Energy based models to determine fracture toughness of thin coated systems by nanoindentation

This project alms to determine the fracture toughness of thin coated systems by nanoindentation. The development of techniques for the assessment of coating toughness lags behind the determination of Young's modulus and hardness of thin coatings. No universal model or technique has been agreed to estimate coating toughness. With the development of complex coating stacks (e.g. multilayered coated systems) and the presence of variable crack patterns, the difficulty of generating a solution by stress analysis based models is dramatically increased. Therefore, there is an urgent need for the development of models to deal with complex coatings and varied cracking patterns. The most successful models in this respect are energy-based. In this thesis the existing models and techniques to assess coating toughness and adhesion have been critically reviewed. The stress analysis based models usually require empirical fitting parameters and they only deal with specific crack patterns. In contrast, the energy based models can deal with different cracking patterns without empirical constants; but they usually require that the crack propagates during loading cycle only, whilst, stress analysis based models do not have such a restriction. Several new models have been developed to assess coating toughness in this work. Two of them are based on excursions in load-displacement (P-c5) curves resulting from fracture during nanoindentation. The first model (Wcdp method) is based on extrapolating the plot of total work during indentation versus displacement. Compared to a literature model based on extrapolating P-c5 curve, this approach removes the influence on fracture dissipated energy from plastic deformation of the substrate. The second is a modified model to estimate the limiting value of coating toughness which could equally give the upper and lower boundary for toughness from nanoindentation performed under load control and displacement control, thus improving on the initial boundary model by Toonder et al which could only provide an upper boundary of coating toughness for nanoindentation under displacement control. However, it is often observed that fracture does not result in an excursion in the P-c5 curve, which requires a different modelling approach. The third model (Wirr-W p model) developed addresses this problem. All the previous models address through-thickness fracture which is widely observed in nanoindentation testing of hard coatings. In addition, another energy based method is proposed to estimate the adhesion of coatings by analysing the extra linear recovery of unloading curve associated with the rebound of the coating during unloading. Models were validated by experiments carried out by a range of nanoindentation techniques. The low load tests were performed by a Hysitron Triboindenter fitted with a sharp cube comer tip and a Berkovich tip. The maximum penetration was in the range of 40-400nm. Higher load tests were performed using a Nanoindenter II ™ fitted with a Berkovich tip in the range of lOmN-SOOmN. Atomic force microscopy (AFM) , highresolution scanning electron microscopy (SEM) and reflected light microscopy have been employed to investigate the fracture behaviour. To examme the models developed in this work, two different coated systems were investigated: one is multilayer optical coatings (total thickness <SOOnm) including ITO, Sn02, ZnO, and TiOxNy on glass, which is the case of harder coating on hard (but relatively softer) substrate and they are the main samples investigated in this project; the other is a Ij.lm fullerene-like CNx coating on various ceramic substrates such as SiC, Si, and Ah03, which is the case of hard (but relatively softer) coating on a harder substrate. Some common brittle bulk materials (e.g. Si) were also tested to examine the applicability of the models. Reasonable toughness values for both coated systems and bulk materials have been obtained by the new models developed in this work. To gain more insight into the fracture mechanisms of coated systems, the threshold of fracture is also an important issue to be addressed. The loading rate may influence the critical load for fracture in brittle materials. It was observed that within the penetration rate range used in this study (lO-40nmls) the higher the penetration rate the higher was the threshold for cracking in the optical multilayer coatings on glass indented by a cube comer tip. When analysing the P-J curves at different loading rates, significantly different behaviour was observed for ITO and Sn02 coatings which is possibly due to a pressureinduced phase transformation in these coatings.EThOS - Electronic Theses Online ServiceOverseas Research Students Award Scheme (ORSAS), Newcastle UniversityGBUnited Kingdo

Exosomes From miRNA-126-Modified Endothelial Progenitor Cells Alleviate Brain Injury and Promote Functional Recovery After Stroke

Aims: We previously showed that the protective effects of endothelial progenitor cells (EPCs)-released exosomes (EPC-EXs) on endothelium in diabetes. However, whether EPC-EXs are protective in diabetic ischemic stroke is unknown. Here, we investigated the effects of EPC-EXs on diabetic stroke mice and tested whether miR-126 enriched EPC-EXs (EPC-EXs miR126 ) have enhanced efficacy. Methods: The db/db mice subjected to ischemic stroke were intravenously administrated with EPC-EXs 2 hours after ischemic stroke. The infarct volume, cerebral microvascular density (MVD), cerebral blood flow (CBF), neurological function, angiogenesis and neurogenesis, and levels of cleaved caspase-3, miR-126, and VEGFR2 were measured on day 2 and 14. Results: We found that (a) injected EPC-EXs merged with brain endothelial cells, neurons, astrocytes, and microglia in the peri-infarct area; (b) EPC-EXs miR126 were more effective than EPC-EXs in decreasing infarct size and increasing CBF and MVD, and in promoting angiogenesis and neurogenesis as well as neurological functional recovery; (c) These effects were accompanied with downregulated cleaved caspase-3 on day 2 and vascular endothelial growth factor receptor 2 (VEGFR2) upregulation till day 14. Conclusion: Our results indicate that enrichment of miR126 enhanced the therapeutic efficacy of EPC-EXs on diabetic ischemic stroke by attenuating acute injury and promoting neurological function recovery

EPC-EXs Improve Astrocyte Survival and Oxidative Stress Through Different Uptaking Pathways in Diabetic Hypoxia Condition

Background: Hyperglycemia contributes to cardiovascular complications in patients with type 2 diabetes. We confirmed that high glucose (HG) induces endothelial dysfunction and cerebral ischemic injury is enlarged in diabetic mice. Stem cell-released exosomes have been shown to protect the brain from ischemic stroke. We have previously shown that endothelial progenitor cells (EPCs)-released exosomes (EPC-EXs) can protect endothelial cells from hypoxia/reoxygenation (H/R) and HG-induced injury. Here, we aim to investigate the effects of EPC-EXs on astrocytes under H/R and HG-induced injury and whether miR-126 enriched EPC-EXs (miR126-EPC-EXs) have enhanced efficacy. Methods: EPC-EX uptake and co-localization were measured by fluorescent microscopy using PKH26 and DAPI staining. miR-126 enrichment was achieved by transfecting with miR-126 mimics and quantified with real-time PCR. After co-incubation, cell death or injury was measured by using LDH (Lactate Dehydrogenase) assay. Oxidative stress/ROS (reactive oxygen species) generation was measured by DHE (Dihydroethidium) staining and lipid peroxidation assay. Results: The EPC-EXs were effectively taken up by the astrocytes in a concentration as well as time-dependent manners and were co-localized within the nucleus as well as the cytoplasm. Pathway uptake inhibitors revealed that the EPC-EXs are effectively taken up by the clathrin-mediated, caveolin-dependent, and micropinocytosis via PI3K/Akt pathway. H/R and HG-induced a cell injury which could be protected by EPC-EXs evidenced by decreased cell cytotoxicity, oxidative stress, and lipid peroxidation. Moreover, miR-126 overexpression could increase the level of miR-126 in astrocytes and enhance the protective effects of EPC-EXs. Conclusions: These results collectively indicate that the EPC-EXs could protect astrocytes against the HG plus H/R-induced damage

EPC-EXs Improve Neuronal Survival and Oxidative Stress Through Different Uptaking Pathways in Diabetic Hypoxia Condition

Background: Hyperglycemia contributes to cardiovascular complications in patients with type 2 diabetes. We confirmed that high glucose (HG) induces endothelial dysfunction and cerebral ischemic injury is enlarged in diabetic mice. Stem cell-released exosomes have been shown to protect the brain from ischemic stroke. We have previously shown that endothelial progenitor cells (EPCs)-released exosomes (EPC-EXs) can protect endothial cells from hypoxia/reoxygenation (H/R) and HG-induced injury. Here, we aim to investigate the effects of EPC-EXs on astrocytes under H/R and HG-induced injury and whether miR-126 enriched EPC-EXs (EPC-EXsmiR126) have enhanced efficacy. Methods: EPC-EX uptake and co-localization were measured by fluorescent microscopy using PKH26 and DAPI staining. miR-126 enrichment was achieved by transfecting with miR-126 mimics and quantified with real-time PCR. After co-incubation, cell death or apoptosis was measured by using flow cytometric analysis and LDH (Lactate Dehydrogenase) assay. Oxidative stress/ROS (reactive oxygen species) generation was measured by DHE (Dihydroethidium) staining and lipid peroxidation assay. Results: The EPC-EXs were effectively taken up by the astrocytes in a concentration as well as time- dependent manners and were co-localized within the nucleus as well as the cytoplasm. Pathway uptake inhibitors revealed that the EPC-EXs are effectively taken up by the clathrin-mediated, caveolin-dependent, and micropinocytosis via PI3K/Akt pathway. H/R and HG-induced a cell injury which could be protected by EPC-EXs evidenced by decreased cell apoptosis, cell cytotoxicity, oxidative stress, and lipid peroxidation. Moreover, miR-126 overexpression could enhance the protective effects of EPC-EXs. Conclusions: These results collectively indicate that the EPC-EXs could protect astrocytes against the HG plus H/R-induced damage

Moderate Exercise Enhances Endothelial Progenitor Cell Exosomes Release and Function

Purpose: Exercise has cardiovascular benefits which might be related to endothelial progenitor cells (EPC). Meanwhile, there is evidence suggesting that EPC-derived exosomes (EPC-EX) promote vascular repair and angiogenesis through their carried microRNA (miR)-126. In this study, we investigated whether exercise could increase the levels of circulating EPC-EX and their miR-126 cargo, and by which promote the protective function of EPC-EX on endothelial cells (EC). Methods: Plasma EPC-EX from sedentary, low, or moderate exercise mice, respectively, denoted as EPC-EXS, EPC-EXL, and EPC-EXM, were isolated using microbead-based sorting techniques and characterized by nanoparticle tracking analysis, Western blot, and quantitative real-time polymerase chain reaction assessments of biomarkers and miR-126. High glucose (25 mM) with hypoxia (1% O2) was used for inducing an EC injury model. The injured EC were treated by coculturing with vehicle, EPC-EXS, EPC-EXL, EPC-EXM, or EPC-EXM + anti–miR-126. After that, EC were used for flow cytometry analysis of apoptosis, assessments of tube formation and migration, and measurements of miR-126 level and its downstream sprouty-related protein-1 (SPRED1) and vascular endothelial growth factor (VEGF). Results: 1) Isolated EPC-EX positively expressed exosomal markers (CD63 and Tsg101) and EPC markers (CD34 and VEGFR2). 2) Exercise intensity dependently elevated plasma level of EPC, EPC-EX/EPC ratio, and miR-126 expression in EPC and EPC-EX. 3) Injured EC displayed apoptosis increment, angiogenic dysfunction and miR-126 reduction. 4) EPC-EXM had better effects than EPC-EXS and EPC-EXL on alleviating those changes of injured EC, accompanied with SPRED1 downregulation and VEGF upregulation. 5) The effects of EPC-EXM were abolished by miR-126 knockdown. Conclusions: Our data demonstrate that exercise can increase EPC-EX release and miR-126 level and enhance the effects of EPC-EX on protecting EC against injury through the SPRED1/VEGF pathway

Dynamics of Droplets Impacting on Aerogel, Liquid Infused, and Liquid-Like Solid Surfaces

Droplets impacting superhydrophobic surfaces have been extensively studied due to their compelling scientific insights and important industrial applications. In these cases, the commonly reported impact regime was that of complete rebound. This impact regime strongly depends on the nature of the superhydrophobic surface. Here, we report the dynamics of droplets impacting three hydrophobic slippery surfaces, which have fundamental differences in normal liquid adhesion and lateral static and kinetic liquid friction. For an air cushion-like (super)hydrophobic solid surface (Aerogel) with low adhesion and low static and low kinetic friction, complete rebound can start at a very low Weber (We) number (∼1). For slippery liquid-infused porous (SLIP) surfaces with high adhesion and low static and low kinetic friction, complete rebound only occurs at a much higher We number (>5). For a slippery omniphobic covalently attached liquid-like (SOCAL) solid surface, with high adhesion and low static friction similar to SLIPS but higher kinetic friction, complete rebound was not observed, even for a We as high as 200. Furthermore, the droplet ejection volume after impacting the Aerogel surface is 100% across the whole range of We numbers tested compared to other surfaces. In contrast, droplet ejection for SLIPs was only observed consistently when the We was above 5–10. For SOCAL, 100% (or near 100%) ejection volume was not observed even at the highest We number tested here (∼200). This suggests that droplets impacting our (super)hydrophobic Aerogel and SLIPS lose less kinetic energy. These insights into the differences between normal adhesion and lateral friction properties can be used to inform the selection of surface properties to achieve the most desirable droplet impact characteristics to fulfill a wide range of applications, such as deicing, inkjet printing, and microelectronics

miR-132-3p Priming Enhances the Effects of Mesenchymal Stromal Cell-Derived Exosomes on Ameliorating Brain Ischemic Injury

Backgrounds/aims: Mesenchymal stromal cell-derived exosomes (MSC-EXs) could exert protective effects on recipient cells by transferring the contained microRNAs (miRs), and miR-132-3p is one of angiogenic miRs. However, whether the combination of MSC-EXs and miR-132-3p has better effects in ischemic cerebrovascular disease remains unknown. Methods: Mouse MSCs transfected with scrambler control or miR-132-3p mimics were used to generate MSC-EXs and miR-132-3p-overexpressed MSC-EXs (MSC-EXsmiR-132-3p). The effects of EXs on hypoxia/reoxygenation (H/R)-injured ECs in ROS generation, apoptosis, and barrier function were analyzed. The levels of RASA1, Ras, phosphorylations of PI3K, Akt and endothelial nitric oxide synthesis (eNOS), and tight junction proteins (Claudin-5 and ZO-1) were measured. Ras and PI3K inhibitors were used for pathway analysis. In transient middle cerebral artery occlusion (tMCAO) mouse model, the effects of MSC-EXs on the cerebral vascular ROS production and apoptosis, cerebral vascular density (cMVD), Evans blue extravasation, brain water content, neurological deficit score (NDS), and infarct volume were determined. Results: MSC-EXs could deliver their carried miR-132-3p into target ECs, which functionally downregulated the target protein RASA1, while upregulated the expression of Ras and the downstream PI3K phosphorylation. Compared to MSC-EXs, MSC-EXsmiR-132-3p were more effective in decreasing ROS production, apoptosis, and tight junction disruption in H/R-injured ECs. These effects were associated with increased levels of phosphorylated Akt and eNOS, which could be abolished by PI3K inhibitor (LY294002) or Ras inhibitor (NSC 23766). In the tMCAO mouse model, the infusion of MSC-EXsmiR-132-3p was more effective than MSC-EXs in reducing cerebral vascular ROS production, BBB dysfunction, and brain injury. Conclusion: Our results suggest that miR-132-3p promotes the beneficial effects of MSC-EXs on brain ischemic injury through protecting cerebral EC functions

Hierarchical rose-petal surfaces delay the early-stage bacterial biofilm growth

A variety of natural surfaces exhibit antibacterial properties; as a result significant efforts in the past decade have been dedicated towards fabrication of biomimetic surfaces that can help control biofilm growth. Examples of such surfaces include rose petals, which possess hierarchical structures like the micro-papillae measuring tens of microns and nano-folds that range in the size of 700 ±100 nm. We duplicated the natural structures on rose-petal surfaces via a simple UV-curable nanocasting technique, and tested the efficacy of these artificial surfaces in preventing biofilm growth using clinically relevant bacteria strains. The rose-petal structured surfaces exhibited hydrophobicity (contact angle~130.8º ±4.3º) and high contact angle hysteresis (~91.0° ±4.9°). Water droplets on rose-petal replicas evaporated following the constant contact line mode, indicating the likely coexistence of both Cassie and Wenzel states (Cassie-Baxter impregnating wetting state). Fluorescent microscopy and image analysis revealed the significantly lower attachment of Staphylococcus epidermidis (86.1± 6.2% less) and Pseudomonas aeruginosa (85.9 ±3.2% less) on the rose-petal structured surfaces, compared with flat surfaces over a period of 2 hours. Extensive biofilm matrix was observed in biofilms formed by both species on flat surfaces after prolonged growth (several days), but was less apparent on rose-petal biomimetic surfaces. In addition, the biomass of S. epidermidis (63.2 ±9.4% less) and P. aeruginosa (76.0 ±10.0% less) biofilms were significantly reduced on the rose-petal structured surfaces, in comparison to the flat surfaces. By comparing P. aeruginosa growth on representative unitary nano-pillars, we demonstrated that hierarchical structures are more effective in delaying biofilm growth. The mechanisms are two-fold: 1) the nano-folds across the hemispherical micro-papillae restrict initial attachment of bacterial cells and delay the direct contacts of cells via cell alignment, and 2) the hemispherical micro-papillae arrays isolate bacterial clusters and inhibit the formation of a fibrous network. The hierarchical features on rose petal surfaces may be useful for developing strategies to control biofilm formation in medical and industrial contexts

Microstructural and Rheological Transitions in Bacterial Biofilms

Abstract Biofilms are aggregated bacterial communities structured within an extracellular matrix (ECM). ECM controls biofilm architecture and confers mechanical resistance against shear forces. From a physical perspective, biofilms can be described as colloidal gels, where bacterial cells are analogous to colloidal particles distributed in the polymeric ECM. However, the influence of the ECM in altering the cellular packing fraction (ϕ) and the resulting viscoelastic behavior of biofilm remains unexplored. Using biofilms of Pantoea sp. (WT) and its mutant (ΔUDP), the correlation between biofilm structure and its viscoelastic response is investigated. Experiments show that the reduction of exopolysaccharide production in ΔUDP biofilms corresponds with a seven‐fold increase in ϕ, resulting in a colloidal glass‐like structure. Consequently, the rheological signatures become altered, with the WT behaving like a weak gel, whilst the ΔUDP displayed a glass‐like rheological signature. By co‐culturing the two strains, biofilm ϕ is modulated which allows us to explore the structural changes and capture a change in viscoelastic response from a weak to a strong gel, and to a colloidal glass‐like state. The results reveal the role of exopolysaccharide in mediating a structural transition in biofilms and demonstrate a correlation between biofilm structure and viscoelastic response