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The propensity of glycosaminoglycans to mediate cell-cell and cell-matrix interactions opens the door to capture cells, including circulating blood cells, onto biomaterial substrates. Chondroitin sulfate (CS)-B is of particular interest, since it interacts with the receptor (EGF)-like module-containing mucin-like hormone receptor-like 2 precursor (EMR2) displayed on the surface of leukocytes and endothelial progenitor cells. Herein, CS-B and its isomer CS-A were covalently immobilized onto heptylamine plasma polymer films via three different binding chemistries to develop platform technology for the capture of EMR2 expressing cells onto solid carriers. Surface characterization verified the successful immobilization of both glycosaminoglycans. The EMR2 expressing human myeloid cell line U937 preferentially bound onto CS-B-modified substrates, and U937 cells preincubated with CS-B in solution exhibited reduced affinity for the substrate. The direct capture of hematopoietic and blood-circulating endothelial cell types via a glycosaminoglycan-binding surface receptor opens an unexplored route for the development of biomaterials targeted at these cell types. Refereed/Peer-reviewed

Current organ transplantation protocols require the rapid transport of freshly isolated donor tissue to the recipient patient at the site where the procedure is to be conducted. During transport, the tissue graft can quickly deteriorate as a result of oxygen starvation. In this study, we report the fabrication of oxygen-releasing coatings for improved tissue preservation. The coatings were prepared via the encapsulation of calcium peroxide or urea peroxide microparticles between layers of octadiene plasma polymer films. By varying the thickness of the plasma polymer coating and type of peroxide, formulations were obtained that generate oxygen upon contact with aqueous solutions, while at the same time limiting the amount of toxic reactive oxygen species produced. The optimized coatings were tested under hypoxic conditions using the MIN6 β-cell line, which resulted in a 3-fold increase in the viability of cultured cells. These thin oxygen-releasing coatings can be deposited on a wide range of surfaces, creating a platform for oxygen delivery with the potential to extend the viability of transported tissues and increase the time frame available for graft transport. Refereed/Peer-reviewed

One of the most significant hurdles to the affordable, accessible delivery of cell therapy is the cost and difficulty of expanding cells to clinically relevant numbers. Immunotherapy to prevent autoimmune disease, tolerate organ transplants or target cancer critically relies on the expansion of specialized T cell populations. We have designed 3D-printed cell culture lattices with highly organized micron-scale architectures, functionalized via plasma polymerization to bind monoclonal antibodies that trigger cell proliferation. This 3D technology platform facilitate the expansion of therapeutic human T cell subsets, including regulatory, effector, and cytotoxic T cells while maintaining the correct phenotype. Lentiviral gene delivery to T cells is enhanced in the presence of the lattices. Incorporation of the lattice format into existing cell culture vessels such as the G-Rex system is feasible. This cell expansion platform is user-friendly and expedites cell recovery and scale-up, making it ideal for translating T cell therapies from bench to bedside. Refereed/Peer-reviewed

A label-free porous silicon (pSi) based, optical biosensor, using both an antibody and aptamer bioreceptor motif has been developed for the detection of insulin. Two parallel biosensors were designed and optimised independently, based on each bioreceptor. Both bioreceptors were covalently attached to a thermally hydrosilylated pSi surface though amide coupling, with unreacted surface area rendered stable and low fouling by incorporation of PEG moieties. The insulin detection ability of each biosensor was determined using interferometric reflectance spectroscopy, using a range of different media both with and without serum. Sensing performance was compared in terms of response value, response time and limit of detection (LOD) for each platform. In order to demonstrate the capability of the best performing biosensor to detect insulin from real samples, an in vitro investigation with the aptamer-modified surface was performed. This biosensor was exposed to buffer conditioned by glucose-stimulated human islets, with the result showing a positive response and a high degree of selectivity towards insulin capture. The obtained results correlated well with the ELISA used in the clinic for assaying glucose-stimulated insulin release from donor islets. We anticipate that this type of sensor can be applied as a rapid point-of-use biosensor to assess the quality of donor islets in terms of their insulin production efficiency, prior to transplantation. Refereed/Peer-reviewed

PolyJet three-dimensional (3D) printing allows for the rapid manufacturing of 3D moulds for the fabrication of cross-linked poly(dimethylsiloxane) microwell arrays (PMAs). As this 3D printing technique has a resolution on the micrometer scale, the moulds exhibit a distinct surface roughness. In this study, the authors demonstrate by optical profilometry that the topography of the 3D printed moulds can be transferred to the PMAs and that this roughness induced cell adhesive properties to the material. In particular, the topography facilitated immobilization of endothelial cells on the internal walls of the microwells. The authors also demonstrate that upon immobilization of endothelial cells to the microwells, a second population of cells, namely, pancreatic islets could be introduced, thus producing a 3D coculture platform Refereed/Peer-reviewed

In the field of stem cell technology, future advancements rely on the effective isolation, scale-up and maintenance of specific stem cell populations and robust procedures for their directed differentiation. The stem cell microenvironment – or niche – encompasses signal inputs from stem cells, supporting cells and from the extracellular matrix. In this context, the contribution of physicochemical surface variables is being increasingly recognised. This paradigm can be exploited to exert control over cellular behaviour. However, the number of parameters at play, and their complex interactions, presents a formidable challenge in delineating how the decisions of cell fate are orchestrated within the niche. Additionally, in the case of mesenchymal stem cells (MSC), more than one type of stem cell niche has been identified. By employing high throughput screening (HTS) strategies, common and specific attributes of each MSC niche can be probed. Here, we explore biological, chemical and physical parameters that are known to influence MSC self-renewal and differentiation. We then review techniques and strategies that allow the HTS of surface properties for conditions that direct stem cell fate, using MSC as a case study. Finally, challenges in recapturing the niche, particularly its three dimensional nature, in surface-based HTS formats are discussed. Refereed/Peer-reviewed

The cell microarray format can recreate a multitude of cell microenvironments on a single chip using only minimal amounts of reagent. In this study, we describe surface modifications to passivate cell microarrays, aiming to adapt the platform to the study of stem cell behavior over long-term culture periods. Functionalization of glass slides with (3-glycidyloxypropyl) trimethoxysilane enabled covalent anchoring of extracellular matrix proteins on microscale spots printed by a robotic contact printer. Subsequently, the surface was passivated by bovine serum albumin (BSA) or poly(ethylene glycol)bisamine (A-PEG) with molecular weights of 3000, 6000, and 10 000 Da. Cloud-point conditions for A-PEG grafting were attained that were compatible with protein deposition. Passivation strategies were assessed by culturing mesenchymal stem cells on the microarray platform. While both BSA and A-PEG passivation initially blocked cell adhesion between the printed spots, only A-PEG grafting was able to maintain cell pattern integrity over the entire culture period of 3 weeks. Refereed/Peer-reviewed

Interfacing mammalian cells with single-walled carbon nanotubes (SWCNTs) has been considered a potential route for various bioengineering applications. Here, the interaction between human neuroblastoma cell line, SK-N-SH, with catalyst-free SWCNT-decorated porous silicon (pSi) substrates is investigated. SWCNT-decorated surfaces were fabricated by chemically attaching carboxy-functional SWCNTs to pSi functionalized via an amino silane. SWCNT attachment was confirmed by atomic force microscopy and Raman spectroscopy. Patterning of the amino silane on pSi by photolithography permitted the creation of patterned SWCNT-decorated pSi substrates. The number of cells attaching to the SWCNT decorated surface was observed to correlate with SWCNT density, implying that vertically aligned SWCNTs allowed the capture of cells. By incorporating a low fouling PEG silanization surface modification into the procedure, cell patterning was achieved on the fabricated SWCNT patterns. These SWCNT-decorated pSi substrates could potentially find application in lab-on-chip devices, particularly as platforms for the electrical stimulation of neuronal cells. Refereed/Peer-reviewed

Porous silicon (pSi) substrates are a promising platform for cell expansion, since pore size and chemistry can be tuned to control cell behavior. In addition, a variety of bioactives can be loaded into the pores and subsequently released to act on cells adherent to the substrate. Here, we construct a cell microarray on a plasma polymer coated pSi substrate that enables the simultaneous culture of human endothelial cells on printed immobilized protein factors, while a second soluble growth factor is released from the same substrate. This allows three elements of candidate pSi scaffold materials - topography, surface functionalization, and controlled factor release - to be assessed simultaneously in high throughput. We show that protein conjugation within printed microarray spots is more uniform on the pSi substrate than on flat glass or silicon surfaces. Active growth factors are released from the pSi surface over a period of several days. Using an endothelial progenitor cell line, we investigate changes in cell behavior in response to the microenvironment. This platform facilitates the design of advanced functional biomaterials, including scaffolds, and carriers for regenerative medicine and cell therapy. Refereed/Peer-reviewed

Porous silicon (pSi) materials are regarded as promising drug delivery vehicles due to their biocompatibility, in vivo degradation, and resorption. We examine pSi films as a platform for the controlled delivery of levothyroxine, as a means to overcome problems with consistent dosage of this drug by oral administration. Oxidized pSi films were modified with 3-(aminopropyl)triethoxysilane (APTES), creating a surface chemistry that increased levothyroxine drug loading capacity by 50% and sustained drug release under physiological conditions for 14 days. Release kinetics from APTES-functionalized films initially followed a zero-order release profile, which is highly desirable for drug delivery. The loading and release profiles of levothyroxine suggest that the film size required to deliver a therapeutic dose is feasible for further consideration as an implantable delivery system. Refereed/Peer-reviewed

Ordered arrays of silicon nano- to microscale pillars are used to enable biomolecular trafficking into primary human cells, consistently demonstrating high transfection efficiency can be achieved with broader and taller pillars than reported to date. Cell morphology on the pillar arrays is often strikingly elongated. Investigation of the cellular interaction with the pillar reveals that cells are suspended on pillar tips and do not interact with the substrate between the pillars. Although cells remain suspended on pillar tips, acute local deformation of the cell membrane was noted, allowing pillar tips to penetrate the cell interior, while retaining cell viability. Refereed/Peer-reviewed

The control of cell-material interactions is the key to a broad range of biomedical interactions. Gradient surfaces have recently been established as tools allowing the high-throughput screening and optimization of these interactions. In this paper, we show that plasma polymer gradients can reveal the subtle influence of surface chemistry on embryonic stem cell behavior and probe the mechanisms by which this occurs. Lateral gradients of surface chemistry were generated by plasma polymerization of diethylene glycol dimethyl ether on top of a substrate coated with an acrylic acid plasma polymer using a tilted slide as a mask. Gradient surfaces were characterized by X-ray photoelectron spectroscopy, infrared microscopy mapping and profilometry. By changing the plasma polymerization time, the gradient profile could be easily manipulated. To demonstrate the utility of these surfaces for the screening of cell-material interactions, we studied the response of mouse embryonic stem (ES) cells to these gradients and compared the performance of different plasma polymerization times during gradient fabrication. We observed a strong correlation between surface chemistry and cell attachment, colony size and retention of stem cell markers. Cell adhesion and colony formation showed striking differences on gradients with different plasma polymer deposition times. Deposition time influenced the depth of the plasma film deposited and the relative position of surface functional group density on the substrate, but not the range of plasma-generated species. Refereed/Peer-reviewed

We report on the synthesis of two carboxy-functional alkene compounds as modifiers of porous silicon (pSi) surface chemistry by means of thermal hydrosilylation. Both alkene compounds have hydrophobic, aliphatic carbon segments, with terminal carboxylic acid functionality appended through a thioether linkage. In one of the alkene linkers, we incorporated a short oligoethylene glycol (OEG) moiety to explore its low-fouling properties when attached to porous silicon. We examined surface stability of the surface-modified porous silicon in aqueous milieu. Albumin and fibronectin were adsorbed and, using carbodiimide chemistry, covalently immobilised to linker-modified porous silicon, and the propensity for mammalian cells to attach to these surfaces investigated. Surface chemistry was characterised by infrared spectroscopy and the stability of porous silicon in an aqueous milieu was investigated by interferometric reflectance spectroscopy (IRS). Surfaces functionalised with the alkene linker containing OEG displayed greater resistance to the adsorption of albumin. This linker also facilitated higher levels of covalent protein immobilisation to the functionalised pSi surface. Higher levels of cell attachment were observed on pSi surfaces with fibronectin covalently immobilised onto the OEG linker, than for fibronectin covalently immobilised on the non-OEG linker. Refereed/Peer-reviewed

Appendix A. Figures with essential colour discrimination: Certain figures in this article, particularly Figures 1, 3 and 4, are difficult to interpret in black and white. The full colour images can be found in the on-line version, at doi:10.1016/j.actbio.2010. 03.031. The suitability of porous silicon (pSi) encapsulated in microfibers of the biodegradable polymer polycaprolactone (PCL) for ophthalmic applications was evaluated, using both a cell attachment assay with epithelial cells and an in vivo assessment of biocompatibility in rats. Microfibers of PCL containing encapsulated pSi particles at two different concentrations (6 and 20 wt.%) were fabricated as non-woven fabrics. Given the dependence of Si particle dissolution kinetics on pSi surface chemistry, two different types of pSi particles (hydride-terminated and surface-oxidized) were evaluated for each of the two particle concentrations. Significant attachment of a human lens epithelial cell line (SRA 01/04) to all four types of scaffolds within a 24 h period was observed. Implantation of Si fabric samples beneath the conjunctiva of rat eyes for 8 weeks demonstrated that the composite materials did not cause visible infection or inflammation, and did not erode the ocular surface. We suggest that these novel composite materials hold considerable promise as scaffolds in tissue engineering with controlled release applications. Refereed/Peer-reviewed

Control over particle self-assembly is a prerequisite for the colloidal templating of lithographical etching masks to define nanostructures. This work integrates and combines for the first time bottom-up and top-down approaches, namely, particle self-assembly at liquid-liquid interfaces and metal-assisted chemical etching, to generate vertically aligned silicon nanowire (VA-SiNW) arrays and, alternatively, arrays of nanoscale pores in a silicon wafer. Of particular importance, and in contrast to current techniques, including conventional colloidal lithography, this approach provides excellent control over the nanowire or pore etching site locations and decouples nanowire or pore diameter and spacing. The spacing between pores or nanowires is tuned by adjusting the specific area of the particles at the liquid-liquid interface before deposition. Hence, the process enables fast and low-cost fabrication of ordered nanostructures in silicon and can be easily scaled up. We demonstrate that the fabricated VA-SiNW arrays can be used as in vitro transfection platforms for transfecting human primary cells. Refereed/Peer-reviewed

Zero-valent, or elemental, silicon nanostructures exhibit a number of properties that render them attractive for applications in nanomedicine. These materials hold significant promise for improving existing diagnostic and therapeutic techniques. This review summarizes some of the essential aspects of the fabrication techniques used to generate these fascinating nanostructures, comparing their material properties and suitability for biomedical applications. We examine the literature in regards to toxicity, biocompatibility and biodistribution of silicon nanoparticles, nanowires and nanotubes, with an emphasis on surface modification and its influence on cell adhesion and endocytosis. In the final part of this review, our attention is focused on current applications of the fabricated silicon nanostructures in nanomedicine, specifically examining drug and gene delivery, bioimaging and biosensing. Refereed/Peer-reviewed

We have examined the link between ionized-gas plasma delivery of reactive oxygen species (ROS) to immortalized keratinocyte (HaCaT) cells and cell fate, defined in terms of cell viability versus death. Phospholipid vesicles were used as cell mimics to measure the possible intracellular ROS concentration, [ROSi], delivered by various plasma treatments. Cells were exposed to a helium cold atmospheric plasma (CAP) jet for different plasma exposure times (5-60 s) and gas flow rates (50-1000 ml min-1). Based upon the [ROSi] data we argue that plasma-generated ROS in the cell culture medium can readily diffuse into real cells. Plasma exposure that equated to an [ROSi] in the range of 3.81 × 10-10-9.47 × 10-8 M, measured at 1 h after the plasma exposure, resulted in increased cell viability at 72 h; whereas a higher [ROSi] at 1 h decreased cell viability after 72 h of culture. This may be because of the manner in which the ROS are delivered by the plasma: HaCaT cells better tolerate a low ROS flux over an extended plasma exposure period of 1 min, compared to a high flux delivered in a few seconds, although the final [ROSi] may be the same. Our results suggest that plasma stimulation of HaCaT cells follows the principle of hormesis. Refereed/Peer-reviewed

Nanomaterials promise to improve disease diagnosis and treatment by enhancing the delivery of drugs, genes, biomolecules and imaging agents to specific subcellular targets. In order to optimize nanomaterial design for this purpose, a comprehensive understanding of how these materials are taken up and transported within the cell is required. In this review, we discuss the endocytic pathways employed by different types of nanoparticles with emphasis on the influence of nanoparticle surface modification. The use of pharmacological inhibition to probe internalization and intracellular trafficking pathways of nanoparticles is critically evaluated. Finally, approaches to target-specific delivery of therapeutics via nanoparticles into the cytoplasm and nucleus are addressed. Refereed/Peer-reviewed

In this study, the ability of porous silicon nanoparticles (PSi NPs) to entrap and deliver nitric oxide (NO) as an effective antibacterial agent is tested against different Gram-positive and Gram-negative bacteria. NO was entrapped inside PSi NPs functionalized by means of the thermal hydrocarbonization (THC) process. Subsequent reduction of nitrite in the presence ofd-glucose led to the production of large NO payloads without reducing the biocompatibility of the PSi NPs with mammalian cells. The resulting PSi NPs demonstrated sustained release of NO and showed remarkable antibacterial efficiency and anti-biofilm-forming properties. These results will set the stage to develop antimicrobial nanoparticle formulations for applications in chronic wound treatment.

Advanced approaches to direct the differentiation of embryonic stem cells are highly sought after. The surface-bound chemical gradient format is a powerful screening approach that can be deployed to study changes in stem cell behavior as a function of subtle changes in surface chemistry. Here, we investigate the spontaneous differentiation of cells derived from differentiating mouse embryoid body (mEB) cells into endoderm, mesoderm, and ectoderm following culture on surface-bound gradients of chemical functional groups in the absence of differentiation-biasing bioactive factors. Gradients were created using a diffusion-controlled plasma polymerization technique. The generated coating ranged from hydrophobic 1,7-octadiene (OD) plasma polymer at one end of the gradient to a more hydrophilic allylamine (AA) plasma polymer on the opposite end. The gradient surface was divided into seven equal regions of progressively increasing AA plasma polymer content and mEB cell response within these regions was compared. Cells adhered preferentially to the central regions of the gradient; however, cell proliferation increased toward AA-plasma-polymer-rich end of the gradient. Variation in the expression of germ layer markers was noted across the gradient surface. High AA:OD plasma polymer ratios triggered cell differentiation toward both mesoderm and ectoderm. Expression of tissue-specific markers, in particular, KRT18, AFP, and TNNT2, was strikingly responsive to subtle changes in surface chemistry, exhibiting vastly different expression levels between adjacent regions. Our results suggest that the surface-bound gradient platform is well suited to screening surface chemistries for use in the field of stem cell technologies and regenerative medicine.

Isatin, 1H-indole-2,3-dione, is a heterocyclic compound of significant importance in medicinal chemistry. It is a synthetically versatile molecule, a precursor for a large number of pharmacologically active compounds. Isatin and its derivatives have aroused great attention in recent years due to their wide variety of biological activities, relevant to application as insecticides and fungicides and in a broad range of drug therapies, including anticancer drugs, antibiotics and antidepressants. The purpose of this review is to provide an overview of the pharmacological activities of isatin and its synthetic and natural derivatives. Molecular modifications to tailor the properties of isatin and its derivatives are also discussed. Refereed/Peer-reviewed

“Maskless” microplasma treatment of passivated surfaces has been developed for the micropatterning of materials surfaces. The micropatterned surfaces are used in the fabrication of arrays for protein and cell-based assays. The advantage of this micropatterning approach is that it can be easily integrated into current manufacturing practices and the resultant micropatterned surfaces used with existing life sciences techniques and instrumentation. Refereed/Peer-reviewed

Gradient surfaces are highly effective tools to screen and optimize cell- surface interactions. Here, the response of embryonic stem (ES) cell colonies to plasma polymer gradient surfaces is investigated. Surface chemistry ranged from pure allylamine (AA) plasma polymer on one end of the gradient to pure octadiene (OD) plasma polymer on the other end. Optimal surface chemistry conditions for retention of pluripotency were identified. Expression of the stem cell markers alkaline phosphatase (AP) and Oct4 varied with the position of the ES cell colonies across the OD-AA plasma polymer gradient. Both markers were more strongly retained on the OD plasma polymer rich regions of the gradients. The observed variation of expression across the plasma polymer gradient increased with duration of stem cell culture. While maximum cell adhesion to the gradient substrate occurred at a nitrogen- to-carbon (N/C ratio) of approximately 0.1, Oct4 and AP expression was best retained at an N/C ratio < 0.04. Stem cell marker expression correlated with colony size and morphology: more compact, multilayered colonies with prominent F-actin staining arose as the N/C ratio decreased. Disruption of actin polymerization using Y-27632 ROCK inhibitor resulted in a collapse of the multilayer colony structure into monolayers with limited cell-cell contact. A corresponding decrease in expression of AP and Oct4 was observed. Oct4 expression along with 3D colony morphology was partially rescued on the OD plasma polymer rich regions of the gradient. Refereed/Peer-reviewed

Chemical gradients of ethyl-6-bromohexanoate (EBH) were generated over porous silicon (pSi) substrates via electrochemical attachment. Immobilised ester moieties were hydrolysed and activated to produce a gradient of functional carboxylic acid groups. After subsequent immobilisation of cyclicRGD(cRGD), the surfaces were used to screen the extent of rat mesenchymal stem cell (MSC) attachment. Mapping of surface chemistry was carried out by means of infrared microscopy and X-ray photoelectron spectroscopy (XPS). MSC culture studies showed that short-term cell attachment responded to the cRGD density present on the gradient surface. Refereed/Peer-reviewed

Recent work used carboxylic acid plasma polymer gradient surfaces to demonstrate a relationship between retention of "stemness" in mouse embryonic stem cells and the degree of cell-surface adhesion (Wells et al., 2009). Over a threshold, increased cell spreading coincided with loss of a stem cell marker. This work examines whether the relationship between cell-surface adhesion and inhibition of stem cell differentiation found on acrylic acid gradients extends to other surface chemistries known to modulate cell attachment. Refereed/Peer-reviewed

Gradient surfaces have become invaluable tools for the high-throughput characterisation of biomolecule- and cellmaterial surface interactions as they allow for the screening and optimisation of surface parameters such as surface chemistry, topography and ligand density in a single experiment. Here, we have generated surface chemistry gradients on oxidised porous silicon (pSi) substrates using silane functionalisation. In these studies, pSi films with a pore size of 15- 30 nm and a layer thickness of around 1.7 μm were utilised. The manufacture of gradient surface chemistries of silanes was performed using a simple dip coating method, whereby an increasing incubation time of the substrate in a solution of the silane led to increasing surface coverage of the silane. In this work, the hydrophobic n-octadecyldimethyl chlorosilane (ODCS) and pentafluorophenyldimethyl chlorosilane (PFPS) were used since they were expected to produce significant changes in wettability upon attachment. Chemical gradients were characterised using infrared (IR) spectroscopy, X-ray photoelectron spectroscopy (XPS) and sessile drop water contact angle measurements. In addition, the surface chemistry of the gradient was mapped using synchrotron IR microscopy. The ODCS gradient displayed sessile drop water contact angles ranging from 12° to 71°, confirming the successful formation of a gradient. IR microscopy and an XPS line scan confirmed the formation of a chemical gradient on the porous substrate. Furthermore, the chemical gradients produced can be used for the high-throughput in vitro screening of protein and cell-surface interactions, leading to the definition of surface chemistry on nanostructured silicon which will afford improved control of biointerfacial interactions.