Tuning conductivity while maintaining mechanical properties in perylene bisimide hydrogels at physiological pH.

By using different salts as a method to achieve gelation of two different amino-acid-functionalised perylene bisimides, we were able to tune reduction potentials while maintaining the mechanical and optical properties of the system all at pH 7.4.

Bioengineered nerve conduits and wraps for peripheral nerve repair of the upper limb.

BACKGROUND: Traumatic peripheral nerve injury is common and incurs significant cost to individuals and society. Healing following direct nerve repair or repair with autograft is slow and can be incomplete. Several bioengineered nerve wraps or devices have become available as an alternative to direct repair or autologous nerve graft. Nerve wraps attempt to reduce axonal escape across a direct repair site and nerve devices negate the need for a donor site defect, required by an autologous nerve graft. Comparative evidence to guide clinicians in their potential use is lacking. We collated existing evidence to guide the clinical application of currently available nerve wraps and conduits. OBJECTIVES: To assess and compare the effects and complication rates of licensed bioengineered nerve conduits or wraps for surgical repair of traumatic peripheral nerve injuries of the upper limb. To compare effects and complications against the current gold surgical standard (direct repair or nerve autograft). SEARCH METHODS: We used standard, extensive Cochrane search methods. The latest search was 26 January 2022. We searched online and, where not accessible, contacted societies' secretariats to review abstracts from the British Surgical Society of the Hand, International Federation of Surgical Societies of the Hand, Federation of European Surgical Societies of the Hand, and the American Society for Peripheral Nerve from October 2007 to October 2018. SELECTION CRITERIA: We included parallel group randomised controlled trials (RCTs) and quasi-RCTs of nerve repair in the upper limb using a bioengineered wrap or conduit, with at least 12 months of follow-up. DATA COLLECTION AND ANALYSIS: We used standard Cochrane procedures. Our primary outcomes were 1. muscle strength and 2. sensory recovery at 24 months or more. Our secondary outcomes were 3. British Medical Research Council (BMRC) grading, 4. integrated functional outcome (Rosén Model Instrument (RMI)), 5. touch threshold, 6. two-point discrimination, 7. cold intolerance, 8. impact on daily living measured using the Disability of Arm Shoulder and Hand Patient-Reported Outcome Measure (DASH-PROM), 9. sensory nerve action potential, 10. cost of the device, and 11. adverse events (any and specific serious adverse events (further surgery)). We used GRADE to assess the certainty of the evidence. MAIN RESULTS: Five studies involving 213 participants and 257 nerve injuries reconstructed with wraps or conduits (129 participants) or standard repair (128 participants) met the inclusion criteria. Of those in the standard repair group, 119 nerve injuries were managed with direct epineurial repair, and nine autologous nerve grafts were performed. One study excluded the outcome data for the repair using an autologous nerve graft from their analysis, as it was the only autologous nerve graft in the study, so data were available for 127 standard repairs. There was variation in the functional outcome measures reported and the time postoperatively at which they were recorded. Mean sensory recovery, assessed with BMRC sensory grading (range S0 to S4, higher score considered better) was 0.03 points higher in the device group (range 0.43 lower to 0.49 higher; 1 RCT, 28 participants; very low-certainty evidence) than in the standard repair group (mean 2.75 points), which suggested little or no difference between the groups, but the evidence is very uncertain. There may be little or no difference at 24 months in mean touch thresholds between standard repair (0.81) and repair using devices, which was 0.01 higher but this evidence is also very uncertain (95% confidence interval (CI) 0.06 lower to 0.08 higher; 1 trial, 32 participants; very low-certainty evidence). Data were not available to assess BMRC motor grading at 24 months or more. Repair using bioengineered devices may not improve integrated functional outcome scores at 24 months more than standard techniques, as assessed by the Rosén Model Instrument (RMI; range 0 to 3, higher scores better); the CIs allow for both no important difference and a better outcome with standard repair (mean RMI 1.875), compared to the device group (0.17 lower, 95% CI 0.38 lower to 0.05 higher; P = 0.13; 2 trials, 60 participants; low-certainty evidence). Data from one study suggested that the five-year postoperative outcome of RMI may be slightly improved after repair using a device (mean difference (MD) 0.23, 95% CI 0.07 to 0.38; 1 trial, 28 participants; low-certainty evidence). No studies measured impact on daily living using DASH-PROM. The proportion of people with adverse events may be greater with nerve wraps or conduits than with standard techniques, but the evidence is very uncertain (risk ratio (RR) 7.15, 95% CI 1.74 to 29.42; 5 RCTs, 213 participants; very low-certainty evidence). This corresponds to 10 adverse events per 1000 people in the standard repair group and 68 per 1000 (95% CI 17 to 280) in the device group. The use of nerve repair devices may be associated with a greater need for revision surgery but this evidence is also very uncertain (12/129 device repairs required revision surgery (removal) versus 0/127 standard repairs; RR 7.61, 95% CI 1.48 to 39.02; 5 RCTs, 256 nerve repairs; very low-certainty evidence). AUTHORS' CONCLUSIONS: Based on the available evidence, this review does not support use of currently available nerve repair devices over standard repair. There is significant heterogeneity in participants, injury pattern, repair timing, and outcome measures and their timing across studies of nerve repair using bioengineered devices, which make comparisons unreliable. Studies were generally small and at high or unclear risk of bias. These factors render the overall certainty of evidence for any outcome low or very low. The data reviewed here provide some evidence that more people may experience adverse events with use of currently available bioengineered devices than with standard repair techniques, and the need for revision surgery may also be greater. The evidence for sensory recovery is very uncertain and there are no data for muscle strength at 24 months (our primary outcome measures). We need further trials, adhering to a minimum standard of outcome reporting (with at least 12 months' follow-up, including integrated sensorimotor evaluation and patient-reported outcomes) to provide high-certainty evidence and facilitate more detailed analysis of effectiveness of emerging, increasingly sophisticated, bioengineered repair devices.

Human Platelet Lysate Acts Synergistically With Laminin to Improve the Neurotrophic Effect of Human Adipose-Derived Stem Cells on Primary Neurons in vitro.

BACKGROUND: Despite the advancements in microsurgical techniques and noteworthy research in the last decade, peripheral nerve lesions have still weak functional outcomes in current clinical practice. However, cell transplantation of human adipose-derived stem cells (hADSC) in a bioengineered conduit has shown promising results in animal studies. Human platelet lysate (hPL) has been adopted to avoid fetal bovine serum (FBS) in consideration of the biosafety concerns inherent with the use of animal-derived products in tissue processing and cell culture steps for translational purposes. In this work, we investigate how the interplay between hPL-expanded hADSC (hADSChPL) and extracellular matrix (ECM) proteins influences key elements of nerve regeneration. METHODS: hADSC were seeded on different ECM coatings (laminin, LN; fibronectin, FN) in hPL (or FBS)-supplemented medium and co-cultured with primary dorsal root ganglion (DRG) to establish the intrinsic effects of cell-ECM contact on neural outgrowth. Co-cultures were performed "direct," where neural cells were seeded in contact with hADSC expanded on ECM-coated substrates (contact effect), or "indirect," where DRG was treated with their conditioned medium (secretome effect). Brain-derived nerve factor (BDNF) levels were quantified. Tissue culture plastic (TCPS) was used as the control substrate in all the experiments. RESULTS: hPL as supplement alone did not promote higher neurite elongation than FBS when combined with DRG on ECM substrates. However, in the presence of hADSC, hPL could dramatically enhance the stem cell effect with increased DRG neurite outgrowth when compared with FBS conditions, regardless of the ECM coating (in both indirect and direct co-cultures). The role of ECM substrates in influencing neurite outgrowth was less evident in the FBS conditions, while it was significantly amplified in the presence of hPL, showing better neural elongation in LN conditions when compared with FN and TCPS. Concerning hADSC growth factor secretion, ELISA showed significantly higher concentrations of BDNF when cells were expanded in hPL compared with FBS-added medium, without significant differences between cells cultured on the different ECM substrates. CONCLUSION: The data suggest how hADSC grown on LN and supplemented with hPL could be active and prone to support neuron-matrix interactions. hPL enhanced hADSC effects by increasing both proliferation and neurotrophic properties, including BDNF release.

Bandpass sorting of heterogeneous cells using a single surface acoustic wave transducer pair.

Separation and sorting of biological entities (viruses, bacteria, and cells) is a critical step in any microfluidic lab-on-a-chip device. Acoustofluidics platforms have demonstrated their ability to use physical characteristics of cells to perform label-free separation. Bandpass-type sorting methods of medium-sized entities from a mixture have been presented using acoustic techniques; however, they require multiple transducers, lack support for various target populations, can be sensitive to flow variations, or have not been verified for continuous flow sorting of biological cells. To our knowledge, this paper presents the first acoustic bandpass method that overcomes all these limitations and presents an inherently reconfigurable technique with a single transducer pair for stable continuous flow sorting of blood cells. The sorting method is first demonstrated for polystyrene particles of sizes 6, 10, and 14.5 µm in diameter with measured purity and efficiency coefficients above 75 ± 6% and 85 ± 9%, respectively. The sorting strategy was further validated in the separation of red blood cells from white blood cells and 1 µm polystyrene particles with 78 ± 8% efficiency and 74 ± 6% purity, respectively, at a flow rate of at least 1 µl/min, enabling to process finger prick blood samples within minutes.

Human platelet lysate as a potential clinical-translatable supplement to support the neurotrophic properties of human adipose-derived stem cells.

BACKGROUND: The autologous nerve graft, despite its donor site morbidity and unpredictable functional recovery, continues to be the gold standard in peripheral nerve repair. Rodent research studies have shown promising results with cell transplantation of human adipose-derived stem cells (hADSC) in a bioengineered conduit, as an alternative strategy for nerve regeneration. To achieve meaningful clinical translation, cell therapy must comply with biosafety. Cell extraction and expansion methods that use animal-derived products, including enzymatic adipose tissue dissociation and the use of fetal bovine serum (FBS) as a culture medium supplement, have the potential for transmission of zoonotic infectious and immunogenicity. Human-platelet-lysate (hPL) serum has been used in recent years in human cell expansion, showing reliability in clinical applications. METHODS: We investigated whether hADSC can be routinely isolated and cultured in a completely xenogeneic-free way (using hPL culture medium supplement and avoiding collagenase digestion) without altering their physiology and stem properties. Outcomes in terms of stem marker expression (CD105, CD90, CD73) and the osteocyte/adipocyte differentiation capacity were compared with classical collagenase digestion and FBS-supplemented hADSC expansion. RESULTS: We found no significant differences between the two examined extraction and culture protocols in terms of cluster differentiation (CD) marker expression and stem cell plasticity, while hADSC in hPL showed a significantly higher proliferation rate when compared with the usual FBS-added medium. Considering the important key growth factors (particularly brain-derived growth factor (BDNF)) present in hPL, we investigated a possible neurogenic commitment of hADSC when cultured with hPL. Interestingly, hADSC cultured in hPL showed a statistically higher secretion of neurotrophic factors BDNF, glial cell-derived growth factor (GDNF), and nerve-derived growth factor (NFG) than FBS-cultured cells. When cocultured in the presence of primary neurons, hADSC which had been grown under hPL supplementation, showed significantly enhanced neurotrophic properties. CONCLUSIONS: The hPL-supplement medium could improve cell proliferation and neurotropism while maintaining stable cell properties, showing effectiveness in clinical translation and significant potential in peripheral nerve research.

A novel poly-ε-lysine based implant, Proliferate®, for promotion of CNS repair following spinal cord injury.

The limited regenerative capacity of the CNS poses formidable challenges to the repair of spinal cord injury (SCI). Two key barriers to repair are (i) the physical gap left by the injury, and (ii) the inhibitory milieu surrounding the injury, the glial scar. Biomaterial implantation into the injury site can fill the cavity, provide a substrate for cell migration, and potentially attenuate the glial scar. We investigated the biological viability of a biocompatible and biodegradable poly-ε-lysine based biomaterial, Proliferate®, in low and high cross-linked forms and when coated with IKVAV peptide, for SCI implantation. We demonstrate altered astrocyte morphology and nestin expression on Proliferate® compared to conventional glass cell coverslips suggesting a less reactive phenotype. Moreover Proliferate® supported myelination in vitro, with myelination observed sooner on IKVAV-coated constructs compared with uncoated Proliferate®, and delayed overall compared with maintenance on glass coverslips. For in vivo implantation, parallel-aligned channels were fabricated into Proliferate® to provide cell guidance cues. Extensive vascularisation and cellular infiltration were observed in constructs implanted in vivo, along with an astrocyte border and microglial response. Axonal ingrowth was observed at the construct border and inside implants in intact channels. We conclude that Proliferate® is a promising biomaterial for implantation following SCI.

Numerical Determination of the Secondary Acoustic Radiation Force on a Small Sphere in a Plane Standing Wave Field.

Two numerical methods based on the Finite Element Method are presented for calculating the secondary acoustic radiation force between interacting spherical particles. The first model only considers the acoustic waves scattering off a single particle, while the second model includes re-scattering effects between the two interacting spheres. The 2D axisymmetric simplified model combines the Gor'kov potential approach with acoustic simulations to find the interacting forces between two small compressible spheres in an inviscid fluid. The second model is based on 3D simulations of the acoustic field and uses the tensor integral method for direct calculation of the force. The results obtained by both models are compared with analytical equations, showing good agreement between them. The 2D and 3D models take, respectively, seconds and tens of seconds to achieve a convergence error of less than 1%. In comparison with previous models, the numerical methods presented herein can be easily implemented in commercial Finite Element software packages, where surface integrals are available, making it a suitable tool for investigating interparticle forces in acoustic manipulation devices.

Controlling fluid flow to improve cell seeding uniformity.

Standard methods for seeding monolayer cell cultures in a multiwell plate or dish do not uniformly distribute cells on the surface. With traditional methods, users find aggregation around the circumference, in the centre, or a combination of the two. This variation is introduced due to the macro scale flow of the cell seeding suspension, and movement of the dish before cells can settle and attach to the surface. Reproducibility between labs, users, and experiments is hampered by this variability in cell seeding. We present a simple method for uniform and user-independent the cell seeding using an easily produced uniform cell seeder (UCS) device. This allows precise control of cell density in a reproducible manner. By containing the cell seeding suspension in a defined volume above the culture surface with the UCS, fluctuations in cell density are minimised. Seeding accuracy, as defined by the actual cell density versus the target seeding density is improved dramatically across users with various levels of expertise. We go on to demonstrate the impact of local variation in cell density on the lineage commitment of human embryonic stem cells (hESCs) towards pancreatic endoderm (PE). Variations in the differentiation profile of cells across a culture well closely mirror variations in cell density introduced by seeding method-with the UCS correcting variations in differentiation efficiency. The UCS device provides a simple and reproducible method for uniform seeding across multiple culture systems.

Particle separation by phase modulated surface acoustic waves.

High efficiency isolation of cells or particles from a heterogeneous mixture is a critical processing step in lab-on-a-chip devices. Acoustic techniques offer contactless and label-free manipulation, preserve viability of biological cells, and provide versatility as the applied electrical signal can be adapted to various scenarios. Conventional acoustic separation methods use time-of-flight and achieve separation up to distances of quarter wavelength with limited separation power due to slow gradients in the force. The method proposed here allows separation by half of the wavelength and can be extended by repeating the modulation pattern and can ensure maximum force acting on the particles. In this work, we propose an optimised phase modulation scheme for particle separation in a surface acoustic wave microfluidic device. An expression for the acoustic radiation force arising from the interaction between acoustic waves in the fluid was derived. We demonstrated, for the first time, that the expression of the acoustic radiation force differs in surface acoustic wave and bulk devices, due to the presence of a geometric scaling factor. Two phase modulation schemes are investigated theoretically and experimentally. Theoretical findings were experimentally validated for different mixtures of polystyrene particles confirming that the method offers high selectivity. A Monte-Carlo simulation enabled us to assess performance in real situations, including the effects of particle size variation and non-uniform acoustic field on sorting efficiency and purity, validating the ability to separate particles with high purity and high resolution.

Microtopographical cues promote peripheral nerve regeneration via transient mTORC2 activation.

Despite microsurgical repair, recovery of function following peripheral nerve injury is slow and often incomplete. Outcomes could be improved by an increased understanding of the molecular biology of regeneration and by translation of experimental bioengineering strategies. Topographical cues have been shown to be powerful regulators of the rate and directionality of neurite regeneration, and in this study we investigated the downstream molecular effects of linear micropatterned structures in an organotypic explant model. Linear topographical cues enhanced neurite outgrowth and our results demonstrated that the mTOR pathway is important in regulating these responses. mTOR gene expression peaked between 48 and 72h, coincident with the onset of rapid neurite outgrowth and glial migration, and correlated with neurite length at 48h. mTOR protein was located to glia and in a punctate distribution along neurites. mTOR levels peaked at 72h and were significantly increased by patterned topography (p<0.05). Furthermore, the topographical cues could override pharmacological inhibition. Downstream phosphorylation assays and inhibition of mTORC1 using rapamycin highlighted mTORC2 as an important mediator, and more specific therapeutic target. Quantitative immunohistochemistry confirmed the presence of the mTORC2 component rictor at the regenerating front where it co-localised with F-actin and vinculin. Collectively, these results provide a deeper understanding of the mechanism of action of topography on neural regeneration, and support the incorporation of topographical patterning in combination with pharmacological mTORC2 potentiation within biomaterial constructs used to repair peripheral nerves. STATEMENT OF SIGNIFICANCE: Peripheral nerve injury is common and functionally devastating. Despite microsurgical repair, healing is slow and incomplete, with lasting functional deficit. There is a clear need to translate bioengineering approaches and increase our knowledge of the molecular processes controlling nerve regeneration to improve the rate and success of healing. Topographical cues are powerful determinants of neurite outgrowth and represent a highly translatable engineering strategy. Here we demonstrate, for the first time, that microtopography potentiates neurite outgrowth via the mTOR pathway, with the mTORC2 subtype being of particular importance. These results give further evidence for the incorporation of microtopographical cues into peripheral nerve regeneration conduits and indicate that mTORC2 may be a suitable therapeutic target to potentiate nerve regeneration.

Nacre Topography Produces Higher Crystallinity in Bone than Chemically Induced Osteogenesis.

It is counterintuitive that invertebrate shells can induce bone formation, yet nacre, or mother of pearl, from marine shells is both osteoinductive and osteointegrative. Nacre is composed of aragonite (calcium carbonate) and induces production of vertebrate bone (calcium phosphate). Exploited by the Mayans for dental implants, this remarkable phenomenon has been confirmed in vitro and in vivo, yet the characteristic of nacre that induces bone formation remains unknown. By isolating nacre topography from its inherent chemistry in the production of polycaprolactone (PCL) nacre replica, we show that, for mesenchymal stem cells, nacre topography is osteoinductive. Gene expression of specific bone marker proteins, osteopontin, osteocalcin, osteonectin, and osterix, is increased 10-, 2-, 1.7-, and 1.8-fold, respectively, when compared to planar PCL. Furthermore, we demonstrate that bone tissue that forms in response to the physical topographical features of nacre has a higher crystallinity than bone formed in response to chemical cues with a full width half-maximum for PO43- Raman shift of 7.6 ± 0.7 for mineral produced in response to nacre replica compared to a much broader 34.6 ± 10.1 in response to standard osteoinductive medium. These differences in mineral product are underpinned by differences in cellular metabolism. This observation can be exploited in the design of bone therapies; a matter that is most pressing in light of a rapidly aging human population.

Contactless Acoustic Manipulation and Sorting of Particles by Dynamic Acoustic Fields.

This paper presents a contactless, acoustic technique to manipulate and sort particles of varying size in both liquid and air media. An acoustic standing wave is generated by the superposition of counterpropagating waves emitted by two opposing emitters. The acoustic radiation force traps the smallest particles at the pressure nodes of the acoustic standing wave. The position of the particles can be manipulated by dynamically changing the phase difference between the two emitters. By applying a dynamic acoustic field (DAF), it is demonstrated that particles can be manipulated spatially and sorted according to size. The discrimination (sorting dynamic range) capability is initially demonstrated in liquid media by separating three different sets of polystyrene particles, ranging in size from 5 to 45µm in diameter. The separation between particles was performed up to a ratio of 5/6 in diameter (20% diameter difference). Finally, the scalability of the DAF method is demonstrated by sorting expanded polystyrene particles of 2 and 5 mm diameter in air.

Production of Nanoscale Vibration for Stimulation of Human Mesenchymal Stem Cells.

Mechanical stimulation is becoming a common technique for manipulating cell behaviour in bioengineering with applications in tissue engineering and possibly regenerative therapy. Living organisms show biological responses in vivo and in vitro to various types of mechanical stimulation including vibration. The development of apparatus to produce vertical motions of nanoscale amplitude is detailed and their effect on mouse endothelial (Le2) and human mesenchymal stem cells (hMSCs) is investigated. Piezo ceramic actuators and aluminium reinforcement were utilised along with laser interferometry to ensure amplitude consistency at the nanometre level across a cell culture substrate. Peak force applied to the cells was estimated to be of nN magnitude at frequencies of 500 and 1000 Hz. Morphological changes in the cytoskeleton were found for both cell types along with increased MSC proliferation after 1 week of stimulation at 500 Hz. Changes in the nuclear size of MSCs after stimulation were also found.

Development of a novel 3D culture system for screening features of a complex implantable device for CNS repair.

Tubular scaffolds which incorporate a variety of micro- and nanotopographies have a wide application potential in tissue engineering especially for the repair of spinal cord injury (SCI). We aim to produce metabolically active differentiated tissues within such tubes, as it is crucially important to evaluate the biological performance of the three-dimensional (3D) scaffold and optimize the bioprocesses for tissue culture. Because of the complex 3D configuration and the presence of various topographies, it is rarely possible to observe and analyze cells within such scaffolds in situ. Thus, we aim to develop scaled down mini-chambers as simplified in vitro simulation systems, to bridge the gap between two-dimensional (2D) cell cultures on structured substrates and three-dimensional (3D) tissue culture. The mini-chambers were manipulated to systematically simulate and evaluate the influences of gravity, topography, fluid flow, and scaffold dimension on three exemplary cell models that play a role in CNS repair (i.e., cortical astrocytes, fibroblasts, and myelinating cultures) within a tubular scaffold created by rolling up a microstructured membrane. Since we use CNS myelinating cultures, we can confirm that the scaffold does not affect neural cell differentiation. It was found that heterogeneous cell distribution within the tubular constructs was caused by a combination of gravity, fluid flow, topography, and scaffold configuration, while cell survival was influenced by scaffold length, porosity, and thickness. This research demonstrates that the mini-chambers represent a viable, novel, scale down approach for the evaluation of complex 3D scaffolds as well as providing a microbioprocessing strategy for tissue engineering and the potential repair of SCI.

Poly(N-acryloylmorpholine): a simple hydrogel system for temporal and spatial control over cell adhesion.

N-acryloylmorpholine (NAM) was photo-polymerized to produce the homopolymer poly(N-acryloylmorpholine) (PNAM). PNAM behaves as a physical hydrogel in aqueous solvents, doubling its dry weight over a 2 h period before undergoing dissolution following a second order exponential decay profile. In vitro cellular experiments using mouse myoblasts showed that PNAM acts as an effective spatial cell barrier for 38 h, with slow migration of cells into the PNAM area occurring between 45 and 73 h after cell seeding. At 80 h myoblasts fully occupied the area initially blocked by PNAM. Immunofluorescent staining of myoblasts adjacent to PNAM showed normal cytoskeletal structure and well developed focal adhesions indicating limited PNAM toxicity. This study shows that PNAM is an easy to synthesize physical hydrogel that acts as a temporal and spatial barrier to cell adhesion.

Directed nerve regeneration enabled by wirelessly powered electrodes printed on a biodegradable polymer.

Wirelessly directed nerve regeneration: inductively powered electrical stimulation circuits on the biodegradable polymer polycaprolactone demonstrate directed regeneration of sensory neurons from a dorsal root ganglion. These circuits, produced using a unique transfer printing process, illustrate progress towards the use of electrical stimulation systems on biodegradable materials to improve peripheral nerve repair functional outcomes.

Osteogenesis of mesenchymal stem cells by nanoscale mechanotransduction.

It is likely that mesenchymal stem cells will find use in many autologous regenerative therapies. However, our ability to control cell stem growth and differentiation is presently limited, and this is a major hurdle to the clinical use of these multipotent cells especially when considering the desire not to use soluble factors or complex media formulations in culture. Also, the large number of cells required to be clinically useful is currently a hurdle to using materials-based (stiffness, chemistry, nanotopography, etc.) culture substrates. Here we give a first demonstration of using nanoscale sinusoidal mechanotransductive protocols (10-14 nm displacements at 1 kHz frequency), "nanokicking", to promote osteoblastogenesis in human mesenchymal stem cell cultures. On the basis of application of the reverse piezo effect, we use interferometry to develop the optimal stem cell stimulation conditions, allowing delivery of nanoscale cues across the entire surface of the Petri dishes used. A combination of immunofluorescence, PCR, and microarray has then been used to demonstrate osteoblastogenesis, and the arrays implicate RhoA as central to osteoblastic differentiation in agreement with materials-based strategies. We validate this with pharmacological inhibition of RhoA kinase. It is easy to envisage such stimulation protocols being up-scaled to form large-scale osteoblast bioreactors as standard cell culture plates and incubators are used in the protocol.

The development of a ε-polycaprolactone scaffold for central nervous system repair.

Potential treatment strategies for the repair of spinal cord injury (SCI) currently favor a combinatorial approach incorporating several factors, including exogenous cell transplantation and biocompatible scaffolds. The use of scaffolds for bridging the gap at the injury site is very appealing although there has been little investigation into the central nervous system neural cell interaction and survival on such scaffolds before implantation. Previously, we demonstrated that aligned microgrooves 12.5-25 µm wide on ε-polycaprolactone (PCL) promoted aligned neurite orientation and supported myelination. In this study, we identify the appropriate substrate and its topographical features required for the design of a three-dimensional scaffold intended for transplantation in SCI. Using an established myelinating culture system of dissociated spinal cord cells, recapitulating many of the features of the intact spinal cord, we demonstrate that astrocytes plated on the topography secrete soluble factors(s) that delay oligodendrocyte differentiation, but do not prevent myelination. However, as myelination does occur after a further 10-12 days in culture, this does not prevent the use of PCL as a scaffold material as part of a combined strategy for the repair of SCI.

Influence of variable substrate geometry on wettability and cellular responses.

In this report, we evaluate the impact of a systematic change to the extracellular environment on cell morphology and functionality by combining the inherent properties of biocompatible polymers such as polydimethylsiloxane and polycaprolactone with a specific surface response. By microstructuring pillars and pits on the substrates, varying spacing and height of the structures, we investigate the role of topography in fibroblast cell adhesion and viability. The change of wetting behaviour was tailored and evaluated in terms of contact angle measurements. It was shown that the range of micro-scale physical cues at the interface between the cells and the surrounding environment affects cell shape and migrations, indicating a tendency to respond differently to higher features of the micro-scale. We found that surface topography seems dominant over material wettability, fibroblasts responded to variations in topography by altering morphology and migrating along the direction of spacing among the features biased by the height of structures and not by the material. It is therefore possible to selectively influence either cell adhesion or morphology by choosing adequate topography of the surface. This work can impact in the design of biomaterials and can be applied to implanted biomedical devices, tissue engineering scaffolds and lab on chip devices.

Label-free segmentation of Co-cultured cells on a nanotopographical gradient.

The function and fate of cells is influenced by many different factors, one of which is surface topography of the support culture substrate. Systematic studies of nanotopography and cell response have typically been limited to single cell types and a small set of topographical variations. Here, we show a radical expansion of experimental throughput using automated detection, measurement, and classification of co-cultured cells on a nanopillar array where feature height changes continuously from planar to 250 nm over 9 mm. Individual cells are identified and characterized by more than 200 descriptors, which are used to construct a set of rules for label-free segmentation into individual cell types. Using this approach we can achieve label-free segmentation with 84% confidence across large image data sets and suggest optimized surface parameters for nanostructuring of implant devices such as vascular stents.