"Affimer" synthetic protein scaffolds block oxidized LDL binding to the LOX-1 scavenger receptor and inhibit ERK1/2 activation.

In multicellular organisms, a variety of lipid-protein particles control the systemic flow of triacylglycerides, cholesterol, and fatty acids between cells in different tissues. The chemical modification by oxidation of these particles can trigger pathological responses, mediated by a group of membrane proteins termed scavenger receptors. The lectin-like oxidized low-density lipoprotein (LOX-1) scavenger receptor binds to oxidized low-density lipoprotein (oxLDL) and mediates both signaling and trafficking outcomes. Here, we identified five synthetic proteins termed Affimers from a phage display library, each capable of binding recombinant LOX-1 extracellular (oxLDL-binding) domain with high specificity. These Affimers, based on a phytocystatin scaffold with loop regions of variable sequence, were able to bind to the plasma membrane of HEK293T cells exclusively when human LOX-1 was expressed. Binding and uptake of fluorescently labeled oxLDL by the LOX-1-expressing cell model was inhibited with subnanomolar potency by all 5 Affimers. ERK1/2 activation, stimulated by oxLDL binding to LOX-1, was also significantly inhibited (p < 0.01) by preincubation with LOX-1-specific Affimers, but these Affimers had no direct agonistic effect. Molecular modeling indicated that the LOX-1-specific Affimers bound predominantly via their variable loop regions to the surface of the LOX-1 lectin-like domain that contains a distinctive arrangement of arginine residues previously implicated in oxLDL binding, involving interactions with both subunits of the native, stable scavenger receptor homodimer. These data provide a new class of synthetic tools to probe and potentially modulate the oxLDL/LOX-1 interaction that plays an important role in vascular disease.

Compartmentalisation of the inflammatory response following aneurysmal subarachnoid haemorrhage.

INTRODUCTION: There is some evidence to suggest that a systemic and central nervous system (CNS) inflammatory response occurs following aneurysmal subarachnoid haemorrhage (aSAH) which may be related to the pathophysiology of early brain injury and delayed ischaemic neurological deficit (DIND). The aim of this study was to measure inflammatory mediator levels in plasma and cerebrospinal fluid (CSF) in the days following aSAH and to determine their association with aSAH, DIND and clinical outcome. MATERIAL AND METHODS: Plasma and CSF samples were obtained prospectively from patients with aSAH on days 1-3, 5, 7 and 9 and profiled for interleukin (IL)-1α, IL-1ß, IL-4, IL-6, IL-8, IL-10, IL-15, IL-17, IL-18, macrophage chemotactic protein (MCP)-1, vascular endothelial growth factor (VEGF) and tumour necrosis factor (TNF)-α. Plasma and CSF samples from non-aSAH patients undergoing spinal anaesthesia were used as controls. RESULTS: The CSF levels of all cytokines investigated except for IL-1α were significantly higher in aSAH compared to controls in the first seven days of ictus. CSF levels of IL-1α (p = 0.014), IL-18 (p = 0.016), IL-6 (p = 0.0006) and IL-8 (p = 0.006) showed significant increases in the days following aSAH. Conversely IL-17 demonstrated a decrease. In particular, IL-4 was higher in the CSF of patients who had DIND at all time-points (p = 0.032). Plasma IL-6 and IL-8 levels were higher, and IL-1α levels lower, than controls at most time-points. All mediators demonstrated persistent elevation in the CSF compared to plasma apart from IL-1α and IL-18 which followed the opposite trend. Day 3 plasma IL-6 levels predicted poor outcome at six months (Exp(B) 1.12 1.03-1.22, P = 0.012), although this association was lost in the second analysis incorporating Fisher grade, WFNS grade and age. CONCLUSION: The post aSAH inflammatory response peaks on days 5-7 post ictus and remains largely compartmentalised within the CNS. IL-4 may have a particular association with DIND although its precise role in the pathophysiology of the disorder remains unclear. IL-6 predicted poor outcome but not independently of clinical grade, suggesting that it may be a surrogate marker of early brain injury.

Assessment of the Utility of a Vascular Early Warning System Device in the Assessment of Peripheral Arterial Disease in Patients with Diabetes and Incompressible Vessels.

BACKGROUND: The objective of this study was to assess the ability of a novel, automated Conformité Européenne marked vascular early warning system (VEWS) device to detect peripheral arterial disease in patients with incompressible ankle arteries and non-measurable ankle brachial pressure index (ABPI) secondary to diabetes. METHODS: Recruited patients had diabetes, recent magnetic resonance angiography evidence of peripheral arterial disease (PAD), and incompressible vessels on ABPI. VEWS indices of each leg were automatically calculated by using optical infrared and red sensors applied to the foot, with readings obtained with the subject's leg both flat and elevated. Indices <1.03 and ≤0.94 were considered upper and lower diagnostic cutoff limits for PAD. Bollinger scores were calculated from the magnetic resonance angiography. A Best Bollinger Score (BBS) of <4 was defined as no significant PAD. RESULTS: All patients had tissue loss. Per protocol analysis of 28 limbs in 14 patients: VEWS had a sensitivity of 94% and specificity 20% for the detection of PAD at <1.03 cutoff and sensitivity 89% and specificity 80% at ≤0.94 cutoff. There was a good correlation between the VEWS index and BBS (-0.637; P = 0.0003). CONCLUSION: VEWS is a safe, simple-to-use, promising tool to assist in the diagnosis of PAD in patients with incompressible vessels due to diabetes.

Design-encoded dual shape-morphing and shape-memory in 4D printed polymer parts toward cellularized vascular grafts.

Current additive manufacturing technologies wherein as-printed simple two-dimensional (2D) structures morph into complex tissue mimetic three-dimensional (3D) shapes are limited to multi-material hydrogel systems, which necessitates multiple fabrication steps and specific materials. This work utilizes a single shape memory thermoplastic polymer (SMP), PLMC (polylactide-co-trimethylene carbonate), to achieve programmable shape deformation through anisotropic design and infill angles encoded during 3D printing. The shape changes were first computationally predicted through finite element analysis (FEA) simulations and then experimentally validated through quantitative correlation. Rectangular 2D sheets could self-roll into complete hollow tubes of specific diameters (ranging from ≈6 mm to ≈10 mm) and lengths (as long as 40 mm), as quantitatively predicted from FEA simulations within one minute at relatively lower temperatures (≈80 °C). Furthermore, shape memory properties were demonstrated post-shape change to exhibit dual shape morphing at temperatures close to physiological levels. The tubes (retained as the permanent shape) were deformed into flat sheets (temporary shape), seeded with endothelial cells (at T < Tg), and thereafter triggered at ≈37 °C back into tubes (permanent shape), utilizing the shape memory properties to yield bioresorbable tubes with cellularized lumens for potential use as vascular grafts with improved long-term patency. Additionally, out-of-plane bending and twisting deformation were demonstrated in complex structures by careful control of infill angles that can unprecedently expand the scope of cellularized biomimetic 3D shapes. This work demonstrates the potential of the combination of shape morphing and SMP behaviors at physiological temperatures to yield next-generation smart implants with precise control over dimensions for tissue repair and regeneration.

A biphasic endothelial stress-survival mechanism regulates the cellular response to vascular endothelial growth factor A.

Vascular endothelial growth factor A (VEGF-A) is an essential cytokine that regulates endothelial function and angiogenesis. VEGF-A binding to endothelial receptor tyrosine kinases such as VEGFR1 and VEGFR2 triggers cellular responses including survival, proliferation and new blood vessel sprouting. Increased levels of a soluble VEGFR1 splice variant (sFlt-1) correlate with endothelial dysfunction in pathologies such as pre-eclampsia; however the cellular mechanism(s) underlying the regulation and function of sFlt-1 are unclear. Here, we demonstrate the existence of a biphasic stress response in endothelial cells, using serum deprivation as a model of endothelial dysfunction. The early phase is characterized by a high VEGFR2:sFlt-1 ratio, which is reversed in the late phase. A functional consequence is a short-term increase in VEGF-A-stimulated intracellular signaling. In the late phase, sFlt-1 is secreted and deposited at the extracellular matrix. We hypothesized that under stress, increased endothelial sFlt-1 levels reduce VEGF-A bioavailability: VEGF-A treatment induces sFlt-1 expression at the cell surface and VEGF-A silencing inhibits sFlt-1 anchorage to the extracellular matrix. Treatment with recombinant sFlt-1 inhibits VEGF-A-stimulated in vitro angiogenesis and sFlt-1 silencing enhances this process. In this response, increased VEGFR2 levels are regulated by the phosphatidylinositol-3-kinase and PKB/Akt signaling pathways and increased sFlt-1 levels by the ERK1/2 signaling pathway. We conclude that during serum withdrawal, cellular sensing of environmental stress modulates sFlt-1 and VEGFR2 levels, regulating VEGF-A bioavailability and ensuring cell survival takes precedence over cell proliferation and migration. These findings may underpin an important mechanism contributing to endothelial dysfunction in pathological states.

A Global Challenge: Sustainability of Submicrometer PEO and PVP Fiber Production.

The field of submicrometer polymeric production currently has a predominant research focus on morphology and application. In comparison, the sustainability of the manufacture of submicrometer polymeric fibers, specifically the energy efficiency, is less explored. The principles of Green Chemistry and Green Engineering outline frameworks for the manufacture of "greener" products, where the most significant principles in the two frameworks are shown to be centered on energy efficiency, material wastage, and the use of non-hazardous materials. This study examines the power consumption during the production of Polyethylene oxide (PEO) and Polyvinylpyrrolidone (PVP) submicrometer fibers under magnitudes of the key forming parameters to generate fibers via pressure spinning. The energy consumption, along with the fiber diameter, and production rate during the manufacture of fibers is predominantly attributed to the characteristics of polymeric solutions utilized.

Trends in Photopolymerizable Bioinks for 3D Bioprinting of Tumor Models.

Three-dimensional (3D) bioprinting technologies involving photopolymerizable bioinks (PBs) have attracted enormous attention in recent times owing to their ability to recreate complex structures with high resolution, mechanical stability, and favorable printing conditions that are suited for encapsulating cells. 3D bioprinted tissue constructs involving PBs can offer better insights into the tumor microenvironment and offer platforms for drug screening to advance cancer research. These bioinks enable the incorporation of physiologically relevant cell densities, tissue-mimetic stiffness, and vascularized channels and biochemical gradients in the 3D tumor models, unlike conventional two-dimensional (2D) cultures or other 3D scaffold fabrication technologies. In this perspective, we present the emerging techniques of 3D bioprinting using PBs in the context of cancer research, with a specific focus on the efforts to recapitulate the complexity of the tumor microenvironment. We describe printing approaches and various PB formulations compatible with these techniques along with recent attempts to bioprint 3D tumor models for studying migration and metastasis, cell-cell interactions, cell-extracellular matrix interactions, and drug screening relevant to cancer. We discuss the limitations and identify unexplored opportunities in this field for clinical and commercial translation of these emerging technologies.

Casein fibres for wound healing.

The name casein is given to a family of phosphoproteins which is commonly found in milk. Until recently, this was a constituent of milk that was commonly discarded; however today, it is widely used in health supplements all over the world. In this work, a high loading (50 wt%) of casein is mixed with a solution of polycaprolactone (PCL) to produce bandage-like fibres with an average fibre diameter of 1.4 ± 0.5 µm, which would be used to cover wounds in a series of tests with diabetic rats. Mouse fibroblast cell viability tests show that the casein-loaded fibres had little cytotoxicity with over 90% observed viability. A 14-day in vivo trial involving three groups of rats, used as control (no treatment), pure PCL fibres and casein-loaded fibres, showed that the casein within the fibres contributed to a significantly more extensive healing process. Histological analysis showed increased development of granulation tissue and follicle regrowth for the casein-loaded fibres. Further analysis showed that casein-loaded fibres have significantly lower levels of TNF-α, TGF-ß IL-1ß, NF-κB and IL-6, contributing to superior healing. The results presented here show an economical and simple approach to advanced wound healing.

Pressure-Spun Fibrous Surgical Sutures for Localized Antibacterial Delivery: Development, Characterization, and In Vitro Evaluation.

Surgical sutures designed to prevent infection are critical in addressing antibiotic-resistant pathogens that cause surgical site infections. Instead of antibiotics, alternative materials such as biocides have been assessed for coating commercially used sutures due to emerging antibiotic resistance concerns worldwide. This study has a new approach to the development of fibrous surgical sutures with the ability to deliver localized antibacterial agents. A new manufacturing process based on pressure spinning was used for the first time in the production of fibrous surgical sutures by physically blending antibacterial triclosan (Tri) agent with poly(lactic-co-glycolic acid) (PLGA) and poly(ethylene oxide) (PEO) polymers. Fibrous surgical sutures with virgin PLGA, virgin PEO, different ratios of PLGA-PEO, and different ratios of Tri-loaded PLGA-PEO fibrous sutures were produced to mimic the FDA- and NICE-approved PLGA-based sutures available in the market and compared for their characteristics. They were also tested simultaneously with commercially available sutures to compare their in vitro biodegradation, antibacterial, drug release, and cytotoxicity properties. After in vitro antibacterial testing for 24 h, sutures having 285 ± 12 µg/mg Tri loading were selected as a model for further testing as they exhibited antibacterial activity against all tested bacteria strains. The selected model of antibacterial fibrous sutures exhibited an initial burst of Tri release within 24 h, followed by a sustained release for the remaining time until the sutures completely degraded within 21 days. The cell viability assay showed that these surgical sutures had no cytotoxic effect on mammalian cells.

Emerging 4D fabrication of next-generation nerve guiding conduits: a critical perspective.

The latest advancements in the field of manufacturing for biomedicine, digital health, targeted therapy, and personalized medicine have fuelled the fabrication of smart medical devices. Four-dimensional (4D) fabrication strategies, which combine the manufacturing of three-dimensional (3D) parts with smart materials and/or design, have proved beneficial in creating customized and self-fitting structures that change their properties on demand with time. These frontier techniques that yield dynamic implants can indeed alleviate various drawbacks of current clinical practices, such as the use of sutures and complex microsurgeries and associated inflammation, among others. Among various clinical applications, 4D fabrication has lately made remarkable progress in the development of next-generation nerve-guiding conduits for treating peripheral nerve injuries (PNIs) by improving the end-to-end co-aptation of transected nerve endings. The current perspective highlights the relevance of 4D fabrication in developing state-of-the-art technologies for the treatment of PNIs. Various 4D fabrication/bio-fabrication techniques for PNI treatment are summarized while identifying the challenges and opportunities for the future. Such advancements hold immense promise for improving the quality of life of patients suffering from nerve damage and the potential for extending the treatment of many other disorders. Although the techniques are being described for PNIs, they will lend themselves suitably to certain cases of cranial nerve injuries as well.

CORM-3, a carbon monoxide-releasing molecule, alters the inflammatory response and reduces brain damage in a rat model of hemorrhagic stroke.

OBJECTIVE: Intracerebral hemorrhage is accompanied by a pronounced inflammatory response that mediates brain damage but is also essential for the tissue reparative process. We assessed the effect of CORM-3, a water-soluble carbon monoxide-releasing molecule possessing anti-inflammatory properties, on inflammation and brain injury after intracerebral hemorrhage. DESIGN: In vivo and in vitro laboratory study. SETTING: Research laboratory. SUBJECTS: Male Sprague-Dawley rats, 250-350 g. INTERVENTIONS: A model of collagenase injection (2 µL) in the brain was established to induce intracerebral hemorrhage. CORM-3 (4 or 8 mg/kg) was administered intravenously at different times as follows: 1) 5 mins before collagenase; 2) 3 hrs after collagenase; and 3) 3 days after collagenase challenge. MEASUREMENTS AND MAIN RESULTS: Saline was used as a negative control. Brain damage, brain water content, and behavioral assessment were evaluated. The inflammatory response was determined at set intervals after intracerebral hemorrhage by counting peripheral neutrophils and lymphocytes, neutrophils, and activated microglia/macrophages in the intracerebral hemorrhage area and measuring plasma tumor necrosis factor-á levels. BV2 microglia and DI-TNC1 astrocytes were exposed to triton (1%) or CORM-3 (10-100 ìM) and cytotoxicity (lactic dehydrogenase assay) measured at 24 hrs. A challenge with collagenase to induce intracerebral hemorrhage caused marked brain damage and modified the levels of inflammatory markers. Pretreatment with CORM-3 significantly prevented injury, modulated inflammation, and reduced plasma tumor necrosis factor-α. CORM-3 given 3 hrs after collagenase significantly increased brain injury and tumor necrosis factor-α production. In contrast, CORM-3 given 3 days after collagenase afforded partial protection, modulated inflammation, and decreased tumor necrosis factor-α starting from the day of application. No dose-dependent effects were observed. CONCLUSIONS: CORM-3 promotes neuroprotection or neurotoxicity after intracerebral hemorrhage depending on the time of administration. Beneficial effects are achieved when CORM-3 is given either before or 3 days after intracerebral hemorrhage, namely, as a prophylactic agent or during the postacute inflammatory phase.

An in vitro model of warm hypoxia-reoxygenation injury in human liver endothelial cells.

BACKGROUND: Ischemia-reperfusion or hypoxia-reoxygenation (H-R) injury adversely affects hepatic function following transplantation and major resection; the death of human sinusoidal endothelial cells (SECs) by apoptosis may play a central role in this process. Caspase-3 is an important intracellular protease in the intrinsic and extrinsic pathways of apoptosis. MATERIALS AND METHODS: SECs and EAhy926 cells were exposed to warm hypoxia at 37°C, followed by reoxygenation at 37°C. Activity of caspase-3 was quantified using Western blotting and colorimetric kinase assays. RESULTS: H-R caused a significant increase in caspase-3 activity compared with controls in both cell types. CONCLUSIONS: Warm H-R injury causes apoptotic cell death of SECs and immortalized cells, but with differing patterns of caspase activity.

Emerging trends in biliary stents: a materials and manufacturing perspective.

Biliary stent technology has come a long way since its inception. There have been significant advancements in the materials used, and design and deployment strategies. Options have expanded from plastic and metallic stents to a wider variety of materials and manufacturing technologies to offer several options to clinicians, including self-expandable metallic stents and bioresorbable stents. Bioresorbable biliary stents are still in the early stages of clinical adoption. This review encompasses the materials currently used for biliary stents and the significant developments in the past few years in the resorbable materials for use as biliary stents. We critically discuss the emerging trends in the development of new resorbable materials for fabricating biliary stents. We then assess the developments in drug-eluting stents and advanced manufacturing technologies that could be leveraged for biliary stents. Challenges in the paths for translation for the future, such as pre-clinical and clinical trials, are highlighted. Finally, we present future directions that could drive the biliary stent market to meet the increasingly complex and diverse clinical needs of patients.

Beneficial Effects of Insulin on Ischemia Reperfusion Injury in Human Skeletal Muscle.

Purpose: Exaggerated leucocyte activity is a crucial step in the pathophysiology of skeletal muscle ischemia-reperfusion injury (IRI). We tested the hypothesis that insulin, via its' anti-leukocyte activity, attenuates skeletal muscle IRI in humans. Materials and Methods: This randomized, blinded, placebo-controlled trial was conducted in patients with skeletal muscle ischemia who required revascularization. Treatment protocols were similar among them except for the insulin group, which received an infusion of insulin at 2.5 U/h. The degree of endothelial adhesiveness; leukocyte activity and pro-inflammatory status via P-selectin, tumor necrosis factor (TNF)-alpha, and myeloperoxidase (MPO) levels in the venous effluent; and clinical outcomes were measured. Results: Twenty-four consenting patients were randomized to the insulin or control group. There were no significant differences between the two groups except for the median serum insulin level, which was higher in the insulin group (P<0.01). No serious intervention-related adverse events were observed. P-selectin (55.04-99.86 pg/mL; P<0.001), MPO (110.8-160.6 pg/mL; P<0.001), and TNF-alpha (12.16-36.01 pg/mL; P<0.001) levels demonstrated a significant increase post-reperfusion in the 'control' group, reaching a peak value at 2 hours post-reperfusion. The increase in all three markers from baseline was significantly diminished in the insulin group at the two-hour (P-selectin, P=0.001; MPO, P=0.001; TNF-alpha, P=0.005) and four-hour (P-selectin, P=0.003; MPO, P=0.002; TNF-alpha, P=0.01) intervals. The differences in clinical outcomes between the insulin and control groups were not statistically significant. Conclusion: In clinical practice, insulin has the potential to attenuate the severity of skeletal muscle IRI inhibiting P-selectin, MPO, and TNF-alpha levels.

Facile One-Pot Method for All Aqueous Green Formation of Biocompatible Silk Fibroin-Poly(Ethylene Oxide) Fibers for Use in Tissue Engineering.

Silk fibroin (SF) fibers are highly regarded in tissue engineering because of their outstanding biocompatibility and tunable properties. A challenge remains in overcoming the trade-off between functioning and biocompatible fibers and the use of cytotoxic, environmentally harmful organic solvents in their processing and formation. The aim of this research was to produce biocompatible SF fibers without the use of cytotoxic solvents, via pressurized gyration (PG). Aqueous SF was blended with poly(ethylene oxide) (PEO) in ratios of 80:20 (labeled SF-PEO 80:20) and 90:10 (labeled SF-PEO 90:10) and spun into fibers using PG, assisted by a range of applied pressures and heat. Pure PEO (labeled PEO-Aq) and SF solubilized in hexafluoro-isopropanol (HFIP) (labeled SF-HFIP) and aqueous SF (labeled SF-Aq) were also prepared for comparison. The resulting fibers were characterized using SEM, TGA, and FTIR. Their in vitro cell behavior was analyzed using a Live/Dead assay and cell proliferation studies with the SaOS-2 human bone osteosarcoma cell line (ATCC, HTB-85) and human fetal osteoblast cells (hFob) (ATCC, CRL-11372) in 2D culture conditions. Fibers in the micrometer range were successfully produced using SF-PEO blends, SF-HFIP, and PEO-Aq. The fiber thickness ranged from 0.71 ± 0.17 µm for fibers produced using SF-PEO 90:10 with no applied pressure to 2.10 ± 0.78 µm for fibers produced using SF-PEO 80:10 with 0.3 MPa applied pressure. FTIR confirmed the presence of SF via amide I and amide II bands in the blend fibers because of a change in structural conformation. No difference was observed in thermogravimetric properties among varying pressures and no significant difference in fiber diameters for pressures. SaOS-2 cells and hFOb cell studies demonstrated higher cell densities and greater live cells on SF-PEO blends when compared to SF-HFIP. This research demonstrates a scalable and green method of producing SF-based constructs for use in bone-tissue engineering applications.

Experimental evaluation of the patient-specific haemodynamics of an aortic dissection model using particle image velocimetry.

Aortic Dissection (AD) is a complex pathology that affects the aorta. Diagnosis, management and treatment remain a challenge as it is a highly patient-specific pathology and there is still a limited understanding of the fluid-mechanics phenomena underlying clinical outcomes. Although in vitro models can allow the accurate study of AD flow fields in physical phantoms, they are currently scarce and almost exclusively rely on over simplifying assumptions. In this work, we present the first experimental study of a patient-specific case of AD. An anatomically correct phantom was produced and combined with a state-of-the-art in vitro platform, informed by clinical data, employed to accurately reproduce personalised conditions. The complex AD haemodynamics reproduced by the platform was characterised by flow rate and pressure acquisitions as well as Particle Image Velocimetry (PIV) derived velocity fields. Clinically relevant haemodynamic indices, that can be correlated with AD prognosis - such as velocity, shear rate, turbulent kinetic energy distributions - were extracted in two regions of interest in the aortic domain. The acquired data highlighted the complex nature of the flow (e.g. recirculation regions, low shear rate in the false lumen) and was in very good agreement with the available clinical data and the CFD results of a study conducted alongside, demonstrating the accuracy of the findings. These results demonstrate that the described platform constitutes a powerful, unique tool to reproduce in vitro personalised haemodynamic conditions, which can be used to support the evaluation of surgical procedures, medical devices testing and to validate state-of-the-art numerical models.

Severe Acute Respiratory Syndrome Type 2-Causing Coronavirus: Variants and Preventive Strategies.

COVID-19 vaccines have constituted a substantial scientific leap in countering severe acute respiratory syndrome type 2-causing coronavirus (SARS-CoV-2), and worldwide implementation of vaccination programs has significantly contributed to the global pandemic effort by saving many lives. However, the continuous evolution of the SARS-CoV-2 viral genome has resulted in different variants with a diverse range of mutations, some with enhanced virulence compared with previous lineages. Such variants are still a great concern as they have the potential to reduce vaccine efficacy and increase the viral transmission rate. This review summarizes the significant variants of SARS-CoV-2 encountered to date (December 2021) and discusses a spectrum of possible preventive strategies, with an emphasis on physical and materials science.

3D direct-write printing of water soluble micromoulds for high-resolution rapid prototyping.

Direct-write printing has contributed tremendously to additive manufacturing; in particular extrusion based printing where it has extended the range of materials for 3D printing and thus enabled use across many more sectors. The printing inks for direct-write printing however, need careful synthesis and invariably undergo extensive preparation before being able to print. Hence, new ink synthesis efforts are required every time a new material is to be printed; this is particularly challenging for low storage modulus (G') materials like silicones, especially at higher resolutions (under 10 µm). Here we report the development of a precise (< 10 µm) 3D printable polymer, with which we 3D print micromoulds which are filled with standard silicones like polydimethylsiloxane (PDMS) and left to cure at room temperature. The proof of concept is demonstrated using a simple water soluble polymer as the mould material. The approach enables micrometre scale silicone structures to be prototyped with ease, away from the cleanroom.

Purification and Analysis of Circulating Lipid Particles.

Lipid particles found in circulating extracellular fluids such as blood or lymph are essential for cellular homeostasis, metabolism and survival. Such particles provide essential lipids and fats which enable cells to synthesize new membranes and regulate different biochemical pathways. Imbalance in lipid particle metabolism can cause pathological states such as atherosclerosis. Here, elevated low-density lipoprotein (LDL) accumulation leads to fat-filled lesions or plaques in arterial walls. In this chapter, we provide a detailed set of protocols for the rapid and safe purification of lipid particles from human blood using high-speed ultracentrifugation. We provide a detailed set of assays for further analysis of the biochemical and cellular properties of these lipid particles. By combining these assays, we can better understand the complex roles of different lipid particles in normal physiology and disease pathology.

Co-Axial Gyro-Spinning of PCL/PVA/HA Core-Sheath Fibrous Scaffolds for Bone Tissue Engineering.

The present study aspires towards fabricating core-sheath fibrous scaffolds by state-of-the-art pressurized gyration for bone tissue engineering applications. The core-sheath fibers comprising dual-phase poly-ε-caprolactone (PCL) core and polyvinyl alcohol (PVA) sheath are fabricated using a novel "co-axial" pressurized gyration method. Hydroxyapatite (HA) nanocrystals are embedded in the sheath of the fabricated scaffolds to improve the performance for application as a bone tissue regeneration material. The diameter of the fabricated fiber is 3.97 ± 1.31 µm for PCL-PVA/3%HA while pure PCL-PVA with no HA loading gives 3.03 ± 0.45 µm. Bead-free fiber morphology is ascertained for all sample groups. The chemistry, water contact angle and swelling behavior measurements of the fabricated core-sheath fibrous scaffolds indicate the suitability of the structures in cellular activities. Saos-2 bone osteosarcoma cells are employed to determine the biocompatibility of the scaffolds, wherein none of the scaffolds possess any cytotoxicity effect, while cell proliferation of 94% is obtained for PCL-PVA/5%HA fibers. The alkaline phosphatase activity results suggest the osteogenic activities on the scaffolds begin earlier than day 7. Overall, adaptations of co-axial pressurized gyration provides the flexibility to embed or encapsulate bioactive substances in core-sheath fiber assemblies and is a promising strategy for bone healing.