Flexible, Transparent, and Cytocompatible Nanostructured Indium Tin Oxide Thin Films for Bio-optoelectronic Applications.

Electrical stimulation has been used successfully for several decades for the treatment of neurodegenerative disorders, including motor disorders, pain, and psychiatric disorders. These technologies typically rely on the modulation of neural activity through the focused delivery of electrical pulses. Recent research, however, has shown that electrically triggered neuromodulation can be further enhanced when coupled with optical stimulation, an approach that can benefit from the development of novel electrode materials that combine transparency with excellent electrochemical and biological performance. In this study, we describe an electrochemically modified, nanostructured indium tin oxide/poly(ethylene terephthalate) (ITO/PET) surface as a flexible, transparent, and cytocompatible electrode material. Electrochemical oxidation and reduction of ITO/PET electrodes in the presence of an ionic liquid based on d-glucopyranoside and bistriflamide units were performed, and the electrochemical behavior, conductivity, capacitance, charge transport processes, surface morphology, optical properties, and cytocompatibility were assessed in vitro. It has been shown that under selected conditions, electrochemically modified ITO/PET films remained transparent and highly conductive and were able to enhance neural cell survival and neurite outgrowth. Consequently, electrochemical modification of ITO/PET electrodes in the presence of an ionic liquid is introduced as an effective approach for tailoring the properties of ITO for advanced bio-optoelectronic applications.

In vitro analysis of a physiological strain sensor formulated from a PEDOT:PSS functionalized carbon nanotube-poly(glycerol sebacate urethane) composite.

Biodegradable strain sensors able to undergo controlled degradation following implantation have recently received significant interest as novel approaches to detect pathological tissue swelling or non-physiological stresses. In this study, the physicomechanical, electrochemical and active pressure sensing behavior of an electrically conductive and biodegradable poly(glycerol sebacate urethane) (PGSU) composite, reinforced with poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) functionalized carbon nanotubes (CNTs), was evaluated in vitro. Analysis of these PGSU-CNTs composites demonstrated that the incorporation of functionalized CNTs into a biodegradable elastomer resulted in enhanced mechanical strength, conductivity and tailored matrix biodegradation. PGSU-CNT composites were subsequently formulated into flexible and active pressure sensors which demonstrated optimal sensitivity to applied 1% uniaxial tensile strains. Finally, cytocompatibility analysis a with primary neural culture confirmed that PGSU-CNT composites exhibited low cytotoxicity, and supported neuron adhesion, viability, and proliferation in vitro.

Electrical percolation in extrinsically conducting, poly(ε-decalactone) composite neural interface materials.

By providing a bidirectional communication channel between neural tissues and a biomedical device, it is envisaged that neural interfaces will be fundamental in the future diagnosis and treatment of neurological disorders. Due to the mechanical mismatch between neural tissue and metallic neural electrodes, soft electrically conducting materials are of great benefit in promoting chronic device functionality. In this study, carbon nanotubes (CNT), silver nanowires (AgNW) and poly(hydroxymethyl 3,4-ethylenedioxythiophene) microspheres (MSP) were employed as conducting fillers within a poly(ε-decalactone) (EDL) matrix, to form a soft and electrically conducting composite. The effect of a filler type on the electrical percolation threshold, and composite biocompatibility was investigated in vitro. EDL-based composites exhibited favourable electrochemical characteristics: EDL/CNT-the lowest film resistance (1.2 ± 0.3 kΩ), EDL/AgNW-the highest charge storage capacity (10.7 ± 0.3 mC cm- 2), and EDL/MSP-the highest interphase capacitance (1478.4 ± 92.4 µF cm-2). All investigated composite surfaces were found to be biocompatible, and to reduce the presence of reactive astrocytes relative to control electrodes. The results of this work clearly demonstrated the ability of high aspect ratio structures to form an extended percolation network within a polyester matrix, resulting in the formulation of composites with advantageous mechanical, electrochemical and biocompatibility properties.

Nanovibrational Stimulation of Mesenchymal Stem Cells Induces Therapeutic Reactive Oxygen Species and Inflammation for Three-Dimensional Bone Tissue Engineering.

There is a pressing clinical need to develop cell-based bone therapies due to a lack of viable, autologous bone grafts and a growing demand for bone grafts in musculoskeletal surgery. Such therapies can be tissue engineered and cellular, such as osteoblasts, combined with a material scaffold. Because mesenchymal stem cells (MSCs) are both available and fast growing compared to mature osteoblasts, therapies that utilize these progenitor cells are particularly promising. We have developed a nanovibrational bioreactor that can convert MSCs into bone-forming osteoblasts in two- and three-dimensional, but the mechanisms involved in this osteoinduction process remain unclear. Here, to elucidate this mechanism, we use increasing vibrational amplitude, from 30 nm (N30) to 90 nm (N90) amplitudes at 1000 Hz and assess MSC metabolite, gene, and protein changes. These approaches reveal that dose-dependent changes occur in MSCs' responses to increased vibrational amplitude, particularly in adhesion and mechanosensitive ion channel expression and that energetic metabolic pathways are activated, leading to low-level reactive oxygen species (ROS) production and to low-level inflammation as well as to ROS- and inflammation-balancing pathways. These events are analogous to those that occur in the natural bone-healing processes. We have also developed a tissue engineered MSC-laden scaffold designed using cells' mechanical memory, driven by the stronger N90 stimulation. These mechanistic insights and cell-scaffold design are underpinned by a process that is free of inductive chemicals.

In vitro attenuation of astrocyte activation and neuroinflammation through ibuprofen-doping of poly(3,4-ethylenedioxypyrrole) formulations.

Neuroinflammation is often associated with poor functional recovery and may contribute to or initiate the development of severe neurological disorders, such as epilepsy, Parkinson's disease or Alzheimer's disease. Ibuprofen (IBU), being one of the most commonly used non-steroidal anti-inflammatory drugs, is known to possess neuroprotective activity and serve as a promising therapeutic for the treatment of neuroinflammation. In this study, the potential of an IBU-loaded poly(3,4-ethylenedioxypyrrole) (PEDOP) matrix has been assessed as a neural interface material with an aim to control astrocyte activation and suppress neuroinflammation in vitro. Three types of drug immobilization protocols were investigated, leading to the fabrication of IBU-loaded PEDOP matrices exhibiting a broad spectrum of electrical characteristics, drug release profiles, as well as biological responses. Among all investigated PEDOP formulations, PEDOP matrices formed through a three-step immobilization protocol exhibited the highest charge storage capacity (30 ± 1 mC/cm2) as well as a double layer capacitance of 645.0 ± 51.1 µF, associated with a relatively enlarged surface area. Demonstrating a total drug loading capacity of 150 µg/ml and a release rate constant of 0.15 1/h, this coating formulation may be employed as a safe electrical conducting drug eluting system.

Boron Nitride Nanotube Addition Enhances the Crystallinity and Cytocompatibility of PVDF-TrFE.

Analysis of the cellular response to piezoelectric materials has been driven by the discovery that many tissue components exhibit piezoelectric behavior ex vivo. In particular, polyvinylidene fluoride and the trifluoroethylene co-polymer (PVDF-TrFE) have been identified as promising piezo and ferroelectric materials with applications in energy harvesting and biosensor devices. Critically, the modulation of the structural and crystalline properties of PVDF-TrFE through annealing processes and the addition of particulate or fibrous fillers has been shown to modulate significantly the materials electromechanical properties. In this study, a PVDF-TrFE/boron-nitride nanotube composite was evaluated by modulated differential scanning calorimetry to assess the effects of boron nitride nanotube addition and thermal annealing on the composite structure and crystal behavior. An increased beta crystal formation [f(ß) = 0.71] was observed following PVDF-TrFE annealing at the first crystallization temperature of 120°C. In addition, the inclusion of boron nitride nanotubes significantly increased the crystal formation behavior [f(ß) = 0.76] and the mechanical properties of the material. Finally, it was observed that BNNT incorporation enhance the adherence and proliferation of human tenocyte cells in vitro.

The Synergistic Effects of Gold Particles and Dexamethasone on the Electrochemical and Biological Performance of PEDOT Neural Interfaces.

Although neural devices have shown efficacy in the treatment of neurodegenerative diseases, their functionality is limited by the inflammatory state and glial scar formation associated with chronic implantation. The aim of this study was to investigate neural electrode performance following functionalization with an anti-inflammatory coating derived from a conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) matrix doped with dexamethasone (Dex) and decorated with Au particles. Pristine PEDOT, PEDOT-Dex and their gold-decorated analogues (PEDOT/Au and PEDOT-Dex/Au) were formulated by electrochemical deposition and characterized with respect to electrode electrochemical properties, surface morphology and biocompatibility towards primary neural cells. Through a process of gold deposition, it was possible to eliminate the initial burst release observed in PEDOT-Dex and maintain a stable, stepwise increase in Dex elution over 7 days. The released amounts of Dex exceeded the concentrations considered as therapeutic for both PEDOT-Dex and PEDOT-Dex/Au. The results clearly indicated that the presence of either Dex or Au particles facilitated the outgrowth of neurites. Finally, it was shown that the application of composite materials, such as PEDOT-Dex/Au, is an efficient way to improve the efficacy of neural interfaces in vitro.

Analysis of a poly(ε-decalactone)/silver nanowire composite as an electrically conducting neural interface biomaterial.

BACKGROUND: Advancement in polymer technologies, facilitated predominantly through chemical engineering approaches or through the identification and utilization of novel renewable resources, has been a steady focus of biomaterials research for the past 50 years. Aliphatic polyesters have been exploited in numerous biomedical applications including the formulation of soft-tissue sutures, bone fixation devices, cardiovascular stents etc. Biomimetic 'soft' polymer formulations are of interest in the design of biological interfaces and specifically, in the development of implantable neuroelectrode systems intended to interface with neural tissues. Critically, soft polymer formulations have been shown to address the challenges associated with the disregulation of mechanotransductive processes and micro-motion induced inflammation at the electrode/tissue interface. In this study, a polyester-based poly(ε-decalactone)/silver nanowire (EDL:Ag) composite was investigated as a novel electrically active biomaterial with neural applications.Neural interfaces were formulated through spin coating of a polymer/nanowire formulation onto the surface of a Pt electrode to form a biocompatible EDL matrix supported by a percolated network of silver nanowires. As-formed EDL:Ag composites were characterized by means of infrared spectroscopy, scanning electron microscopy and electrochemical methods, with their cytocompatibility assessed using primary cultures of a mixed neural population obtained from the ventral mesencephalon of Sprague-Dawley rat embryos. RESULTS: Electrochemical characterization of various EDL:Ag composites indicated EDL:Ag 10:1 as the most favourable formulation, exhibiting high charge storage capacity (8.7 ± 1.0 mC/cm2), charge injection capacity (84.3 ± 1.4 µC/cm2) and low impedance at 1 kHz (194 ± 28 Ω), outperforming both pristine EDL and bare Pt electrodes. The in vitro biological evaluation showed that EDL:Ag supported significant neuron viability in culture and to promote neurite outgrowth, which had the average length of 2300 ± 6 µm following 14 days in culture, 60% longer than pristine EDL and 120% longer than bare Pt control substrates. CONCLUSIONS: EDL:Ag nanocomposites are shown to serve as robust neural interface materials, possessing favourable electrochemical characteristics together with high neural cytocompatibility.

Chirality-sorted carbon nanotube films as high capacity electrode materials.

Carbon nanomaterials show great promise for a wide range of applications due to their excellent physicochemical and electrical properties. Since their discovery, the state-of-the-art has expanded the scope of their application from scientific curiosity to impactful solutions. Due to their tunability, carbon nanomaterials can be processed into a wide range of formulations and significant scope exists to couple carbon structures to electronic and electrochemical applications. In this paper, the electrochemical performance of various types of CNT films, which differ by the number of walls, diameter, chirality and surface chemistry is presented. Especially, chirality-sorted (6,5)- and (7,6)-based CNT films are shown to possess a high charge storage capacity (up to 621.91 mC cm-2), areal capacitance (262 mF cm-2), significantly increased effective surface area and advantageous charge/discharge characteristics without addition of any external species, and outperform many other high capacity materials reported in the literature. The results suggest that the control over the CNT structure can lead to the manufacture of macroscopic CNT devices precisely tailored for a wide range of applications, with the focus on energy storage devices and supercapacitors. The sorted CNT macroassemblies show great potential for energy storage technologies to come from R&D laboratories into real life.

The Functional Response of Mesenchymal Stem Cells to Electron-Beam Patterned Elastomeric Surfaces Presenting Micrometer to Nanoscale Heterogeneous Rigidity.

Cells directly probe and respond to the physicomechanical properties of their extracellular environment, a dynamic process which has been shown to play a key role in regulating both cellular adhesive processes and differential cellular function. Recent studies indicate that stem cells show lineage-specific differentiation when cultured on substrates approximating the stiffness profiles of specific tissues. Although tissues are associated with a range of Young's modulus values for bulk rigidity, at the subcellular level, tissues are comprised of heterogeneous distributions of rigidity. Lithographic processes have been widely explored in cell biology for the generation of analytical substrates to probe cellular physicomechanical responses. In this work, it is shown for the first time that that direct-write e-beam exposure can significantly alter the rigidity of elastomeric poly(dimethylsiloxane) substrates and a new class of 2D elastomeric substrates with controlled patterned rigidity ranging from the micrometer to the nanoscale is described. The mechanoresponse of human mesenchymal stem cells to e-beam patterned substrates was subsequently probed in vitro and significant modulation of focal adhesion formation and osteochondral lineage commitment was observed as a function of both feature diameter and rigidity, establishing the groundwork for a new generation of biomimetic material interfaces.

Stimulation of 3D osteogenesis by mesenchymal stem cells using a nanovibrational bioreactor.

Bone grafts are one of the most commonly transplanted tissues. However, autologous grafts are in short supply, and can be associated with pain and donor-site morbidity. The creation of tissue-engineered bone grafts could help to fulfil clinical demand and provide a crucial resource for drug screening. Here, we show that vibrations of nanoscale amplitude provided by a newly developed bioreactor can differentiate a potential autologous cell source, mesenchymal stem cells (MSCs), into mineralized tissue in 3D. We demonstrate that nanoscale mechanotransduction can stimulate osteogenesis independently of other environmental factors, such as matrix rigidity. We show this by generating mineralized matrix from MSCs seeded in collagen gels with stiffness an order of magnitude below the stiffness of gels needed to induce bone formation in vitro. Our approach is scalable and can be compatible with 3D scaffolds.

Publisher Correction: Stimulation of 3D osteogenesis by mesenchymal stem cells using a nanovibrational bioreactor.

In the version of this Article originally published, in Fig. 4f, the asterisk was missing; in Fig. 6a-c, the labels 'Wnt/ß-catenin signalling', 'Wnt/Ca+ pathway' and 'ERK' and their associated lines/arrows were missing; and in Fig. 6d and in the sentence beginning "In MSCs that were...", 'myosin' and 'nanostimulated', respectively, were spelt incorrectly. These errors have now been corrected in all versions of the Article.

Nanoscale neuroelectrode modification via sub-20 nm silicon nanowires through self-assembly of block copolymers.

Neuroprosthetic technologies for therapeutic neuromodulation have seen major advances in recent years but these advances have been impeded due to electrode failure or a temporal deterioration in the device recording or electrical stimulation potential. This deterioration is attributed to an intrinsic host tissue response, namely glial scarring or gliosis, which prevents the injured neurons from sprouting, drives neurite processes away from the neuroelectrode and increases signal impedance by increasing the distance between the electrode and its target neurons. To address this problem, there is a clinical need to reduce tissue encapsulation of the electrodes in situ and improve long-term neuroelectrode function. Nanotopographical modification has emerged as a potent methodology for the disruption of protein adsorption and cellular adhesion in vitro. This study investigates the use of block copolymer self-assembly technique for the generation of sub-20 nm nanowire features on silicon substrates. Critically, these nanostructures were observed to significantly reduce electrical impedance and increase conductivity. Human neuroblastoma SH-SY5Y cells cultured on nanowire substrates for up to 14 days were associated with enhanced focal adhesion reinforcement and a reduction in proliferation. We conclude that nanowire surface modulation may offer significant potential as an electrode functionalization strategy.

Diatoms: a biotemplating approach to fabricating drug delivery reservoirs.

Biotemplating is a rapidly expanding subfield that utilizes nature-inspired systems and structures to create novel functional materials, and it is through these methods that the limitations of current engineering practices may be advanced. The diatom is an exceptional template for drug delivery applications, owing largely to its highly-ordered pores, large surface area, species-specific architecture, and flexibility for surface modifications. Diatoms have been studied in a wide range of biomedical applications and their potential as the next frontier of drug delivery has yet to be fully exploited. In this editorial, the authors aim to review the use of diatoms in the delivery of poorly water-soluble drugs as reported in the literature, discuss the progress and advancements that have been made thus far, identify the shortcomings and limitations in the field, and, lastly, present their expert opinion and convey the future outlook on biotemplating approaches for drug delivery.

Biofunctionalisation of electrically conducting polymers.

During a single decade of research, evidence has emerged that glial scar formation around the electro-tissue interface drives neural loss and increases the signal impedance of the electrodes, compromising the efficiency of the stimulating systems. Studies with conducting polymers (CPs) as electrode coatings have shown enhanced tissue integration and electrode performance in situ through biochemical and physicomechanical functionalisation. In this review, recent findings on CP modifications are provided in the context of neurospecific biomaterials, shedding light on the valuable impact of multifunctionalised strategies for biomedical applications.

The role of microtopography in cellular mechanotransduction.

Mechanotransduction is crucial for cellular processes including cell survival, growth and differentiation. Topographically patterned surfaces offer an invaluable non-invasive means of investigating the cell response to such cues, and greater understanding of mechanotransduction at the cell-material interface has the potential to advance development of tailored topographical substrates and new generation implantable devices. This study focuses on the effects of topographical modulation of cell morphology on chromosomal positioning and gene regulation, using a microgrooved substrate as a non-invasive mechanostimulus. Intra-nuclear reorganisation of the nuclear lamina was noted, and the lamina was required for chromosomal repositioning. It appears that larger chromosomes could be predisposed to such repositioning. Microarrays and a high sensitivity proteomic approach (saturation DiGE) were utilised to identify transcripts and proteins that were subject to mechanoregulated changes in abundance, including mediators of chromatin remodelling and DNA synthesis linked to the changes in nucleolar morphology and the nucleoskeleton.

Using immuno-scanning electron microscopy for the observation of focal adhesion-substratum interactions at the nano- and microscale in S-phase cells.

It is becoming clear that the nano/microtopography of a biomaterial in vivo is of first importance in influencing focal adhesion formation and subsequent cellular behaviour. When considering next-generation biomaterials, where the material's ability to elicit a regulated cell response will be key to device success, focal adhesion analysis is an useful indicator of cytocompatibility and can be used to determine functionality. Here, a methodology is described to allow simultaneous high-resolution imaging of focal adhesion sites and the material topography using field emission scanning electron microscopy. Furthermore, through the use of BrdU pulse labelling and immunogold detection, S-phase cells can be selected from a near-synchronised population of cells to remove artefacts due to cell cycle phase. This is a key factor in adhesion quantification as there is natural variation in focal adhesion density as cells progress through the cell cycle, which can skew the quantitative analysis of focal adhesion formation on fabricated biomaterials.

Nanotopographical control of stem cell differentiation.

Stem cells have the capacity to differentiate into various lineages, and the ability to reliably direct stem cell fate determination would have tremendous potential for basic research and clinical therapy. Nanotopography provides a useful tool for guiding differentiation, as the features are more durable than surface chemistry and can be modified in size and shape to suit the desired application. In this paper, nanotopography is examined as a means to guide differentiation, and its application is described in the context of different subsets of stem cells, with a particular focus on skeletal (mesenchymal) stem cells. To address the mechanistic basis underlying the topographical effects on stem cells, the likely contributions of indirect (biochemical signal-mediated) and direct (force-mediated) mechanotransduction are discussed. Data from proteomic research is also outlined in relation to topography-mediated fate determination, as this approach provides insight into the global molecular changes at the level of the functional effectors.

The use of nanoscale topography to modulate the dynamics of adhesion formation in primary osteoblasts and ERK/MAPK signalling in STRO-1+ enriched skeletal stem cells.

The physiochemical characteristics of a material with in vivo applications are critical for the clinical success of the implant and regulate both cellular adhesion and differentiated cellular function. Topographical modification of an orthopaedic implant may be a viable method to guide tissue integration and has been shown in vitro to dramatically influence osteogenesis, inhibit bone resorption and regulate integrin mediated cell adhesion. Integrins function as force dependant mechanotransducers, acting via the actin cytoskeleton to translate tension applied at the tissue level to changes in cellular function via intricate signalling pathways. In particular the ERK/MAPK signalling cascade is a known regulator of osteospecific differentiation and function. Here we investigate the effects of nanoscale pits and grooves on focal adhesion formation in human osteoblasts (HOBs) and the ERK/MAPK signalling pathway in mesenchymal populations. Nanopit arrays disrupted adhesion formation and cellular spreading in HOBs and impaired osteospecific differentiation in skeletal stem cells. HOBs cultured on 10 microm wide groove/ridge arrays formed significantly less focal adhesions than cells cultured on planar substrates and displayed negligible differentiation along the osteospecific lineage, undergoing up-regulations in the expression of adipospecific genes. Conversely, osteospecific function was correlated to increased integrin mediated adhesion formation and cellular spreading as noted in HOBS cultured on 100 microm wide groove arrays. Here osteospecific differentiation and function was linked to focal adhesion growth and FAK mediated activation of the ERK/MAPK signalling pathway in mesenchymal populations.