo transplantation and morphological evaluations revealed a high bone tissue formation and osteogenic cells commitment in group 3 and 4 when compared to other groups. From these results, the cell-laden PCAMSC scaffold offers a significant advantage in the TM regeneration in a rat subacute TM perforation model. It may offer attractive opportunities in the conservative clinical treatment. This study demonstrated that scaffold of group 3 and 4 significantly improved bone tissue regeneration in animal model and successfully showed new bone deposition in situ compared to the control scaffold (group 5) and group 1 and 2. Based on the results, we believe that the bioprinted scaffold may become a novel treatment for tissue regeneration approach therapy.

Scale Invariant Disordered Nanotopography Promotes Hippocampal Neuron Development and Maturation with Involvement of Mechanotransductive Pathways.

The identification of biomaterials which promote neuronal maturation up to the generation of integrated neural circuits is fundamental for modern neuroscience. The development of neural circuits arises from complex maturative processes regulated by poorly understood signaling events, often guided by the extracellular matrix (ECM). Here we report that nanostructured zirconia surfaces, produced by supersonic cluster beam deposition of zirconia nanoparticles and characterized by ECM-like nanotopographical features, can direct the maturation of neural networks. Hippocampal neurons cultured on such cluster-assembled surfaces displayed enhanced differentiation paralleled by functional changes. The latter was demonstrated by single-cell electrophysiology showing earlier action potential generation and increased spontaneous postsynaptic currents compared to the neurons grown on the featureless unnaturally flat standard control surfaces. Label-free shotgun proteomics broadly confirmed the functional changes and suggests furthermore a vast impact of the neuron/nanotopography interaction on mechanotransductive machinery components, known to control physiological in vivo ECM-regulated axon guidance and synaptic plasticity. Our results indicate a potential of cluster-assembled zirconia nanotopography exploitable for the creation of efficient neural tissue interfaces and cell culture devices promoting neurogenic events, but also for unveiling mechanotransductive aspects of neuronal development and maturation.

Nanopattern-induced osteogenic differentiation of stem cells - A systematic review.

The use of nanotopography to induce cellular responses represents a novel and rapidly growing area of research. Nevertheless, the findings and trends so far are difficult to identify and discuss mostly due to a non-systematic research approach. The present manuscript is providing a systematic review focused on nanopattern-induced osteogenic differentiation of mesenchymal stem cells. The coverage of the most relevant aspects including nanopatterns fabrication methods, their effects on osteogenic differentiation of mesenchymal stem cells as well as the related effects on adhesion and cell morphology has enabled an integrated discussion including the potential mechanotransduction mechanisms involved. Furthermore, a clear distinction between the studies that use only surface nanotopographies and the ones that mix nanotopographical features with osteogenic supplements has been made. This delineation is essential for revealing and understanding the role of biomaterial's nanotopography per se on stem cells differentiation based on which novel osteoinductive biomaterials can be developed.

Multidimensional nanomaterials for the control of stem cell fate.

Current stem cell therapy suffers low efficiency in giving rise to differentiated cell lineages, which can replace the original damaged cells. Nanomaterials, on the other hand, provide unique physical size, surface chemistry, conductivity, and topographical microenvironment to regulate stem cell differentiation through multidimensional approaches to facilitate gene delivery, cell–cell, and cell–ECM interactions. In this review, nanomaterials are demonstrated to work both alone and synergistically to guide selective stem cell differentiation. From three different nanotechnology families, three approaches are shown: (1) soluble microenvironmental factors; (2) insoluble physical microenvironment; and (3) nano-topographical features. As regenerative medicine is heavily invested in effective stem cell therapy, this review is inspired to generate discussions in the potential clinical applications of multi-dimensional nanomaterials.

A Radially Organized Multipatterned Device as a Diagnostic Tool for the Screening of Topographies in Tissue Engineering Biomaterials.

Micro- and nanotextured biomaterial surfaces have been widely studied for their capacity to drive the regeneration of organized tissues. Nanotopographical features in the shape of groove-ridge patterns aim at mimicking the extracellular matrix organization. However, to date, a wide array of groove and ridge sizes has been described. In this work, we therefore tested a device composed of a multipatterned array consisting of six patterns of radially arranged parallel nanogrooves, with a pitch ranging from 0 to 1000 nm and a depth ranging from 0 to 170 nm, to be used as a tool for the expeditious and simultaneous screening of surface topographies aiming the regeneration of anisotropically organized tissues such as ligament. The topographies were reproduced in (1) epoxy resin or (2) membranes produced by the crosslinking of platelet lysate (PL) with genipin (gPL). Both materials were seeded with periodontal ligament cells (PDLCs) and the proliferation, migration, as well as cell alignment were assessed. The effect of topography in PDLCs was only evident in terms of cell organization, resulting in a highly anisotropic organization of the cells for the 1000 and 600 nm patterns, and in an increased isotropic organization for shallower topographies. Overall, our results suggest that this multipatterned system can be a valuable diagnostic tool for biomaterials aiming at the regeneration of anisotropically organized tissues, such as periodontal ligament.

Spatial Control of Cell-Nanosurface Interactions by Tantalum Oxide Nanodots for Improved Implant Geometry.

Nanotopological cues can be exploited to understand the nature of interactions between cells and their microenvironment to generate superior implant geometries. Nanosurface parameters which modulate the cell behavior and characteristics such as focal adhesions, cell morphology are not clearly understood. Here, we studied the role of different nanotopographic dimensions in modulating the cell behavior, characteristics and ultimately the cell fate and accordingly, a methodology to improve implant surface geometry is proposed. Tantalum oxide nanodots of 50, 100nm dot diameter with an inter-dot spacing of 20, 70nm and heights 40, 100nm respectively, were engineered on Silicon substrates. MG63 cells were cultured for 72 hours and the modulation in morphology, focal adhesions, cell extensible area, cell viability, transcription factors and genes responsible for bone protein secretion as a function of the nanodot diameter, inter-dot distance and nanodot height were evaluated. Nanodots of 50nm diameter with a 20nm inter-dot spacing and 40nm height enhanced cell spreading area by 40%, promoted cell viability by 70% and upregulated transcription factors and genes twice as much, as compared to the 100nm nanodots with 70nm inter-dot spacing and 100nm height. Favorable interactions between cells and all dimensions of 50nm nanodot diameter were observed, determined with Scanning electron microscopy and Immunofluorescence staining. Nanodot height played a vital role in controlling the cell fate. Dimensions of nanodot features which triggered a transition in cell characteristics or behavior was also defined through statistical analysis. The findings of this study provide insights in the parameters of nanotopographic features which can vitally control the cell fate and should therefore be taken into account when designing implant geometries.

Enhanced biocompatibility of TiO2 surfaces by highly reactive plasma

In the present study the biological response to various nanotopographic features after gaseous plasma treatment were studied. The usefulness of nanostructured surfaces for implantable materials has already been acknowledged, while less is known on the combined effect of nanostructured plasma modified surfaces. In the present work the influence of oxygen plasma treatment on nanostructured titanium oxide (TiO2) surfaces was studied. Characterization of the TiO2 surface chemical composition and morphological features was analyzed after plasma modification by x-ray photoelectron spectroscopy and by scanning electron microscopy while surface wettability was studied with measuring the water contact angle. Cell adhesion and morphology was assessed from images taken with scanning electron microscopy, whereas cell viability was measured with a calorimetric assay. The obtained results showed that oxygen plasma treatment of TiO2 nanotube surfaces significantly influences the adhesion and morphology of osteoblast-like cells in comparison to untreated nanostructured surfaces. Marked changes in surface composition of plasma treated surfaces were observed, as plasma treatment removed hydrocarbon contamination and removed fluorine impurities, which were present due to the electrochemical anodization process. However no differences in wettability of untreated and plasma treated surfaces were noticed. Treatment with oxygen plasma stimulated osteoblast-like cell adhesion and spreading on the nanostructured surface, suggesting the possible use of oxygen plasma surface treatment to enhance osteoblast-like cell response.

Enhancement of Capture Sensitivity for Circulating Tumor Cells in a Breast Cancer Patient's Blood by Silicon Nanowire Platform.

The separation of circulating tumor cells (CTCs) from the blood of cancer patients with high sensitivity is an essential technique for selecting chemotherapeutic agents at a patient-by-patient level. Recently, various research groups have reported a nanostructure-based platform for rare cell capture due to its high surface area and 3D nanotopographic features. However, evaluation of capture sensitivity based on chemical modification of the nanostructure surface has not yet been performed. Here, we evaluated the capture sensitivity for CTCs from the blood of three patients diagnosed with stage IV metastatic breast cancer by using the following three platforms: streptavidin-conjugated silicon nanowire (STR-SiNW), poly-l-lysine-coated silicon nanowire (PLL-SiNW), and poly-l-lysine-coated glass (PLL-glass). The number of evaluated CTCs on STR-SiNW, PLL-SiNW, and PLL-glass were 16.2 ± 5.5 cells, 7.3 ± 2.9 cells, and 4.7 ± 1.5 cells, respectively, per 0.5 ml. Therefore, we suggest that the STR-SiNW platform is highly adaptable for the quantitative evaluation of CTCs from the blood of cancer patients in the clinical setting.

ZnO Nanostructure Templates as a Cost-Efficient Mass-Producible Route for the Development of Cellular Networks.

The development of artificial surfaces which can regulate or trigger specific functions of living cells, and which are capable of inducing in vivo-like cell behaviors under in vitro conditions has been a long-sought goal over the past twenty years. In this work, an alternative, facile and cost-efficient method for mass-producible cellular templates is presented. The proposed methodology consists of a cost-efficient, two-step, all-wet technique capable of producing ZnO-based nanostructures on predefined patterns on a variety of substrates. ZnO—apart from the fact that it is a biocompatible material—was chosen because of its multifunctional nature which has rendered it a versatile material employed in a wide range of applications. Si, Si3N4, emulated microelectrode arrays and conventional glass cover slips were patterned at the micrometer scale and the patterns were filled with ZnO nanostructures. Using HeLa cells, we demonstrated that the fabricated nanotopographical features could promote guided cellular adhesion on the pre-defined micron-scale patterns only through nanomechanical cues without the need for further surface activation or modification. The basic steps of the micro/nanofabrication are presented and the results from the cell adhesion experiments are discussed, showing the potential of the suggested methodology for creating low-cost templates for engineered cellular networks.

Sub-nanometer resolution of an organic semiconductor crystal surface using friction force microscopy in water

Organic semiconductors (OSC) are attracting much interest for (opto)electronic applications, such as photovoltaics, LEDs, sensors or solid state lasers. In particular, crystals formed by small -conjugated molecules have shown to be suitable for constructing OSC devices. However, the (opto)electronic properties are complex since they depend strongly on both the mutual orientation of molecules as well as the perfection of bulk crystal surfaces. Hence, there is an urgent need to control nano-topographic OSC features in real space. Here we show that friction force microscopy in water is a very suitable technique to image the free surface morphology of an OSC single crystal (TDDCS) with sub-nanometer resolution. We demonstrate the power of the method by direct correlation to the structural information extracted from combined single crystal (SC-) and specular (s-) XRD studies, which allows us to identify the pinning centers encountered in the stick-slip motion of the probing tip with the topmost methyl groups on the TDDCS surface.

Nanotopography Promotes Pancreatic Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells.

Although previous studies suggest that nanotopographical features influence properties and behaviors of stem cells, only a few studies have attempted to derive clinically useful somatic cells from human pluripotent stem cells using nanopatterned surfaces. In the present study, we report that polystyrene nanopore-patterned surfaces significantly promote the pancreatic differentiation of human embryonic and induced pluripotent stem cells. We compared different diameters of nanopores and showed that 200 nm nanopore-patterned surfaces highly upregulated the expression of PDX1, a critical transcription factor for pancreatic development, leading to an approximately 3-fold increase in the percentage of differentiating PDX1(+) pancreatic progenitors compared with control flat surfaces. Furthermore, in the presence of biochemical factors, 200 nm nanopore-patterned surfaces profoundly enhanced the derivation of pancreatic endocrine cells producing insulin, glucagon, or somatostatin. We also demonstrate that nanopore-patterned surface-induced upregulation of PDX1 is associated with downregulation of TAZ, suggesting the potential role of TAZ in nanopore-patterned surface-mediated mechanotransduction. Our study suggests that appropriate cytokine treatments combined with nanotopographical stimulation could be a powerful tool for deriving a high purity of desired cells from human pluripotent stem cells.