Multi-casting approach for vascular networks in cellularized hydrogels

Vascularization is essential for living tissue and remains a major challenge in the field of tissue engineering. A lack of a perfusable channel network within a large and densely populated tissue engineered construct leads to necrotic core formation, preventing fabrication of functional tissues and organs. We report a new method for producing a hierarchical, three-dimensional (3D) and perfusable vasculature in a large, cellularized fibrin hydrogel. Bifurcating channels, varying in size from 1 mm to 200-250 µm, are formed using a novel process in which we convert a 3D printed thermoplastic material into a gelatin network template, by way of an intermediate alginate hydrogel. This enables a CAD-based model design, which is highly customizable, reproducible, and which can yield highly complex architectures, to be made into a removable material, which can be used in cellular environments. Our approach yields constructs with a uniform and high density of cells in the bulk, made from bioactive collagen and fibrin hydrogels. Using standard cell staining and immuno-histochemistry techniques, we showed good cell seeding and the presence of tight junctions between channel endothelial cells, and high cell viability and cell spreading in the bulk hydrogel.This research was supported by the European Research Council (ERC, grant no. 240446), and an Engineering for Clinical Practice Grant from the Department of Engineering, University of Cambridge. A.W.J. acknowledges the support of the Engineering and Physical Sciences Research Council (EPSRC) through a PhD studentship (EP/L504920/1). R.A.B. gratefully acknowledges financial support from the National Institute for Health Research

Cell response to plasma electrolytic oxidation surface-modified low-modulus β-type titanium alloys.

Plasma electrolytic oxidation (PEO) has been demonstrated to be an effective surface treatment for enhancing the osteoconduction and osseointegration of commercially pure α-Ti (CP α-Ti) dental implant materials for clinical application. To explore the feasibility of extending the application of PEO to low-modulus β-type titanium alloys for load-bearing orthopaedic implants, a thorough understanding of the effect of substrate material on the biological performance of the PEO-treated surface is required. A 10 kW 50 Hz KeroniteTM processing unit was used to modify the surface of low-modulus near β-Ti13Nb13Zr and β-Ti45Nb substrates. CP α-Ti and (α + β)-Ti6Al4V were also used in parallel as reference materials. In vitro culture of foetal human osteoblast (fHOb) cells on PEO-treated low-modulus near β-Ti13Nb13Zr and β-Ti45Nb alloys revealed comparable behaviour to that seen with CP α-Ti and (α + β)-Ti6Al4V with respect to metabolic activity, collagen production, matrix formation and matrix mineralisation. No difference was observed in TNF-α and IL-10 cytokine release from CD14+ monocytes as markers of inflammatory response across samples. Cell interdigitation into the porous structure of the PEO coatings was demonstrated and cell processes remained adherent to the porous structure despite rigorous sonication. This study shows that PEO technology can be used to modify the surface of low-modulus β-type titanium alloys with porous structure facilitating osseointegration, without impeding osteoblast activity or introducing an untoward inflammatory response.European Commission FP7 International training Network National Institute for Health Research Cambridge Biomedical Research Centr

Bioactive conformable hydrogel-carbonated hydroxyapatite nanocomposite coatings on Ti-6Al-4V substrates

A series of nanocomposite coatings was produced, comprising a hydrogel polymer, poly(2-hydroxyethyl methacrylate)/poly(ε-caprolactone) (PHEMA/PCL) matrix with nanoscale carbonated hydroxyapatite (nCHA) filler particles. The weight fraction of the filler was varied from 0 to 20% and the composites were applied as coatings onto Ti-6Al-4V substrates. The filler distribution and surface morphology were investigated by AFM, and the mechanical stability of the coatings was characterised using nanoindentation in both dry and wet conditions. The cellular response to the coatings was also examined in vitro using human osteoblast (HOB) cells. It was found that interfacial cracking occurred for composites containing greater than 10 wt.% nCHA and that 10 wt.% nCHA composite coatings appear to offer the greatest coating stability and bioactivity compared with the other composite coatings. It was concluded that the nCHA-containing PHEMA/PCL composite coatings had the potential to provide a soft, low modulus interface between metal implants and bone

Cell structure, stiffness and permeability of freeze-dried collagen scaffolds in dry and hydrated states.

UNLABELLED: Scaffolds for tissue engineering applications should be highly permeable to support mass transfer requirements while providing a 3-D template for the encapsulated biological cells. High porosity and cell interconnectivity result in highly compliant scaffolds. Overstraining occurs easily with such compliant materials and can produce misleading results. In this paper, the cell structure of freeze-dried collagen scaffolds, in both dry and hydrated states, was characterised using X-ray tomography and 2-photon confocal microscopy respectively. Measurements have been made of the scaffold's Young's modulus using conventional mechanical testing and a customised see-saw testing configuration. Specific permeability was measured under constant pressure gradient and compared with predictions. The collagen scaffolds investigated here have a coarse cell size (∼100-150 μm) and extensive connectivity between adjacent cells (∼10-30 μm) in both dry and hydrated states. The Young's modulus is very low, of the order of 10 kPa when dry and 1 kPa when hydrated. There is only a single previous study concerning the specific permeability of (hydrated) collagen scaffolds, despite its importance in nutrient diffusion, waste removal and cell migration. The experimentally measured value reported here (5 × 10(-)(10)m(2)) is in good agreement with predictions based on Computational Fluid Dynamics simulation and broadly consistent with the Carman-Kozeny empirical estimate. It is however about three orders of magnitude higher than the single previously-reported value and this discrepancy is attributed at least partly to the high pressure gradient imposed in the previous study. STATEMENT OF SIGNIFICANCE: The high porosity and interconnectivity of tissue engineering scaffolds result in highly compliant structures (ie large deflections under low applied loads). Characterisation is essential if these scaffolds are to be systematically optimised. Scaffold overstraining during characterisation can lead to misleading results. In this study, the stiffness (in dry and hydrated states) and specific permeability of freeze-dried collagen scaffolds have been measured using techniques customised for low stiffness structures. The scaffold cell structure is investigated using X-ray computed tomography, which has been applied previously to visualise such materials, without extracting any structural parameters or simulating fluid flow. These are carried out in this work. 2-photon confocal microscopy is used for the first time to study the structure in hydrated state.This research was supported by the European Research Council (Grant No. 240446) and the EPSRC (EP/E025862/1). Financial support for MCV and RAB has been provided via the WD Armstrong studentship and the National Institute for Health Research (NIHR), respectively.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.actbio.2016.01.04

Collagen scaffolds as a tool for understanding the biological effect of silicates

Dietary silicon is essential in the maintenance of bone and cartilage. However, the mechanism by which silicon, in the form of silicates, triggers a biological response has never been uncovered. Here we demonstrate the incorporation of orthosilicic acid (Si(OH)4), the form of silicon in the body, within collagen scaffolds for use as an in vitro platform to identify key genes affected by silicates. Ice-templated collagen–silicate scaffolds, containing 0.21 wt% silicon, were validated by examining the mRNA levels for an array of genes in human osteoblasts and mesenchymal stromal cells (MSC) after 48 h in culture. Several novel genes, such as tumor necrosis factor alpha (TNF), were identified as having potential links to orthosilicic acid, verifying that collagen–silicate scaffolds are a versatile platform for identifying novel mechanisms in which silicates regulate musculoskeletal tissue.The authors gratefully acknowledge the financial support of the Gates Cambridge Trust , ERC Advanced Grant 320598 3D-E and from the National Institute for Health Research. RJ is supported by the Medical Research Council (Grant number MC_US_A090_0008/Unit Programme number U1059).This is the final published version. It first appeared at http://www.sciencedirect.com/science/article/pii/S0167577X15300203#

The Turbulent Structure of the Arctic Summer Boundary Layer During The Arctic Summer Cloud‐Ocean Study

The mostly ice covered Arctic Ocean is dominated by low‐level liquid‐ or mixed‐phase clouds. Turbulence within stratocumulus is primarily driven by cloud top cooling that induces convective instability. Using a suite of in situ and remote sensing instruments we characterize turbulent mixing in Arctic stratocumulus, and for the first time we estimate profiles of the gradient Richardson number at relatively high resolution in both time (10 min) and altitude (10 m). It is found that the mixing occurs both within the cloud, as expected, and by wind shear instability near the surface. About 75% of the time these two layers are separated by a stably stratified inversion at 100–200 m altitude. Exceptions are associated with low cloud bases that allow the cloud‐driven turbulence to reach the surface. The results imply that turbulent coupling between the surface and the cloud is sporadic or intermittent

The transient voltage response of ReBCO coated conductors exhibiting dynamic resistance

Abstract: Dynamic resistance can be observed in a superconducting tape carrying a DC current which is exposed to an oscillating magnetic field. This effect is attributed to the interaction between the transport current and moving fluxons, and can occur in various superconducting components including high temperature superconducting (HTS) flux pumps, fast-ramping magnets and HTS rotating machines. Although conventionally expressed in terms of a DC ‘resistance,’ the phenomenon is inherently transient in nature, and the voltage drop across the superconductor follows a time-dependent periodic waveform. Here we present experimental measurements of the dynamic resistance of different REBCO tapes carrying a DC current and exposed to an oscillating perpendicular field. Measurements of both the transient voltage waveforms and the time-averaged DC resistances are compared with numerical finite element simulations obtained using the H-formulation. We observe clear variations between the voltage response from different tapes, which can be understood in terms of their differing Jc(B, θ) dependence. In particular, a key feature of the experimentally measured waveforms is the emergence of a split ‘double peak’ at higher applied fields. Graphical visualisations of the finite element data show that this coincides with a periodic increase in Jc(B, θ) throughout the tape. This occurs during each cycle at those times when the applied field falls below the shielding threshold of the tape (as the penetrating field within the tape then approaches zero). Our findings show that models which assume a constant Jc irrespective of local field strength cannot capture the full range of behaviour observed by experiment. This emphasises the importance of employing experimentally measured Jc(B, θ) data when simulating transient effects in HTS materials

Numerical Modelling of Dynamic Resistance in a Parallel-Connected Stack of HTS Coated-Conductor Tapes

Dynamic resistance is observed in type-II superconductors carrying a DC transport current while simultaneously exposed to an alternating magnetic field. The appearance of a nonzero resistance is attributed to the interaction between the transport current and moving fluxons. This effect is relevant to many superconductor applications such as high-temperature-superconductor (HTS) flux pumps, DC / AC magnets, synchronous machines, and persistent current switches. Here, we present a finite element method (FEM) analysis of both the time averaged dynamic resistance and the instantaneous current sharing behaviour in a cable comprised of a stack of four YBCO thin films connected in parallel. Numerical modelling was performed using the H-formulation method implemented in the commercial software COMSOL. The model employs experimentally measured values of the angular dependence of the critical current Ic(B, θ) and the flux creep exponent n(B, θ). A single threshold field is observed, above which a finite dynamic resistance is observed in all tapes simultaneously. The time-averaged dynamic resistance of individual tapes tends to be larger for the exterior tapes than the interior tapes, but this difference decreases as the total transport current in the cable increases. We attribute this to shielding currents flowing in the exterior tapes during the majority of the cycle, which displace net DC current into the interior tapes. However, the relative proportion of DC transport current flowing in the exterior and interior tapes is also observed to vary periodically once per half cycle of the applied field. This is due to the periodic trapping of return screening currents in the interior tapes.New Zealand MBIE Endeavour Grant No. RTVU1707 and NZ Royal Society Marsden Grant No. MFP-VUW180

Damage and energy absorption behaviour of composite laminates under impact loading using different impactor geometries

The present paper compares the damage and energy absorption behaviour of composites subjected to low-velocity impact using different frontal geometries for the impactor, with the composites possessing a layup of [02/902]2s. In this study, the rigid impactors with either round-nosed or flat-ended frontal geometry are employed to perform drop-weight tests at various impact energies ranging from 10 to 30 J. The measured loading response and energy absorption are analysed and compared. Additionally, the types and extent of impact-induced damage in the composite specimens are assessed via ultrasonic C-scan, optical microscopy (OM) and scanning electron microscopy (SEM) studies. It is shown that the impact energy threshold for damage initiation is greater than 20 J when using the flat-ended impactor but is less than 10 J when using the round-nosed impactor. In both cases, delamination initiates between the plies in the composite laminate. However, for the flat-ended impactor, the damage behaviour of the fibres exhibits kinking fracture, which differs from the pull-out fibre-fracture caused by the round-nosed impactor. These differences in behaviour are attributed to impactor/composite contact geometry effects which leads to different extents of indentation damage, which in turn directly affects the degree of delamination and fibre damage in the composite

Effect of Rotation on Scaffold Motion and Cell Growth in Rotating Bioreactors

Efficient use of different bioreactor designs to improve cell growth in three-dimensional scaffolds requires an understanding of their mechanism of action. To address this for rotating wall vessel bioreactors, fluid and scaffold motion were investigated experimentally at different rotation speeds and vessel fill volumes. Low cost bioreactors with single and dual axis rotation were developed to investigate the effect of these systems on human osteoblast proliferation in free floating and constrained collagen-glycosaminoglycan porous scaffolds. A range of scaffold motions (free fall, periodic oscillation, and orbital motion) were observed at the rotation speeds and vessel fluid/air ratios used, with 85% fluid fill and an outer vessel wall velocity of ∼14 mm s$^{−1}$ producing a scaffold in a free fall state. The cell proliferation results showed that after 14 and 21 days of culture, this combination of fluid fill and speed of rotation produced significantly greater cell numbers in the scaffolds than when lower or higher rotation speeds (p  0.05).This research was supported by the European Research Council (Grant No. 240446) and the EPSRC (EP/E025862/1). Financial support for M.C.V. and R.A.B. has been provided through the WD Armstrong studentship and the National Institute for Health Research, respectively