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

Tuning structural relaxations, mechanical properties, and degradation timescale of PLLA during hydrolytic degradation by blending with PLCL-PEG

Poly-L-lactide (PLLA) is a popular choice for medical devices due to its bioresorbability and superior mechanical properties compared with other polymers. However, although PLLA has been investigated for use in bioresorbable cardiovascular stents, it presents application-specific limitations which hamper device therapies. These include low toughness and strength compared with metals used for this purpose, and slow degradation. Blending PLLA with novel polyethylene glycol functionalised poly(L-lactide-co-$\varepsilon$-caprolactone) (PLCL-PEG) materials has been investigated here to tailor the mechanical properties and degradation behaviour of PLLA. This exciting approach provides a foundation for a next generation of bioresorbable materials whose properties can be rapidly tuned. The degradation of PLLA was significantly accelerated by addition of PLCL-PEG. After 30 days of degradation, several structural changes were observed in the polymer blends, which were dependent on the level of PLCL-PEG addition. Blends with low PLCL-PEG content displayed enthalpy relaxation, resulting in embrittlement, while blends with high PLCL-PEG content displayed crystallisation, due to enhanced chain mobility brought on by chain scission, also causing embrittlement. Moderate PLCL-PEG additions (10% PLCL(70:30)-PEG and 20 - 30% PLCL(80:20)-PEG) stabilised the structure, reducing the extent of enthalpy relaxation and crystallisation and thus retaining ductility. Compositional optimisation identified a sweet spot for this blend strategy, whereby the ductility was enhanced while maintaining strength. Our results indicate that blending PLLA with PLCL-PEG provides an effective method of tuning the degradation timescale and mechanical properties of PLLA, and provides important new insight into the mechanisms of structural relaxations that occur during degradation, and strategies for regulating these.Lucideon Lt

The 3D printing of freestanding PLLA thin layers and improving first layer consistency through the introduction of sacrificial PVA

Fused filament fabrication (FFF) is an inexpensive way of producing objects through a programmed layer-by-layer deposition. For multi-layer, macro-scaled prints, acceptable printing is achieved provided, amongst other factors, first layer adhesion is sufficient to fix a part to the surface during printing. However, in the deposition of structures with a single or few layers, first layer consistency is significantly more important and is an issue that has been previously overlooked. As layer-to-bed adhesion is prioritised in first layer printing, thin layer structures are difficult to remove without damage. The deposition of controllable thin structures has potential in tissue engineering through the use of bioactive filaments and incorporation of microfeatures into complex, patient-specific scaffolds. This paper presents techniques to progress the deposition of thin, reproducible structures. The linear thickness variation of 3D-printed single PVA and PLLA layers is presented as a function of extrusion factor and the programmed vertical distance moved by the nozzle between layers (the layer separation). A sacrificial PVA layer is shown to significantly improve first layer consistency, reducing the onus on fine printer calibration in the deposition of single layers. In this way, the linear variation in printed single PLLA layers with bed deviation is drastically reduced. Further, this technique is used to demonstrate the printing of freestanding thin layers of ~25 µm in thickness.</jats:p

The Far-Infrared Properties of Spatially Resolved AKARI Observations

We present the spatially resolved observations of IRAS sources from the Japanese infrared astronomy satellite AKARI All-Sky Survey during the performance verification (PV) phase of the mission. We extracted reliable point sources matched with IRAS point source catalogue. By comparing IRAS and AKARI fluxes, we found that the flux measurements of some IRAS sources could have been over or underestimated and affected by the local background rather than the global background. We also found possible candidates for new AKARI sources and confirmed that AKARI observations resolved IRAS sources into multiple sources. All-Sky Survey observations are expected to verify the accuracies of IRAS flux measurements and to find new extragalactic point sources.Comment: 11 pages, 7 figures, accepted publication in PASJ AKARI special issu

Regulation of cell survival by sphingosine-1-phosphate receptor S1P1 via reciprocal ERK-dependent suppression of bim and PI-3-kinase/protein kinase C-mediated upregulation of Mcl-1

Although the ability of bioactive lipid sphingosine-1-phosphate (S1P) to positively regulate anti-apoptotic/pro-survival responses by binding to S1P1 is well known, the molecular mechanisms remain unclear. Here we demonstrate that expression of S1P1 renders CCL39 lung fibroblasts resistant to apoptosis following growth factor withdrawal. Resistance to apoptosis was associated with attenuated accumulation of pro-apoptotic BH3-only protein Bim. However, although blockade of extracellular signal-regulated kinase (ERK) activation could reverse S1P1-mediated suppression of Bim accumulation, inhibition of caspase-3 cleavage was unaffected. Instead S1P1-mediated inhibition of caspase-3 cleavage was reversed by inhibition of phosphatidylinositol-3-kinase (PI3K) and protein kinase C (PKC), which had no effect on S1P1 regulation of Bim. However, S1P1 suppression of caspase-3 was associated with increased expression of anti-apoptotic protein Mcl-1, the expression of which was also reduced by inhibition of PI3K and PKC. A role for the induction of Mcl-1 in regulating endogenous S1P receptor-dependent pro-survival responses in human umbilical vein endothelial cells was confirmed using S1P receptor agonist FTY720-phosphate (FTY720P). FTY720P induced a transient accumulation of Mcl-1 that was associated with a delayed onset of caspase-3 cleavage following growth factor withdrawal, whereas Mcl-1 knockdown was sufficient to enhance caspase-3 cleavage even in the presence of FTY720P. Consistent with a pro-survival role of S1P1 in disease, analysis of tissue microarrays from ER+ breast cancer patients revealed a significant correlation between S1P1 expression and tumour cell survival. In these tumours, S1P1 expression and cancer cell survival were correlated with increased activation of ERK, but not the PI3K/PKB pathway. In summary, pro-survival/anti-apoptotic signalling from S1P1 is intimately linked to its ability to promote the accumulation of pro-survival protein Mcl-1 and downregulation of pro-apoptotic BH3-only protein Bim via distinct signalling pathways. However, the functional importance of each pathway is dependent on the specific cellular context

High-speed camera characterization of voluntary eye blinking kinematics.

Blinking is vital to maintain the integrity of the ocular surface and its characteristics such as blink duration and speed can vary significantly, depending on the health of the eyes. The blink is so rapid that special techniques are required to characterize it. In this study, a high-speed camera was used to record and characterize voluntary blinking. The blinking motion of 25 healthy volunteers was recorded at 600 frames per second. Master curves for the palpebral aperture and blinking speed were constructed using palpebral aperture versus time data taken from the high-speed camera recordings, which show that one blink can be divided into four phases; closing, closed, early opening and late opening. Analysis of data from the high-speed camera images was used to calculate the palpebral aperture, peak blinking speed, average blinking speed and duration of voluntary blinking and compare it with data generated by other methods previously used to evaluate voluntary blinking. The advantages of the high-speed camera method over the others are discussed, thereby supporting the high potential usefulness of the method in clinical research

Improved Culture-Based Isolation of Differentiating Endothelial Progenitor Cells from Mouse Bone Marrow Mononuclear Cells

Numerous endothelial progenitor cell (EPC)-related investigations have been performed in mouse experiments. However, defined characteristics of mouse cultured EPC have not been examined. We focused on fast versus slow adherent cell population in bone marrow mononuclear cells (BMMNCs) in culture and examined their characteristics. After 24 h-culture of BMMNCs, attached (AT) cells and floating (FL) cells were further cultured in endothelial differentiation medium separately. Immunological and molecular analyses exhibited more endothelial-like and less monocyte/macrophage-like characteristics in FL cells compared with AT cells. FL cells formed thick/stable tube and hypoxia or shear stress overload further enhanced these endothelial-like features with increased angiogenic cytokine/growth factor mRNA expressions. Finally, FL cells exhibited therapeutic potential in a mouse myocardial infarction model showing the specific local recruitment to ischemic border zone and tissue preservation. These findings suggest that slow adherent (FL) but not fast attached (AT) BMMNCs in culture are EPC-rich population in mouse

UBR2 of the N-End Rule Pathway Is Required for Chromosome Stability via Histone Ubiquitylation in Spermatocytes and Somatic Cells

The N-end rule pathway is a proteolytic system in which its recognition components (N-recognins) recognize destabilizing N-terminal residues of short-lived proteins as an essential element of specific degrons, called N-degrons. The RING E3 ligases UBR2 and UBR1 are major N-recognins that share size (200 kDa), conserved domains and substrate specificities to N-degrons. Despite the known function of the N-end rule pathway in degradation of cytosolic proteins, the major phenotype of UBR2-deficient male mice is infertility caused by arrest of spermatocytes at meiotic prophase I. UBR2-deficient spermatocytes are impaired in transcriptional silencing of sex chromosome-linked genes and ubiquitylation of histone H2A. In this study we show that the recruitment of UBR2 to meiotic chromosomes spatiotemporally correlates to the induction of chromatin-associated ubiquitylation, which is significantly impaired in UBR2-deficient spermatocytes. UBR2 functions as a scaffold E3 that promotes HR6B/UbcH2-dependent ubiquitylation of H2A and H2B but not H3 and H4, through a mechanism distinct from typical polyubiquitylation. The E3 activity of UBR2 in histone ubiquitylation is allosterically activated by dipeptides bearing destabilizing N-terminal residues. Insufficient monoubiquitylation and polyubiquitylation on UBR2-deficient meiotic chromosomes correlate to defects in double strand break (DSB) repair and other meiotic processes, resulting in pachytene arrest at stage IV and apoptosis. Some of these functions of UBR2 are observed in somatic cells, in which UBR2 is a chromatin-binding protein involved in chromatin-associated ubiquitylation upon DNA damage. UBR2-deficient somatic cells show an array of chromosomal abnormalities, including hyperproliferation, chromosome instability, and hypersensitivity to DNA damage-inducing reagents. UBR2-deficient mice enriched in C57 background die upon birth with defects in lung expansion and neural development. Thus, UBR2, known as the recognition component of a major cellular proteolytic system, is associated with chromatin and controls chromatin dynamics and gene expression in both germ cells and somatic cells