Real-time monitoring of the dynamics and interactions of bacteria and the early-stage formation of biofilms.

Bacterial biofilms are complex colonies of bacteria adhered to a static surface and/or one to another. Bacterial biofilms exhibit high resistance to antimicrobial agents and can cause life-threatening nosocomial infections. Despite the effort of the scientific community investigating the formation and growth of bacterial biofilms, the preliminary interaction of bacteria with a surface and the subsequent early-stage formation of biofilms is still unclear. In this study, we present real-time, label-free monitoring of the interaction of Escherichia coli and Pseudomonas aeruginosa bacteria with untreated glass control surfaces and surfaces treated with benzalkonium chloride, a chemical compound known for its antimicrobial properties. The proof of principle investigation has been performed in a standard inverted optical microscope exploiting the optical phenomenon of caustics as a tool for monitoring bacterial diffusion and early adhesion and associated viability. The enhanced resolving power of the optical set-up allowed the monitoring and characterization of the dynamics of the bacteria, which provided evidence for the relationship between bacterial adhesion dynamics and viability, as well as the ability to form a biofilm. Viable bacteria adhered to the surface exhibited noticeable sliding or rotary dynamics while bacteria killed by surface contact remained static once adhered to the surface. This difference in dynamics allowed the early detection of biofilm formation and offers the potential to quantify the efficiency of antimicrobial surfaces and coatings.

Settling dynamics of nanoparticles in simple and biological media.

The biological response of organisms exposed to nanoparticles is often studied in vitro using adherent monolayers of cultured cells. In order to derive accurate concentration-response relationships, it is important to determine the local concentration of nanoparticles to which the cells are actually exposed rather than the nominal concentration of nanoparticles in the cell culture medium. In this study, the sedimentation-diffusion process of different sized and charged gold nanoparticles has been investigated in vitro by evaluating their settling dynamics and by developing a theoretical model to predict the concentration depth profile of nanoparticles in solution over time. Experiments were carried out in water and in cell culture media at a range of controlled temperatures. The optical phenomenon of caustics was exploited to track nanoparticles in real time in a conventional optical microscope without any requirement for fluorescent labelling that potentially affects the dynamics of the nanoparticles. The results obtained demonstrate that size, temperature and the stability of the nanoparticles play a pivotal role in regulating the settling dynamics of nanoparticles. For gold nanoparticles larger than 60 nm in diameter, the initial nominal concentration did not accurately represent the concentration of nanoparticles local to the cells. Finally, the theoretical model proposed accurately described the settling dynamics of the nanoparticles and thus represents a promising tool to support the design of in vitro experiments and the study of concentration-response relationships.

Cost effective optimised synthetic surface modification strategies for enhanced control of neuronal cell differentiation and supporting neuronal and Schwann cell viability.

Enriching a biomaterial surface with specific chemical groups has previously been considered for producing surfaces that influence cell response. Silane layer deposition has previously been shown to control mesenchymal stem cell adhesion and differentiation. However, it has not been used to investigate neuronal or Schwann cell responses in vitro to date. We report on the deposition of aminosilane groups for peripheral neurons and Schwann cells studying two chain lengths: (a) 3-aminopropyl triethoxysilane (short chain-SC) and (b) 11-aminoundecyltriethoxysilane (long chain-LC) by coating glass substrates. Surfaces were characterised by water contact angle, AFM and XPS. LC-NH2 was produced reproducibly as a homogenous surface with controlled nanotopography. Primary neuron and NG108-15 neuronal cell differentiation and primary Schwann cell responses were investigated in vitro by S100ß, p75, and GFAP antigen expression. Both amine silane surface supported neuronal and Schwann cell growth; however, neuronal differentiation was greater on LC aminosilanes versus SC. Thus, we report that silane surfaces with an optimal chain length may have potential in peripheral nerve repair for the modification and improvement of nerve guidance devices.

Nitric Oxide Releasing Titanium Surfaces for Antimicrobial Bone-Integrating Orthopedic Implants.

Titanium implants in orthopedic applications can fail due to infection and impaired integration into the host. Most research efforts that facilitate osseointegration of the implant have not considered infection, and vice versa. Moreover, most infection control measures involve the use of conventional antibiotics which contributes to the global epidemic of antimicrobial resistance. Nitric oxide (NO) is a promising alternative to antibiotics, and while researchers have investigated NO releasing coatings, there are few reports on the function/robustness or the mechanism of NO release. Our comprehensive mechanistic study has allowed us to design, characterize, and optimize NO releasing coatings to achieve maximum antimicrobial efficacy toward bacteria with minimum cytotoxicity to human primary osteoblasts in vitro. As the antibiotic era is coming to an end and the future of infection control continues to demand new alternatives, the coatings described herein represent a promising therapeutic strategy for use in orthopedic surgeries.

The influence of inter-particle forces on diffusion at the nanoscale.

Van der Waals and electrostatic interactions are the dominant forces acting at the nanoscale and they have been reported to directly influence a range of phenomena including surface adhesion, friction, and colloid stability but their contribution on nanoparticle diffusion dynamics is still not clear. In this study we evaluated experimentally the changes in the diffusion coefficient of nanoparticles as a result of varying the magnitude of Van der Waals and electrostatic forces. We controlled the magnitude of these forces by varying the ionic strength of a salt solution, which has been shown to be a parameter that directly controls the forces, and found by tracking single nanoparticles dispersed in solutions with different salt molarity that the diffusion of nanoparticles increases with the magnitude of the electrostatic forces and Van der Waals forces. Our results demonstrate that these two concurrently dynamic forces play a pivotal role in driving the diffusion process and must be taken into account when considering nanoparticle behaviour.

Antimicrobial Nitric Oxide Releasing Contact Lens Gels for the Treatment of Microbial Keratitis.

Microbial keratitis is a serious sight threatening infection affecting approximately two million individuals worldwide annually. While antibiotic eye drops remain the gold standard treatment for these infections, the significant problems associated with eye drop drug delivery and the alarming rise in antimicrobial resistance has meant that there is an urgent need to develop alternative treatments. In this work, a nitric oxide releasing contact lens gel displaying broad spectrum antimicrobial activity against two of the most common causative pathogens of microbial keratitis is described. The contact lens gel is composed of poly-ε-lysine (pεK) functionalized with nitric oxide (NO) releasing diazeniumdiolate moieties which enables the controlled and sustained release of bactericidal concentrations of NO at physiological pH over a period of 15 h. Diazeniumdiolate functionalization was confirmed by Fourier transform infrared (FTIR), and the concentration of NO released from the gels was determined by chemiluminescence. The bactericidal efficacy of the gels against Pseudomonas aeruginosa and Staphylococcus aureus was ascertained, and between 1 and 4 log reductions in bacterial populations were observed over 24 h. Additional cell cytotoxicity studies with human corneal epithelial cells (hCE-T) also demonstrated that the contact lens gels were not cytotoxic, suggesting that the developed technology could be a viable alternative treatment for microbial  keratitis.

Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies.

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive ('additive') top-down fabrication of nanometer-scale features. Historically, a major drawback of scanning probe lithography has been the intrinsically low throughput of single probe systems. This has been tackled by the use of arrays of multiple probes to enable increased nanolithography throughput. In order to implement such parallelized nanolithography, the accurate alignment of probe arrays with the substrate surface is vital, so that all probes make contact with the surface simultaneously when lithographic patterning begins. This protocol describes the utilization of polymer pen lithography to produce nanometer-scale features over centimeter-sized areas, facilitated by the use of an algorithm for the rapid, accurate, and automated alignment of probe arrays. Here, nanolithography of thiols on gold substrates demonstrates the generation of features with high uniformity. These patterns are then functionalized with fibronectin for use in the context of surface-directed cell morphology studies.

The optimization and production of stable homogeneous amine enriched surfaces with characterized nanotopographical properties for enhanced osteoinduction of mesenchymal stem cells.

Silane modification has been proposed as a powerful biomaterial surface modification tool. This is the first comprehensive investigation into the effect of silane chain length on the resultant properties of -NH2 silane monolayers and the associated osteoinductive properties of the surface. A range of -NH2 presenting silanes, chain length 3-11, were introduced to glass coverslips and characterized using water contact angles, atomic force microscopy, X-ray photoelectron spectroscopy, and Ninhydrin assays. The ability of the variation in chain length to form a homogenous layer across the entirety of the surfaces was also assessed. The osteoinductive potential of the resultant surfaces was evaluated by real-time polymerase chain reaction, immunocytochemistry, and von Kossa staining. Control of surface chemistry and topography was directly associated with changes in chain length. This resulted in the identification of a specific, chain length 11 (CL11) which significantly increased the osteoinductive properties of the modified materials. Only CL11 surfaces had a highly regular nano-topography/roughness which resulted in the formation of an appetite-like layer on the surface that induced a significantly enhanced osteoinductive response (increased expression of osteocalcin, CBFA1, sclerostin, and the production of a calcified matrix) across the entirety of the surface. © 2018 The Authors Journal of Biomedical Materials Research Part A Published by Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1862-1877, 2018.

Photoresponsive Hydrogels with Photoswitchable Mechanical Properties Allow Time-Resolved Analysis of Cellular Responses to Matrix Stiffening.

As cell function and phenotype can be directed by the mechanical characteristics of the surrounding matrix, hydrogels have become important platforms for cell culture systems, with properties that can be tuned by external stimuli, such as divalent cations, enzymatic treatment, and pH. However, many of these stimuli can directly affect cell behavior, making it difficult to distinguish purely mechanical signaling events. This study reports on the development of a hydrogel that incorporates photoswitchable cross-linkers, which can reversibly alter their stiffness upon irradiation with the appropriate wavelength of light. Furthermore, this study reports the response of bone-marrow-derived mesenchymal stem cells (MSCs) on these hydrogels that were stiffened systematically by irradiation with blue light. The substrates were shown to be noncytotoxic, and crucially MSCs were not affected by blue-light exposure. Time-resolved analysis of cell morphology showed characteristic cell spreading and increased aspect ratios in response to greater substrate stiffness. This hydrogel provides a platform to study mechanosignaling in cells responding to dynamic changes in stiffness, offering a new way to study mechanotransduction signaling pathways and biological processes, with implicit changes to tissue mechanics, such as development, ageing, and fibrosis.

Correction: Introducing dip pen nanolithography as a tool for controlling stem cell behaviour: unlocking the potential of the next generation of smart materials in regenerative medicine.

Correction for 'Introducing dip pen nanolithography as a tool for controlling stem cell behaviour: unlocking the potential of the next generation of smart materials in regenerative medicine' by Judith M. Curran et al., Lab Chip, 2010, 10, 1662-1670.

Defining the Properties of an Array of -NH2-Modified Substrates for the Induction of a Mature Osteoblast/Osteocyte Phenotype from a Primary Human Osteoblast Population Using Controlled Nanotopography and Surface Chemistry.

Accelerating the integration of a joint replacement or the healing of a bone fracture, particularly a complicated non-union fracture, would improve patient welfare and decrease healthcare costs. Currently, an autologous bone graft is the gold standard method for the treatment of complicated non-union fractures, but it is not always possible to harvest such a graft. A proactive highly inductive so-called smart material approach is pertinent in these cases. In this study, the surface chemistry of a previously approved material with desirable bulk material properties was modified to investigate its potential as an economical and effective alternative. The objective was to create stable synthetic chemical coatings that could guide cells along the osteogenic lineage required to generate mineralised tissue that would induce and accelerate bone healing. Primary human osteoblast-like cells were cultured in vitro for 7, 14 and 28 days on amine-terminated (chain length in the range 3-11) silane-modified glass surfaces with controlled nanotopography, to determine how surface chemistry and nanotopography change osteoblast function. The materials were characterised using atomic force microscopy (AFM), scanning electron microscopy (SEM), water contact angle (WCA) and a novel ninhydrin assay. The cells were analysed using qRT-PCR, von Kossa tinctural staining for mineralisation, and visualised using both transmitted white light and electron microscopy. Bone-like nodules, quantified using microscopy, only formed on the short-chain (chain length 3 and 4) amines after 7 days, as did the up-regulation of sclerostin, suggestive of a more mature osteoblast phenotype. In this paper, we report more rapid nodule formation than has previously been observed, without the addition of exogenous factors in the culture medium. This suggests that the coating would improve the integration of implants with bone or be the basis of a smart biomaterial that would accelerate the bone regeneration process.

Human mesenchymal stem cell response to poly(ε-caprolactone/poly(methyl methacrylate) demixed thin films.

Advances in material sciences have enabled the fabrication of biomaterials which are able to provide the requisite cues to stimulate cells to behave in a specific way. Nanoscale surface topographies are well known to be able to positively influence cell-substrate interactions. This study reports on a novel series of poly(ε-caprolactone) PCL and poly(methyl methacrylate) demixed nanotopographic films as non-biological cell-stimulating cues. The topographic features observed ranged from nanoislands to nanopits. PMMA was observed to segregate to the air interface, while PCL preferred the substrate interface. Preliminary response of human mesenchymal stem cells to these surfaces indicated that the substrate with nanoisland topography has the potential to differentiate to osteogenic, chondrogenic and adipogenic lineages.

The osteogenic response of mesenchymal stem cells to an injectable PLGA bone regeneration system.

The enrichment of substrates/surfaces with selected functional groups, methyl (-CH3), allyl amine (-NH2), allyl alcohol (-OH) and acrylic acid (-COOH), can be used to trigger mesenchymal stem (MSC) cell differentiation into specified lineages, minimising the need for exogenous biological supplementation. We present the successful translation of this research phenomenon to an injectable two phase injectable PLGA system, utilising plasma techniques, for the repair of bone defects. Modified microspheres were characterised using water contact angel (WCA), X-ray Photon Spectroscopy (XPS) and scanning electron microscopy (SEM). When cultured in contact with MSCs in vitro, the ability of the modified particles, within the 2 phase system, to induce differentiation was characterised using quantitative assays for cell viability and histological analysis for key markers of differentiation throughout the entirety of the three dimensional scaffold. Biological analysis proved that selected modified microspheres have the ability to induce MSC osteogenic (-NH2 modified scaffolds) and chondrogenic (-OH modified scaffolds) differentiation throughout the entirety of the formed scaffold. Therefore optimised plasma modification of microspheres is an effective tool for the production of injectable systems for the repair of bone and cartilage defects.

Introducing dip pen nanolithography as a tool for controlling stem cell behaviour: unlocking the potential of the next generation of smart materials in regenerative medicine.

Reproducible control of stem cell populations, regardless of their original source, is required for the true potential of these cells to be realised as medical therapies, cell biology research tools and in vitro assays. To date there is a lack of consistency in successful output when these cells are used in clinical trials and even simple in vitro experiments, due to cell and material variability. The successful combination of single chemistries in nanoarray format to control stem cell, or any cellular behaviour has not been previously reported. Here we report how homogenously nanopatterned chemically modified surfaces can be used to initiate a directed cellular response, particularly mesenchymal stem cell (MSC) differentiation, in a highly reproducible manner without the need for exogenous biological factors and heavily supplemented cell media. Successful acquisition of these data should lead to the optimisation of cell selective properties of materials, further enhancing the role of nanopatterned substrates in cell biology and regenerative medicine. The successful design and comparison of homogenously molecularly nanopatterned surfaces and their direct effect on human MSC adhesion and differentiation are reported in this paper. Planar gold surfaces were patterned by dip pen nanolithography (DPN) to produce arrays of nanodots with optimised fixed diameter of 70 nanometres separated by defined spacings, ranging from 140 to 1000 nm with terminal functionalities of simple chemistries including carboxyl, amino, methyl and hydroxyl. These nanopatterned surfaces exhibited unprecedented control of initial cell interactions and subsequent control of cell phenotype and offer significant potential for the future.

Nanoscale definition of substrate materials to direct human adult stem cells towards tissue specific populations.

The development of homogenously nano-patterned chemically modified surfaces that can be used to initiate a cellular response, particularly stem cell differentiation, in a highly controlled manner without the need for exogenous biological factors has never been reported, due to that fact that precisely defined and reproducible systems have not been available that can be used to study cell/material interactions and unlock the potential of a material driven cell response. Until now material driven stem cell (furthermore any cell) responses have been variable due to the limitations in definition and reproducibility of the underlying substrate and the lack of true homogeneity of modifications that can dictate a cellular response at a sub-micron level that can effectively control initial cell interactions of all cells that contact the surface. Here we report the successful design and use of homogenously molecularly nanopatterned surfaces to control initial stem cell adhesion and hence function. The highly specified nano-patterned arrays were compared directly to silane modified bulk coated substrates that have previously been proven to initiate mesenchymal stem cell (MSC) differentiation in a heterogenous manner, the aim of this study was to prove the efficiency of these previously observed cell responses could be enhanced by the incorporation of nano-patterns. Nano-patterned surfaces were prepared by Dip Pen Nanolithography (DPN) to produce arrays of 70 nm sized dots separated by defined spacings of 140, 280 and 1000 nm with terminal functionalities of carboxyl, amino, methyl and hydroxyl and used to control cell growth. These nanopatterned surfaces exhibited unprecedented control of initial cell interactions and will change the capabilities for stem cell definition in vitro and then cell based medical therapies. In addition to highlighting the ability of the materials to control stem cell functionality on an unprecedented scale this research also introduces the successful scale-up of DPN and the novel chemistries and systems to facilitate the production of homogeneously patterned substrates (5 mm2) that are applicable for use in in vitro cell conditions over prolonged periods for complete control of material driven cell responses.

The guidance of human mesenchymal stem cell differentiation in vitro by controlled modifications to the cell substrate.

Material-driven control of bone-marrow-derived mesenchymal stem cell (MSC) behaviour and differentiation is a very exciting possibility. The aim of this study was to use silane-modified surfaces to control MSC adhesion and differentiation in vitro and evaluate the use of such techniques to control MSC behaviour both in basal and stimulated conditions. A range of characterised clean glass silane-modified surfaces, methyl (-CH(3)), amino (-NH(2)), silane (-SH), hydroxyl (-OH) and carboxyl (-COOH), were produced and cultured in contact with human MSC, in conjunction with a clean glass (TAAB) control, for time periods up to 28 days in basal, chondrogenic and osteogenic stimulated media. The samples were analysed for levels of viable cell adhesion, morphology and the production of various differentiation and transcription markers using both fluorescent immunohistochemistry (collagen I, II, osteocalcin, CBFA1) and real-time polymerase chain reaction (PCR) (collagen I, II, osteocalcin, osteopontin, osteonectin, CBFA1 and Sox 9). Analysis of the results demonstrated that the range of materials could be broken down into three distinct categories. Firstly, the -TAAB control and -CH(3) surfaces maintained the MSC phenotype; secondly, the -NH(2) and -SH-modified surfaces promoted and maintained osteogenesis both in the presence and absence of biological stimuli. These surfaces did not support long-term chondrogenesis under any test conditions. Finally, the -OH and -COOH-modified surfaces promoted and maintained chondrogenesis under both basal and chondrogenic stimulated conditions, but did not support osteogenesis. These results demonstrate that intricate material properties such as surface chemistry and energy can influence MSC behaviour in vitro. These results have implications not only in promoting the efficiency of tissue-engineered constructs, but also to the wider field of MSC isolation, maintenance and expansion.

The use of poly(l-lactide) and RGD modified microspheres as cell carriers in a flow intermittency bioreactor for tissue engineering cartilage.

The use of biodegradable microcarriers as initial supports for tissue engineering has been demonstrated to be advantageous for maintaining a differentiated cell phenotype; the high surface area also allows rapid cell expansion. Poly l-lactide (PLLA) is a significant member of a group of polymers regarded as bioresorbable and has been widely used for manufacturing 3D scaffolds for tissue engineering. In this study, the hypothesis that PLLA microspheres could be surface modified using RGD peptide sequences to improve the cell adhesion and function of those cells in contact with PLLA was tested. Using this type of approach it may be possible to generate larger structures that contain a high cell number relative to the amount of polymer, whilst remaining free from mass transport limitations. PLLA microspheres were prepared using an oil-in-water solvent-evaporation technique and then an RGD-motif was incorporated onto the microspheres surface by conjugation to improve cell attachment and function. Both PLLA and GRGDSPK modified PLLA microspheres were used as cell microcarriers for chondrocytes cultured in a flow intermittency bioreactor. At the same time, the degradation of the microspheres has been studied after 7, 14, 21, 28, 35, 49 and 56 days. The molecular weight of the PLLA microspheres was determined by Gel Permeation Chromatography. The morphology was assessed by scanning electron microscopy, and the thermal properties determined by Differential Scanning Calorimetry. It was demonstrated that the RGD modified and pure PLLA microspheres degraded gradually at a steady rate over the experimental period, which would provide a controlled degradation profile, both could serve as cell microcarriers because of their thermal and mechanical stabilities. The microspheres with RGD surface modification enhanced cell adhesion and increased the cell numbers in the microspheres aggregates.

The differentiation of bone marrow mesenchymal stem cells into chondrocyte-like cells on poly-L-lactic acid (PLLA) scaffolds.

While intervertebral disc (IVD) degeneration is associated with the majority of cases of low back pain, current treatments are symptomatic rather than curative. Tissue engineering offers a treatment that both cures the problem of disc degeneration and restores normal disc function. One of the major problems for any tissue engineering strategy, however, is ensuring that both the cells and matrices used are suitable for the target tissue. In this study, we have developed and studied a potential system for tissue engineering of the nucleus pulposus (NP) of the severely degenerate IVD. While cells from degenerate discs are not suitable for tissue engineering, bone-marrow-derived mesenchymal stem cells, which are capable of differentiating into chondrocyte-like cells such as those found within the NP of the disc, offer a potential source of cells. We have used transfection with adenoviral SOX-9, a transcription factor involved in differentiation of MSCs along the chondrogenic lineage, combined with culture in a specialised medium, to differentiate monolayer MSCs to NP-like (chondrocyte-like) cells, as shown by real-time quantitative polymerase chain reaction for NP-marker genes. We have also replicated these findings on porous, biodegradable three-dimensional (3D) poly-l-lactic acid scaffolds and shown expression and deposition of NP matrix markers such as type II collagen and aggrecan. We are therefore proposing pre-differentiation of human MSCs and seeding on porous, biodegradable 3D synthetic polymer scaffolds as a realistic tissue engineering strategy for regeneration of the degenerate human IVD.