Biodegradable magnesium materials regulate ROS-RNS balance in pro-inflammatory macrophage environment.

The relationship between reactive oxygen and nitrogen species (ROS-RNS) secretion and the concomitant biocorrosion of degradable magnesium (Mg) materials is poorly understood. We found that Mg foils implanted short term in vivo (24 h) displayed large amounts of proinflammatory F4/80+/iNOS + macrophages at the interface. We sought to investigate the interplay between biodegrading Mg materials (98.6% Mg, AZ31 & AZ61) and macrophages (RAW 264.7) stimulated with lipopolysaccharide (RAW 264.7LPS) to induce ROS-RNS secretion. To test how these proinflammatory ROS-RNS secreting cells interact with Mg corrosion in vitro, Mg and AZ61 discs were suspended approximately 2 mm above a monolayer of RAW 264.7 cells, either with or without LPS. The surfaces of both materials showed acute (24 h) changes when incubated in the proinflammatory RAW 264.7LPS environment. Mg discs incubated with RAW 264.7LPS macrophages showed greater corrosion pitting, while AZ61 showed morphological and elemental bulk product changes via scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDX). X-ray photoelectron spectroscopy (XPS) analysis showed a reduction in the Ca/P ratio of the surface products for AZ61 disc incubated with RAW 264.7LPS, but not the Mg discs. Moreover, RAW 264.7LPS macrophages were found to be more viable in the acute biodegradative environment generated by Mg materials, as demonstrated by calcein-AM and cleaved (active) caspase-3 staining (CC3). LPS stimulation caused an increase in ROS-RNS, and a decrease in antioxidant peroxidase activity. Mg and AZ61 were found to change this ROS-RNS balance, independently of physiological antioxidant mechanisms. The findings highlight the complexity of the cellular driven acute inflammatory responses to different biodegradable Mg, and how it can potentially affect performance of these materials.

Impact of spatial and temporal stability of flow vortices on vascular endothelial cells.

PURPOSE: Intracranial aneurysms (IAs) are pathological dilations of cerebrovascular vessels due to degeneration of the mechanical strength of the arterial wall, precluded by altered cellular functionality. The presence of swirling hemodynamic flow (vortices) is known to alter vascular endothelial cell (EC) morphology and protein expression indicative of IAs. Unfortunately, less is known if vortices with varied spatial and temporal stability lead to differing levels of EC change. The aim of this work is to investigate vortices of varying spatial and temporal stability impact on ECs. METHODS: Vortex and EC interplay was investigated by a novel combination of parallel plate flow chamber (PPFC) design and computational analysis. ECs were exposed to laminar (7.5 dynes/[Formula: see text] wall shear stress) or low (<1 dynes/[Formula: see text]) stress vortical flow using PPFCs. Immunofluorescent imaging analyzed EC morphology, while ELISA tests quantified VE-cadherin (cell-cell adhesion), VCAM-1 (macrophage-EC adhesion), and cleaved caspase-3 (apoptotic signal) expression. PPFC flow was simulated, and vortex stability was calculated via the temporally averaged degree of (volume) overlap (TA-DVO) of vortices within a given area. RESULTS: EC morphological changes were independent of vortex stability. Increased stability promoted VE-cadherin degradation (correlation coefficient r = [Formula: see text]0.84) and 5-fold increased cleaved caspase-3 post 24 h in stable (TA-DVO 0.736 ± 0.05) vs unstable (TA-DVO 0.606 [Formula: see text]0.2) vortices. ECs in stable vortices displayed a 4.5-fold VCAM-1 increase than unstable counterparts after 12 h. CONCLUSION: This work demonstrates highly stable disturbed flow imparts increased inflammatory signaling, degraded cell-cell adhesion, and increased cellular apoptosis than unstable vortices. Such knowledge offers novel insight toward understanding IA development and rupture.

Enhancement of Lymphangiogenesis by Human Mesenchymal Stem Cell Sheet.

Preparation of human mesenchymal stem cell (hMSC) suspension for lymphedema treatment relies on conventional enzymatic digestion methods, which severely disrupts cell-cell and cell-extracellular matrix (ECM) connections, and drastically impairs cell retention and engraftment after transplantation. The objective of the present study is to evaluate the ability of hMSC-secreted ECM to augment lymphangiogenesis by using an in vitro coculturing model of hMSC sheets with lymphatic endothelial cells (LECs) and an in vivo mouse tail lymphedema model. Results demonstrate that the hMSC-secreted ECM augments the formation of lymphatic capillary-like structure by a factor of 1.2-3.6 relative to the hMSC control group, by serving as a prolymphangiogenic growth factor reservoir and facilitating cell regenerative activities. hMSC-derived ECM enhances MMP-2 mediated matrix remodeling, increases the synthesis of collagen IV and laminin, and promotes lymphatic microvessel-like structure formation. The injection of rat MSC sheet fragments into a mouse tail lymphedema model confirms the benefits of the hMSC-derived ECM by stimulating lymphangiogenesis and wound closure.

Fabrication of a Completely Biological and Anisotropic Human Mesenchymal Stem Cell-Based Vascular Graft.

Tissue-engineered small-diameter vascular grafts are required to match mechanical properties as well as cellular and extracellular architecture of native blood vessels. Although various engineering technologies have been developed, the most reliable strategy highlights the needs for incorporating completely biological components and anisotropic cellular and biomolecular organization into the tissue-engineered vascular graft (TEVG). Based on the antithrombogenic, immunoregulatory, and regenerative properties of human mesenchymal stem cells (hMSCs), this chapter provides a step-by-step protocol for generating a completely biological and anisotropic TEVG that comprises of hMSCs and highly aligned extracellular matrix (ECM) nanofibers. The hMSCs were grown on an aligned nanofibrous ECM scaffold derived from an oriented human dermal fibroblast (hDF) sheet and then wrapped around a temporary mandrel to form a tubular assembly, followed by a maturation process in a rotating wall vessel (RWV) bioreactor. The resulting TEVG demonstrates anisotropic structural and mechanical properties similar to that of native blood vessels. A completely biological, anisotropic, and mechanically strong TEVG that incorporates immunoregulatory hMSCs is promising to meet the urgent needs of a surgical intervention for bypass grafting.

Engineering the Lymphatic Network: A Solution to Lymphedema.

Secondary lymphedema is a life-long disorder characterized by chronic tissue swelling and inflammation that obstruct interstitial fluid circulation and immune cell trafficking. Regenerating lymphatic vasculatures using various strategies represents a promising treatment for lymphedema. Growth factor injection and gene delivery have been developed to stimulate lymphangiogenesis and augment interstitial fluid resorption. Using bioengineered materials as growth factor delivery vehicles allows for a more precisely targeted lymphangiogenic activation within the injured site. The implantation of prevascularized lymphatic tissue also promotes in situ lymphatic capillary network formation. The engineering of larger scale lymphatic tissues, including lymphatic collecting vessels and lymph nodes constructed by bioengineered scaffolds or decellularized animal tissues, offers alternatives to reconnecting damaged lymphatic vessels and restoring lymph circulation. These approaches provide lymphatic vascular grafting materials to reimpose lymphatic continuity across the site of injury, without creating secondary injuries at donor sites. The present work reviews molecular mechanisms mediating lymphatic system development, approaches to promoting lymphatic network regeneration, and strategies for engineering lymphatic tissues, including lymphatic capillaries, collecting vessels, and nodes. Challenges of advanced translational applications are also discussed.

Preservation of microvascular integrity and immunomodulatory property of prevascularized human mesenchymal stem cell sheets.

Prevascularization is essential to ensure the viability, functionality, and successful integration of tissue-engineered three-dimensional (3D) constructs with surrounding host tissues after transplantation. Human mesenchymal stem cell (hMSC) sheet can be prevascularized by coculturing with endothelial cells (ECs), and then be further used as building blocks for engineering 3D complex tissues. In addition, predifferentiation of hMSCs into a tissue-specific lineage in vitro has been proven to promote graft engraftment and regeneration. However, it is unclear if the prevascularized hMSC sheets can still maintain their microvascular integrity as well as the immune-regulatory properties after their tissue-specific differentiation. The objective of this study was to investigate the effects of differentiation cues on the microvascular structure, angiogenic factor secretion, and immunogenic responses of prevascularized hMSC sheets. The results showed that upon coculturing with ECs, hMSC sheets successfully formed microvascular network, while maintaining hMSCs' multi-lineage differentiation capability. The next step, osteogenic and adipogenic induction, damaged the preformed microvascular structures and compromised the angiogenic factor secretion ability of hMSCs. Nonetheless, this effect was mitigated by adjusting the concentration of differentiation factors. The subcutaneous transplantation in an immunocompetent rat model demonstrated that the osteogenic differentiated prevascularized hMSC sheet preserved its microvascular structure and immunomodulatory properties comparable to the undifferentiated prevascularized hMSC sheets. This study suggested that a balanced and optimal differentiation condition can effectively promote the tissue-specific predifferentiation of prevascularized hMSC sheet while maintaining its immunomodulatory and tissue integration properties.

Dual Switch Mechanism of Erythropoietin as an Antiapoptotic and Pro-Angiogenic Determinant in the Retina.

Constant or intense light degenerates the retina and retinal pigment epithelial cells. Light generates reactive oxygen species and nitric oxide leading to initial reactions of retinal degeneration. Apoptosis is the primary mechanism of abnormal death of photoreceptors, retinal ganglion cells, or retinal pigment epithelium (RPE) in degenerative retinal diseases, including diabetic retinopathy and age-related macular degeneration. The current study evaluated the function of erythropoietin (EPO) on angiogenesis and apoptosis in the retina and RPE under oxidative stress. We determined the pro-angiogenic and antiapoptotic mechanism of EPO under stress conditions using a conditional EPO knockdown model using siRNA, EPO addition, proteomics, immunocytochemistry, and bioinformatic analysis. Our studies verified that EPO protected retinal cells from light-, hypoxia-, hyperoxia-, and hydrogen peroxide-induced apoptosis through caspase inhibition, whereas up-regulated angiogenic reactions through vascular endothelial growth factor (VEGF) and angiotensin pathway. We demonstrated that the EPO expression in the retina and subsequent serine/threonine/tyrosine kinase phosphorylations might be linked to oxidative stress response tightly to determining angiogenesis and apoptosis. Neuroprotective roles of EPO may involve the balance between antiapoptotic and pro-angiogenic signaling molecules, including BCL-xL, c-FOS, caspase-3, nitric oxide, angiotensin, and VEGF receptor. Our data indicate a new therapeutic application of EPO toward retinal degeneration based on the dual roles in apoptosis and angiogenesis at the molecular level under oxidative stress.

Lactate-enhanced-qSOFA (LqSOFA) score is superior to the other four rapid scoring tools in predicting in-hospital mortality rate of the sepsis patients.

BACKGROUND: The rising prevalence of early therapy for sepsis has led to the demand for rapid risk-stratification tools that can estimate the risk of in-hospital mortality for sepsis patients and the need for intensive care unit (ICU) admission. A robust risk-stratification tool is crucial for in-time sepsis treatment. This study aimed to compare the abilities of five rapid scoring systems, i.e., LqSOFA score, qSOFA score, SIRS, MEDS, and MEWS, in predicting the mortality in hospital and ICU admission for sepsis patients. METHODS: A retrospective observational clinical study was conducted in West China Hospital. Our cases included all patients admitted to the hospital with a diagnosis of sepsis (sepsis-3). We calculated five rapid prediction scores for the enrolled cases. We then compared each rapid score's ability to predict in-hospital mortality and ICU admission. RESULTS: A total of 821 of mixed sepsis patients by sepsis-3 definition were included. The all-cause hospital mortality rate was 21.1%. The LqSOFA score presented the most significant discrimination with an area under the receiver operating characteristic curve (AUC) of 0.751. The AUC of the LqSOFA score for mortality in the hospital was significantly higher than qSOFA (AUC 0.717), SIRS (AUC 0.704), MEDS (AUC 0.670), and MEWS (AUC 0.685). CONCLUSIONS: LqSOFA is a superior prognostic tool for predicting mortality in the hospital. It may provide more exact information for hospital mortality than the other 4 rapid scores in treating sepsis patients.

Oleic Acid, Cholesterol, and Linoleic Acid as Angiogenesis Initiators.

The current study determined the natural angiogenic molecules using an unbiased metabolomics approach. A chick chorioallantoic membrane (CAM) model was used to examine pro- and antiangiogenic molecules, followed by gas chromatography-mass spectrometry (GCMS) analysis. Vessel formation was analyzed quantitatively using the angiogenic index (p < 0.05). At embryonic day one, a white streak or circle area was observed when vessel formation begins. GCMS analysis and database search demonstrated that angiogenesis may initiate when oleic, cholesterol, and linoleic acids increased in the area of angiogenic reactions. The gain of function study was conducted by the injection of cholesterol and oleic acid into a chick embryo to determine the role of each lipid in angiogenesis. We propose that oleic acid, cholesterol, and linoleic acid are natural molecules that set the platform for the initiation stage of angiogenesis before other proteins including the vascular endothelial growth factor, angiopoietin, angiotensin, and erythropoietin join as the angiome in sprout extension and vessel maturation.

Biomimetic hydrogels with spatial- and temporal-controlled chemical cues for tissue engineering.

Biomimetic hydrogels have emerged as the most useful tissue engineering scaffold materials. Their versatile chemistry can recapitulate multiple physical and chemical features to integrate cells, scaffolds, and signaling molecules for tissue regeneration. Due to their highly hydrophilic nature hydrogels can recreate nutrient-rich aqueous environments for cells. Soluble regulatory molecules can be incorporated to guide cell proliferation and differentiation. Importantly, the controlled dynamic parameters and spatial distribution of chemical cues in hydrogel scaffolds are critical for cell-cell communication, cell-scaffold interaction, and morphogenesis. Herein, we review biomimetic hydrogels that provide cells with spatiotemporally controlled chemical cues as tissue engineering scaffolds. Specifically, hydrogels with temporally controlled growth factor-release abilities, spatially controlled conjugated bioactive molecules/motifs, and targeting delivery and reload properties for tissue engineering applications are discussed in detail. Examples of hydrogels that possess clinically favorable properties, such as injectability, self-healing ability, stimulus-responsiveness, and pro-remodeling features, are also covered.

Mechanistic dissection of diabetic retinopathy using the protein-metabolite interactome.

PURPOSE: The current study aims to determine the molecular mechanisms of diabetic retinopathy (DR) using the protein-protein interactome and metabolome map. We examined the protein network of novel biomarkers of DR for direct (physical) and indirect (functional) interactions using clinical target proteins in different models. METHODS: We used proteomic tools including 2-dimensional gel electrophoresis, mass spectrometry analysis, and database search for biomarker identification using in vivo murine and human model of diabetic retinopathy and in vitro model of oxidative stress. For the protein interactome and metabolome mapping, various bioinformatic tools that include STRING and OmicsNet were used. RESULTS: We uncovered new diabetic biomarkers including prohibitin (PHB), dynamin 1, microtubule-actin crosslinking factor 1, Toll-like receptor (TLR 7), complement activation, as well as hypothetical proteins that include a disintegrin and metalloproteinase (ADAM18), vimentin III, and calcium-binding C2 domain-containing phospholipid-binding switch (CAC2PBS) using a proteomic approach. Proteome networks of protein interactions with diabetic biomarkers were established using known DR-related proteome data. DR metabolites were interconnected to establish the metabolome map. Our results showed that mitochondrial protein interactions were changed during hyperglycemic conditions in the streptozotocin-treated murine model and diabetic human tissue. CONCLUSIONS: Our interactome mapping suggests that mitochondrial dysfunction could be tightly linked to various phases of DR pathogenesis including altered visual cycle, cytoskeletal remodeling, altered lipid concentration, inflammation, PHB depletion, tubulin phosphorylation, and altered energy metabolism. The protein-metabolite interactions in the current network demonstrate the etiology of retinal degeneration and suggest the potential therapeutic approach to treat DR.

Development of an Injectable Nitric Oxide Releasing Poly(ethylene) Glycol-Fibrin Adhesive Hydrogel.

Fibrin microparticles were incorporated into poly(ethylene) glycol (PEG)-fibrinogen hydrogels to create an injectable, composite that could serve as a wound healing support and vehicle to deliver therapeutic factors for tissue engineering. Nitric oxide (NO), a therapeutic agent in wound healing, was loaded into fibrin microparticles by blending S-Nitroso-N-acetyl penicillamine (SNAP) with a fibrinogen solution. The incorporation of microparticles affected swelling behavior and improved tissue adhesivity of composite hydrogels. Controlled NO release was induced via photolytic and thermal activation, and modulated by weight percent of particles incorporated. These NO-releasing composites were non-cytotoxic in culture. Cells maintained morphology, viability, and proliferative character. Fibrin microparticles loaded with SNAP and incorporated into a PEG-fibrinogen matrix, creates a novel injectable composite hydrogel that offers improved tissue adhesivity and inducible NO-release for use as a regenerative support for wound healing and tissue engineering applications.

Magnetoelastic galfenol as a stent material for wirelessly controlled degradation rates.

The gold standard of care for coronary artery disease, a leading cause of death for in the world, is balloon angioplasty in conjunction with stent deployment. However, implantation injuries and long-term presence of foreign material often promotes significant luminal tissue growth, leading to a narrowing of the artery and severely restricted blood flow. A promising method to mitigate this process is the use of biodegradable metallic stents, but thus far they have either degraded too slowly (iron) or disappeared prematurely (magnesium). The present work investigates the use of a unique type of magnetic material, galfenol (iron-gallium), for postoperative wireless control of stent degradation rates. Due to its magnetoelastic property, galfenol experiences longitudinal micron-level elongations when exposed to applied magnetic fields, allowing generation of a microstirring effect that affect its degradation behavior. In vitro indirect cytotoxicity tests on primary rat aortic smooth muscle cells indicated that galfenol byproducts must be concentrated approximately seven times from collected 60 day degradation medium to cause ∼15% of death from all cells. Surface and cross-sectional characterization of the material indicate that galfenol (Fe80 Ga20 ) degradation rates (∼0.55% per month) are insufficient for stenting applications. While this material may not be ideal for comprising the entire stent, there is potential for use in combination with other materials. Furthermore, the ability to control degradation rates postimplantation opens new possibilities for biodegradable stents; additional magnetoelastic materials should be investigated for use in stenting applications. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 232-241, 2019.

Biomimetic recyclable microgels for on-demand generation of hydrogen peroxide and antipathogenic application.

Microgels that can generate antipathogenic levels of hydrogen peroxide (H2O2) through simple rehydration in solutions with physiological pH are described herein. H2O2 is a widely used disinfectant but the oxidant is hazardous to store and transport. Catechol, an adhesive moiety found in mussel adhesive proteins, was incorporated into microgels, which generated 1-5 mM of H2O2 for up to four days as catechol autoxidized. The sustained release of low concentrations of H2O2 was antimicrobial against both gram-positive (Staphylococcus epidermidis) and gram-negative (Escherichia coli) bacteria and antiviral against both non-enveloped porcine parvovirus (PPV) and enveloped bovine viral diarrhea virus (BVDV). The amount of released H2O2 is several orders of magnitude lower than H2O2 concentration previously reported for antipathogenic activity. Most notably, these microgels reduced the infectivity of the more biocide resistant non-envelope virus by 3 log reduction value (99.9% reduction in infectivity). By controlling the oxidation state of catechol, microgels can be repeatedly activated and deactivated for H2O2 generation. These microgels do not contain a reservoir for storing the reactive H2O2 and can potentially function as a lightweight and portable dried powder source for the disinfectant for a wide range of applications. STATEMENT OF SIGNIFICANCE: Researchers have designed bioadhesives and coatings using the adhesive moiety catechol to mimic the strong adhesion capability of mussel adhesive proteins. During catechol autoxidation, hydrogen peroxide (H2O2) is generated as a byproduct. Here, catechol was incorporated into microgels, which can generate millimolar levels of H2O2 by simply hydrating the microgels in a solution with physiological pH. The sustained release of H2O2 was both antimicrobial and antiviral, inactivating even the more biocide resistant non-enveloped virus. These microgels can be repeatedly activated and deactivated for H2O2 generation by incubating them in solutions with different pH. This simplicity and recyclability will enable this biomaterial to function as a lightweight and portable source for the disinfectant for a wide range of applications.

Nonthermal ice nucleation observed at distorted contact lines of supercooled water drops.

Ice nucleation is the crucial step for ice formation in atmospheric clouds and therefore underlies climatologically relevant precipitation and radiative properties. Progress has been made in understanding the roles of temperature, supersaturation, and material properties, but an explanation for the efficient ice nucleation occurring when a particle contacts a supercooled water drop has been elusive for over half a century. Here, we explore ice nucleation initiated at constant temperature and observe that mechanical agitation induces freezing of supercooled water drops at distorted contact lines. Results show that symmetric motion of supercooled water on a vertically oscillating substrate does not freeze, no matter how we agitate it. However, when the moving contact line is distorted with the help of trace amounts of oil or inhomogeneous pinning on the substrate, freezing can occur at temperatures much higher than in a static droplet, equivalent to ∼10^{10} increase in nucleation rate. Several possible mechanisms are proposed to explain the observations. One plausible explanation among them, decreased pressure due to interface curvature, is explored theoretically and compared with the observational results quasiquantitatively. Indeed, the observed freezing-temperature increase scales with contact line speed in a manner consistent with the pressure hypothesis. Whatever the mechanism, the experiments demonstrate a strong preference for ice nucleation at three-phase contact lines compared to the two-phase interface, and they also show that movement and distortion of the contact line are necessary contributions to stimulating the nucleation process.

Recent Developments in Tough Hydrogels for Biomedical Applications.

A hydrogel is a three-dimensional polymer network with high water content and has been attractive for many biomedical applications due to its excellent biocompatibility. However, classic hydrogels are mechanically weak and unsuitable for most physiological load-bearing situations. Thus, the development of tough hydrogels used in the biomedical field becomes critical. This work reviews various strategies to fabricate tough hydrogels with the introduction of non-covalent bonds and the construction of stretchable polymer networks and interpenetrated networks, such as the so-called double-network hydrogel. Additionally, the design of tough hydrogels for tissue adhesive, tissue engineering, and soft actuators is reviewed.

Melatonin Modulates Prohibitin and Cytoskeleton in the Retinal Pigment Epithelium.

The retinal pigment epithelium (RPE) plays imperative roles in normal retinal function by photoreceptor protection from light and phagocytosis of rod and cone outer segments during disc shedding. Melatonin is the free radical scavenger and circadian determinant to protect the RPE and retina from oxidative stress and regulate the circadian clock. The current study tested the hypothesis whether melatonin could affect cytoskeletal structure within RPE. Our Western blot analysis demonstrated that melatonin treatment up-regulated prohibitin 3-fold compared to control. ß-tubulin levels were also up-regulated by melatonin but to a lesser extent. Initial cell shape of ARPE-19 is epitheloid, however, after 30-minute treatment with melatonin, RPE cells undergo a morphological change to a fusiform shape with spindle outgrowth. Cells return to epitheloid shape after 12 hours in untreated medium. Melatonin treated cells showed increased and dissimilar distribution of prohibitin and ß-tubulin compared to non-treated cells, thus altered cytoskeletal and mitochondrial structure in the RPE. Our data implies that melatonin may play a protective role under oxidative stress, which is shown by the marker prohibitin in terms of increased expression and nuclear distribution. During the protective process, cells change their morphology. Our results suggest that melatonin treatment could be beneficial to protect mitochondria under oxidative stress and treat certain ocular diseases, including age-related macular degeneration.

Interactome Mapping Guided by Tissue-Specific Phosphorylation in Age-Related Macular Degeneration.

The current study aims to determine the molecular mechanisms of age-related macular degeneration (AMD) using the phosphorylation network. Specifically, we examined novel biomarkers for oxidative stress by protein interaction mapping using in vitro and in vivo models that mimic the complex and progressive characteristics of AMD. We hypothesized that the early apoptotic reactions could be initiated by protein phosphorylation in region-dependent (peripheral retina vs. macular) and tissue-dependent (retinal pigment epithelium vs. retina) manner under chronic oxidative stress. The analysis of protein interactome and oxidative biomarkers showed the presence of tissue- and region-specific post-translational mechanisms that contribute to AMD progression and suggested new therapeutic targets that include ubiquitin, erythropoietin, vitronectin, MMP2, crystalline, nitric oxide, and prohibitin. Phosphorylation of specific target proteins in RPE cells is a central regulatory mechanism as a survival tool under chronic oxidative imbalance. The current interactome map demonstrates a positive correlation between oxidative stress-mediated phosphorylation and AMD progression and provides a basis for understanding oxidative stress-induced cytoskeletal changes and the mechanism of aggregate formation induced by protein phosphorylation. This information could provide an effective therapeutic approach to treat age-related neurodegeneration.

In Vitro Cytotoxicity, Adhesion, and Proliferation of Human Vascular Cells Exposed to Zinc.

Zinc (Zn) and its alloys have recently been introduced as a new class of biodegradable metals with potential application in biodegradable vascular stents. Although an in vivo feasibility study pointed to outstanding biocompatibility of Zn-based implants in vascular environments, a thorough understanding of how Zn and Zn2+ affect surrounding cells is lacking. In this comparative study, three vascular cell types-human endothelial cells (HAEC), human aortic smooth muscle cells (AoSMC), and human dermal fibroblasts (hDF)-were studied to advance the understanding of Zn/Zn2+-cell interactions. Aqueous cytotoxicity using a Zn2+ insult assay resulted in LD50 values of 50 µM for hDF, 70 µM for AoSMC, and 265 µM for HAEC. Direct cell contact with the metallic Zn surface resulted initially in cell attachment, but was quickly followed by cell death. After modification of the Zn surface using a layer of gelatin-intended to mimic a protein layer seen in vivo-the cells were able to attach and proliferate on the Zn surface. Further experiments demonstrated a Zn dose-dependent effect on cell spreading and migration, suggesting that both adhesion and cell mobility may be hindered by free Zn2+.

Direct measurement of actual levels of nitric oxide (NO) in cell culture conditions using soluble NO donors.

Applying soluble nitric oxide (NO) donors is the most widely used method to expose cells of interest to exogenous NO. Because of the complex equilibria that exist between components in culture media, the donor compound and NO itself, it is very challenging to predict the dose and duration of NO cells actually experience. To determine the actual level of NO experienced by cells exposed to soluble NO donors, we developed the CellNO Trap, a device that allows continuous, real-time monitoring of the level of NO adherent cells produce and/or experience in culture without the need to alter cell culturing procedures. Herein, we directly measured the level of NO that cells grown in the CellNO Trap experienced when soluble NO donors were added to solutions in culture wells and we characterized environmental conditions that effected the level of NO in in vitro culture conditions. Specifically, the dose and duration of NO generated by the soluble donors S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (GSNO), S-nitrosocysteine (CysNO) and the diazeniumdiolate diethyltriamine (DETA/NO) were investigated in both phosphate buffered saline (PBS) and cell culture media. Other factors that were studied that potentially affect the ultimate NO level achieved with these donors included pH, presence of transition metals (ion species), redox level, presence of free thiol and relative volume of media. Then murine smooth muscle cell (MOVAS) with different NO donors but with the same effective concentration of available NO were examined and it was demonstrated that the cell proliferation ratio observed does not correlate with the half-lives of NO donors characterized in PBS, but does correlate well with the real-time NO profiles measured under the actual culture conditions. This data demonstrates the dynamic characteristic of the NO and NO donor in different biological systems and clearly illustrates the importance of tracking individual NO profiles under the actual biological conditions.