Hank's Buffered Salt Solution Research Articles

Reactivity of Titanium Dental Implant Surfaces in Simulated Body Fluid.

Osseointegration of titanium (Ti) implants in bone is crucial for dental implant treatment. Bacterial challenge possibly leading to peri-implantitis threatens long-term success. For the improvement of osseointegration, an understanding of materials and tissue intervention is needed. This in vitro study analyzed changes of different implant surfaces exposed to simulated body fluid (SBF). Implants were analyzed by scanning electron microscopy/X-ray photoelectron spectroscopy. Supernatants (SNs) were assessed using inductively coupled plasma-mass spectrometry (ICP-MS). Additional calcium (Ca) and phosphate (P) crystals developed (Hank's buffered salt solution (HBSS)) on implants with layered surfaces. ICP of SN demonstrated a decreased Ca/P ratio. After incubation with human serum (HS), layers appeared flattened containing <1% Ca/P. The etched/machined implants showed the formation of a surface transformation layer or coating consisting of crystalline Ca/P precipitations and a decrease in the Ca/P ratio in the supernatant. Incubation in HS induced noncrystalline coverage, and increased Ti/Ca/P was detected in supernatants. HBSS induced crystals on surfaces of modified implants and crystalline covers on nonmodified implants containing Ca/P. The serum did not show the development of HA-like structures but showed dissolving effects. Titanium surfaces show completely altered behavior when incubated in serum-containing SBF.

Direct-Coupled Electroretinogram (DC-ERG) for Recording the Light-Evoked Electrical Responses of the Mouse Retinal Pigment Epithelium.

The retinal pigment epithelium (RPE) is a specialized monolayer of cells strategically located between the retina and the choriocapillaris that maintain the overall health and structural integrity of the photoreceptors. The RPE is polarized, exhibiting apically and basally located receptors or channels, and performs vectoral transport of water, ions, metabolites, and secretes several cytokines. In vivo noninvasive measurements of RPE function can be made using direct-coupled ERGs (DC-ERGs). The methodology behind the DC-ERG was pioneered by Marmorstein, Peachey, and colleagues using a custom-built stimulation recording system and later demonstrated using a commercially available system. The DC-ERG technique uses glass capillaries filled with Hank's buffered salt solution (HBSS) to measure the slower electrical responses of the RPE elicited from light-evoked concentration changes in the subretinal space due to photoreceptor activity. The prolonged light stimulus and length of the DC-ERG recording make it vulnerable to drift and noise resulting in a low yield of useable recordings. Here, we present a fast, reliable method for improving the stability of the recordings while reducing noise by using vacuum pressure to reduce/eliminate bubbles that result from outgassing of the HBSS and electrode holder. Additionally, power line artifacts are attenuated using a voltage regulator/power conditioner. We include the necessary light stimulation protocols for a commercially available ERG system as well as scripts for analysis of the DC-ERG components: c-wave, fast oscillation, light peak, and off response. Due to the improved ease of recordings and rapid analysis workflow, this simplified protocol is particularly useful in measuring age-related changes in RPE function, disease progression, and in the assessment of pharmacological intervention.

In-situ formation, anti-corrosion and hardness values of Ti-6Al-4V biomaterial with niobium via laser deposition

Ti-6Al-4V alloy biomaterials have low hardness, wear resistance, high corrosion rate and toxicity as results of release of aluminium and vanadium ions that led to the premature failure of the implant. In order to overcome some of these problems, laser cladding of Ti6Al4V alloy with Niobium was conducted. Three laser parameters (laser power, beam diameter and laser scan speed) were used to ascertain the proper operating condition for this laser process. Rofin Nd: yag laser was used in the laser cladding. The electrochemical study was conducted using Hank's buffered salt solution an environment similar to the human body. The optimum improvement in corrosion resistance resulted to 81.79% when compared with substrate. It has been established that operating parameter for laser cladding of Nb on Ti6Al4V were obtained at: laser power (1,000 W), beam diameter (1 mm) and scan speed (0.3 m/min).

Degradation mechanism of magnesium alloy stent under simulated human micro-stress environment

In this study, a vascular stent made of WE43 magnesium alloy was used as a research object and placed in a special physical simulation device constructed independently. This device provided a platform for the study of the degradation of the stent in a dynamic environment. The simulated body fluid of Hank's buffered salt solution flowing inside it would not only make the stent corroded but also apply cyclic shear stress to it, which get closer to the micro-stress environment in human blood vessels. In addition, by means of computer numerical simulation software, ANSYS Fluent 15.0, the fluid-structure interaction (FSI) model was established to simulate the wall shear stress (WSS) exerted by the flowing blood on stent in the blood vessel. Combined with the results of numerical simulation and physical simulation experiments, the degradation mechanism of magnesium alloy sent in an environment similar to the human blood vessels was studied.

XPS and STM study of TiO2(110)-(1 × 1) surfaces immersed in simulated body fluid

Rutile titanium dioxide (TiO2) (110)-(1 × 1) surfaces prepared in ultrahigh vacuum (UHV) were removed from the UHV, immersed in Hank's buffered salt solution (HBSS), and then re-introduced to the UHV to be examined for an atomic-level study of the osteoconductivity of TiO2. X-ray photoelectron spectroscopy (XPS) showed the adsorption of phosphate ions and divalent Ca ions from the HBSS. The density of the phosphate ions increased with the immersion time for up to 1 min and remained constant with further immersion time up to 1 week. Unlike in the case of the phosphate ions, the density of the Ca ions decreased with the immersion time after reaching the maximum value at 1 min immersion. Consequently, calcium phosphate was not formed on the (1 × 1) surface in the 1-week immersion. Scanning tunneling microscopy revealed molecule-sized spots covering the surfaces immersed for 1 min and longer. On the basis of density analysis by XPS, the spots were hypothesized to represent phosphate ions. Phosphate ions on the (1 × 1) surface are unable to undergo arrangement needed to facilitate the epitaxial growth of the brushite.

Sol Gel Glass Coating on Magnesium Alloys to be Used As Temporary Implants: Electrochemical and Surface Study in Simulated Body Fluid

Surface modification of metallic materials is the approach for developing functional properties to assure good biocompatibility, enhance tissue acceptance, and/ or inhibit corrosion rate. It opens a wide spectrum of possibilities oriented to develop the functional properties needed for an implant acceptance when the bare material is not enough to assure good biocompatibility and tissue regeneration. Further, the interface between the implant and the physiological environment becomes the key factor which conditions the prosthesis success or failure. The aim of this work is to generate a chemically modified surface of two commercial magnesium alloys with a 58S and 68S sol-gel made glass coating, obtained from organic precursors such as silicon and phosphate alkoxides and calcium lactate. The produced bioactive layer is sintered at low temperatures in order to maintain the substrate integrity. The glassy coating composition belongs to the SiO2-P2O5-CaO system and has the double goal of promote bone healing and retard the hydrogen evolution coming from the anodic reaction. AZ31B (Al 3%, Zn 1%, Mn 0.2%) and AZ91D (Al 9%, Zn 1%, Fe 0.005%, Mn 0.33%, Ni 0.002%) were used as substrates. Plane samples were polished until 2500 grit SiC paper and cleaned prior to coat. 58S and 68S glasses (60/70 mol% SiO2, 36/26 mol% CaO, 4/4 mol% P2O5, respectively) was prepared by sol-gel method using tetramethyl orthosilicate, methyltriethoxysilane, triethyl phosphite and calcium L-lactate hydrate as precursors, HNO31N was used as catalyser, ethylene glycol to improve the alkoxide condensation degree and methanol dissolve the calcium lactate. Micro-Raman assays were performed to analyze deposits composition (Invia Reflex Confocal, Renishaw RM 2000, UK) with a 785 nm wavelength laser. X-ray photoelectron spectroscopy (XPS) was selected to perform an elemental analysis of surfaces prior and after immersion of 72 h in HBSS. Measurements were made using a commercial VG ESCA 3000 system, using Mg Kα (1253.6 eV) radiation and an overall energy resolution of 0.8 eV. High resolution scans with 0.1 eV steps were conducted over Mg 2p, Ca 2p, P 2p, Si 2p and O 1s. In the entire cases surface charging effects were compensated by referencing the binding energy (BE) to the C 1s line of residual carbon set at 284.5 eV. Coatings visual integrity and deposits produced during immersion tests were examined by scanning electronic microscopy (SEM, JEOL JSM-6460LV, Japan) prior to immersion and after 24 h. Electrochemical assays were performed in a GAMRY Ref 600 electrochemical unit (Gamry, USA) with a conventional three electrode cell, using a saturated calomel electrode (SCE, Radiometer Copenhagen) as reference electrode, a platinum wire as a counter electrode and the coated and bare magnesium alloys as working electrodes. Electrochemical impedance spectroscopy (EIS) tests were registered also at the Ecorr with amplitude of 0.01V rms sweeping frequencies from 20000 to 0.005 Hz. Polarization curves for each sample were also recorded with an initial and final voltage of -1.8V (vs. reference) and 1 mV/s of scan rate. Hank Buffered Salt Solution (HBSS) at 37 ºC was used as electrolyte. Electrochemical test show that the coatings are able to retard anodic reaction being more efficiently on AZ91 alloy. Regarding the compositions, the coating with less calcium content (68S) is able to form more continuous films that the 58S with higher corrosion resistance after immersion in HBSS (Figure 1). Both coatings show signs of deposit of hydroxyapatite related compounds after immersion, as a first sign of bioactivity in vitro. Figure 1

Microstructures, mechanical properties, and degradation behaviors of heat-treated Mg-Sr alloys as potential biodegradable implant materials.

In previous studies, Mg-Sr alloys exhibited great biocompatibility with regard to test animals, and enhanced peri-implant bone formation. The objective of the present study was to investigate the effects of heat treatments on the mechanical and corrosion properties of Mg-Sr alloys. Various heat-treated Mg-xSr (x = 0.5, 1, and 2wt%, nominal composition) alloys were prepared using homogenization and aging treatments. Mechanical tests were performed at room temperature on the as-cast, homogenized, and peak-aged alloys. As the Sr content increased, the volume fraction of Mg17Sr2 phases within the as-cast alloys increased; in addition, the mechanical strength of the alloys initially increased and subsequently decreased, while the ductility decreased. Following the homogenization treatment, the mechanical strength of the alloys decreased, and the ductility increased. Nano-sized Mg17Sr2 phases were re-precipitated during the aging treatment. The age-hardening response at 160°C was enhanced as the Sr content increased. Following the aging treatment, there was an increase in the mechanical strength of the alloys; however, there was a slight reduction in the ductility. Immersion tests were conducted at 37°C for 360h, using Hank's buffered salt solution (HBSS), to study the degradation behavior of the alloys. As the Sr content of the Mg-Sr alloys increased, the corrosion rate (CR) increased owing to the galvanic effect. The homogenization treatment consequently reduced the CR dramatically, and the aging treatment had a slight effect on the CR. The peak-aged Mg-1Sr (wt%) alloy exhibited the best combination of properties. The tensile yield strength (TYS), ultimate tensile strength (UTS), elongation, compressive yield strength (CYS), ultimate compressive strength (UCS), compressibility, and CR of the as-cast Mg-1Sr (wt%) alloy were 56.0MPa, 92.67MPa, 1.27%, 171.4MPa, 243.6MPa, 22.3%, and 1.76mm/year, respectively. The respective results obtained for the peak-aged Mg-1Sr (wt%) alloys were 69.7MPa, 135.6MPa, 3.22%, 183.1MPa, 273.6MPa, 27.6%, and 1.33mm/year. Following immersion in HBSS, the primary corrosion products of the peak-aged Mg-1Sr (wt%) alloy were Mg(OH)2, MgO, MgCO3, Mg3(PO4)2, MgHPO4, and Mg(H2PO4)2, which enhanced the corrosion resistance by forming a composite corrosion film.

In Vitro Assessment of Human Islet Vulnerability to Instant Blood-Mediated Inflammatory Reaction (IBMIR) and Its Use to Demonstrate a Beneficial Effect of Tissue Culture.

Culture of human pancreatic islets is now routinely carried out prior to clinical islet allotransplantation, using conditions that have been developed empirically. One of the major causes of early islet destruction after transplantation is the process termed instant blood-mediated inflammatory reaction (IBMIR). The aim of this study was to develop in vitro methods to investigate IBMIR and apply them to the culture conditions used routinely in our human islet isolation laboratory. Freshly isolated or precultured (24 h, 48 h) human islets were incubated in either ABO-compatible allogeneic human blood or Hank's buffered salt solution (HBSS) for 1 h at 37°C. Tissue factor (TF) expression and leukocyte migration were assessed by light microscopy. TF was also quantified by ELISA. To assess β-cell function, glucose-stimulated insulin secretion (GSIS) assay was carried out. The extent of islet β-cell damage was quantified using a proinsulin assay. Islets cultured for 24 h had higher GSIS when compared to freshly isolated or 48-h precultured islets. Freshly isolated islets had significantly higher TF content than 24-h and 48-h precultured islets. Incubation of freshly isolated human islets in allogeneic human blood released 6.5-fold higher level of proinsulin in comparison to freshly isolated human islets in HBSS. The high level of proinsulin released was significantly attenuated when precultured islets (24 h or 48 h) were exposed to fresh blood. Histological examination of fresh islets in blood clot showed that some islets were fragmented, showing signs of extraislet insulin leakage and extensive neutrophil infiltration and necrosis. These features were markedly reduced when the islets were cultured for 24 h. These results suggest that our standard 24-h islet culture is markedly beneficial in attenuating IBMIR, as evidenced by increased GSIS, lower content of TF, decrease islet fragmentation, and proinsulin release.

Differential autophagic cell death under stress with ectopic cytoplasmic and mitochondrial-specific PPP2R2B in human neuroblastoma cells

Protein phosphatase 2A is one of four major classes of serine/threonine phosphatases. Overexpression of brain-specific regulatory subunit PPP2R2 in neuron cells is implicated in pathogenesis. The alternative splicing of PPP2R2B encodes two isoforms. They are subunit of cytoplasmic specific Bβ1 and mitochondria-targeted Bβ2. The two constructs were transfected into human neuroblastoma cells, SK-N-SH, respectively, and the stable clones overexpressing either Bβ1 or Bβ2 established. We have reported that Bβ2 clones are sensitive to reactive oxygen species (ROS) treatment by inducing autophagic cell death. To study more on the onset of neuropathogenesis under strain, both clones were exposed to different environmental stress, e.g. starvation and endoplasmic reticulum (ER) stress. To learn how PPP2R2B overexpression responds to starvation, cells were incubated in Hank's buffered salt solution of deprived nutrient. Cell death was induced in Bβ1 clones after 6h starvation, but not in Bβ2 clones. The pharmacological inhibitor, Bafilomycin A1, rescued the cell death while suppressing autophagy. On the other hand, to assess how cells respond to ER stress, the cells were treated with 0.1μM of N-glycosylation inhibitor, tunicamycin (TM). In contrast with Bβ1, the apoptotic cell death appeared in Bβ2 after 48h treatment. The formation of autophagolysosome was detected in Bβ2 following 12h treatment with TM as evidenced by lysotracker and GFP-LC3 staining for fluorescence microscopy analysis. The autophagy inhibitor, 3-methyladenine, salvaged the final apoptosis. The stable cell lines with ectopically transfected PPP2R2B genes encoding isoforms of brain-specific regulatory subunit exhibit distinct apoptosis under different stressors. The induced autophagic apoptotic cell death is related to mitochondrial membrane potential drop and ROS generation. Disturbance of autophagy alleviates the induced cell death. The results promised a good model for understanding the onset in pathogenesis under stress in neuron cells with aberrant PPP2R2B expression.

Troubleshooting the dichlorofluorescein assay to avoid artifacts in measurement of toxicant-stimulated cellular production of reactive oxidant species

IntroductionThe dichlorofluorescein (DCF) assay is a popular method for measuring cellular reactive oxidant species (ROS). Although caveats have been reported with the DCF assay and other compounds, the potential for artifactual results due to cell-free interactions between the DCF compound and toxicants has hardly been explored. We evaluated the utility of the DCF assay for measuring ROS generation by the toxicants mono-(2-ethylhexyl) phthalate (MEHP), and tetrabromobisphenol A (TBBPA). MethodsDCF fluorescence was measured spectrofluorometrically after a 1-h incubation of toxicants with 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA). MEHP was incubated with carboxy-H2DCFDA in cell-free solutions of Hank's buffered salt solution (HBSS), or in Royal Park Memorial Institute (RPMI) medium with or without fetal bovine serum. TBBPA was incubated with carboxy-H2DCFDA in cell-free HBSS and with human trophoblast cells (HTR8/SVneo cells). ResultsMEHP did not increase fluorescence in solutions of carboxy-H2DCFDA in HBSS or RPMI medium without serum. However, MEHP (90 and 180μM) increased DCF fluorescence in cell-free RPMI medium containing serum. Furthermore, serum-free and cell-free HBSS containing 25μM TBBPA exhibited concentration-dependent increased fluorescence with 5–100μM carboxy-H2DCFDA (p<0.05), but not 1μM carboxy-H2DCFDA. In addition, we observed increased fluorescence in HTR8/SVneo cell cultures exposed to TBBPA (0.5–25μM) (p<0.05), as we had observed in cell-free buffer. DiscussionMEHP demonstrated an interaction with serum in cell-free generation of DCF fluorescence, whereas TBBPA facilitated conversion of carboxy-H2DCFDA to the fluorescent DCF moiety in the absence of serum. Because TBBPA increased fluorescence in the absence of cells, the increased DCF fluorescence observed with TBBPA in the presence of cells cannot be attributed to cellular ROS and may, instead, be the result of chemical activation of carboxy-H2DCFDA to the fluorescent DCF moiety. These data illustrate the importance of including cell-free controls when using the DCF assay to study toxicant-stimulated cellular production of ROS.

Nutrient deprivation induces the Warburg effect through ROS/AMPK-dependent activation of pyruvate dehydrogenase kinase

The Warburg effect is known to be crucial for cancer cells to acquire energy. Nutrient deficiencies are an important phenomenon in solid tumors, but the effect on cancer cell metabolism is not yet clear. In this study, we demonstrate that starvation of HeLa cells by incubation with Hank's buffered salt solution (HBSS) induced cell apoptosis, which was accompanied by the induction of reactive oxygen species (ROS) production and AMP-activated protein kinase (AMPK) phosphorylation. Notably, HBSS starvation increased lactate production, cytoplasmic pyruvate content and decreased oxygen consumption, but failed to change the lactate dehydrogenase (LDH) activity or the glucose uptake. We found that HBSS starvation rapidly induced pyruvate dehydrogenase kinase (PDK) activation and pyruvate dehydrogenase (PDH) phosphorylation, both of which were inhibited by compound C (an AMPK inhibitor), NAC (a ROS scavenger), and the dominant negative mutant of AMPK. Our data further revealed the involvement of ROS production in AMPK activation. Moreover, DCA (a PDK inhibitor), NAC, and compound C all significantly decreased HBSS starvation-induced lactate production accompanied by enhancement of HBSS starvation-induced cell apoptosis. Not only in HeLa cells, HBSS-induced lactate production and PDH phosphorylation were also observed in CL1.5, A431 and human umbilical vein endothelial cells. Taken together, we for the first time demonstrated that a low-nutrient condition drives cancer cells to utilize glycolysis to produce ATP, and this increases the Warburg effect through a novel mechanism involving ROS/AMPK-dependent activation of PDK. Such an event contributes to protecting cells from apoptosis upon nutrient deprivation.

Roflumilast enhances the renal protective effects of retinoids in an HIV-1 transgenic mouse model of rapidly progressive renal failure

Retinoic acid decreases proteinuria and glomerulosclerosis in several animal models of kidney disease by protecting podocytes from injury. Our recent in vitro studies suggest that all-trans retinoic acid induces podocyte differentiation by activating the retinoic acid receptor-α (RARα)/cAMP/PKA/CREB pathway. When used in combination with all-trans retinoic acid, an inhibitor of phosphodiesterase 4 further enhanced podocyte differentiation by increasing intracellular cAMP. Additionally, we found that Am580, a specific RARα agonist, has similar renal protective effects as all-trans retinoic acid in a rederived colony of HIV-1 transgenic mice with rapidly progressive renal failure (HIV-Tg) that mimics human HIV-associated nephropathy. Treatment with either the inhibitor of phoshodiesterase 4, roflumilast, or Am580 significantly reduced proteinuria, attenuated kidney injury, and improved podocyte differentiation in these HIV-Tg mice. Additional renal protective effects were found when roflumilast was combined with Am580. Consistent with the in vitro data, glomeruli from HIV-Tg mice treated with both Am580 and roflumilast had more active phosphorylated CREB than with either agent alone. Thus, phosphodiesterase 4 inhibitors could be used in combination with RARα agonists to provide additional renal protection.

Treatment with chicken-egg-white or whole-egg extracts maintains and enhances the survival and differentiation of spleen cells

The identification of egg extracts with the ability to maintain and enhance the survival and differentiation of cells would be widely useful in cellular biology research. In this study, we compared the different abilities of spleen cells to survive and differentiate in vivo after permeabilization by five different types of egg extracts. Five types of egg extracts were prepared. The spleen cells from male GFP-transgenic mice were permeabilized by the extracts for 30min, cultured for 12days, and then transfused into irradiated female mice. At varying days after transplantation, the percentage of GFP-expressing surviving spleen cells was detected in the peripheral blood by flow cytometry. At 120days after transplantation, bone marrow cells from the female mice were analyzed for the presence of cells containing the Y chromosome. Surviving GFP-positive spleen cells that had been permeabilized with either chicken-egg-white or whole-egg extracts could be detected in the female mice after transplantation. A lower percentage of GFP-positive cells was also detected after permeabilization by the other extracts tested, and no GFP-positive cells were found in the female mouse transfused with spleen cells permeabilized with Hank's Buffered Salt Solution (HBSS) as a control. At 120days after transplantation, the percentage of cells containing a Y chromosome in the bone marrow positively correlated with the percentage of GFP-positive cells in the peripheral blood. After permeabilization by chicken-egg-white or whole-egg extracts, spleen cells demonstrated significantly enhanced survival and differentiation functions compared with the spleen cells treated with the other egg extracts tested. These results show that chicken-egg-white and whole-egg extracts have roles in maintaining and enhancing the survival and differentiation of spleen cells. Therefore, these two types of extracts may be of future use in maintaining the function of stem cells.

Evolutionary Constraints of Phosphorylation in Eukaryotes, Prokaryotes, and Mitochondria

High accuracy mass spectrometry has proven to be a powerful technology for the large scale identification of serine/threonine/tyrosine phosphorylation in the living cell. However, despite many described phosphoproteomes, there has been no comparative study of the extent of phosphorylation and its evolutionary conservation in all domains of life. Here we analyze the results of phosphoproteomics studies performed with the same technology in a diverse set of organisms. For the most ancient organisms, the prokaryotes, only a few hundred proteins have been found to be phosphorylated. Applying the same technology to eukaryotic species resulted in the detection of thousands of phosphorylation events. Evolutionary analysis shows that prokaryotic phosphoproteins are preferentially conserved in all living organisms, whereas-site specific phosphorylation is not. Eukaryotic phosphosites are generally more conserved than their non-phosphorylated counterparts (with similar structural constraints) throughout the eukaryotic domain. Yeast and Caenorhabditis elegans are two exceptions, indicating that the majority of phosphorylation events evolved after the divergence of higher eukaryotes from yeast and reflecting the unusually large number of nematode-specific kinases. Mitochondria present an interesting intermediate link between the prokaryotic and eukaryotic domains. Applying the same technology to this organelle yielded 174 phosphorylation sites mapped to 74 proteins. Thus, the mitochondrial phosphoproteome is similarly sparse as the prokaryotic phosphoproteomes. As expected from the endosymbiotic theory, phosphorylated as well as non-phosphorylated mitochondrial proteins are significantly conserved in prokaryotes. However, mitochondrial phosphorylation sites are not conserved throughout prokaryotes, consistent with the notion that serine/threonine phosphorylation in prokaryotes occurred relatively recently in evolution. Thus, the phosphoproteome reflects major events in the evolution of life.

XPS Studies of Magnesium Surfaces after Exposure to Dulbecco's Modified Eagle Medium, Hank's Buffered Salt Solution, and Simulated Body Fluid

AbstractMagnesium‐based biomaterials are gaining increasing interest, while in vitro corrosion tests are not standardized yet. Moreover, the effects of different corrosion media on the corrosion products are still not fully understood. To compare and evaluate the three main corrosion media applied in most in vitro studies, an XPS investigation of magnesium surfaces was carried out after exposure of the specimens to Dulbecco's modified eagle medium (DMEM), Hank's buffered salt solution (HBSS), and simulated body fluid (SBF). The effects of rinsing the specimens after immersion were also determined. XPS investigations especially on the Mg 2p state showed that MgO, Mg(OH)2, and MgCO3 species were the dominant corrosion products presenting in all specimens despite of the different corrosion media. However, the ratio of corrosion products depends on the medium composition. It was also shown that rinsing specimens after immersion experiments is a necessary procedure when surface analysis is employed afterward.

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank's Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1°C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank's Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1°C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Palmitoylation of the S0-S1 Linker Regulates Cell Surface Expression of Voltage- and Calcium-activated Potassium (BK) Channels

S-Palmitoylation is rapidly emerging as an important post-translational mechanism to regulate ion channels. We have previously demonstrated that large conductance calcium- and voltage-activated potassium (BK) channels are palmitoylated within an alternatively spliced (STREX) insert. However, these studies also revealed that additional site(s) for palmitoylation must exist outside of the STREX insert, although the identity or the functional significance of these palmitoylated cysteine residues are unknown. Here, we demonstrate that BK channels are palmitoylated at a cluster of evolutionary conserved cysteine residues (Cys-53, Cys-54, and Cys-56) within the intracellular linker between the S0 and S1 transmembrane domains. Mutation of Cys-53, Cys-54, and Cys-56 completely abolished palmitoylation of BK channels lacking the STREX insert (ZERO variant). Palmitoylation allows the S0-S1 linker to associate with the plasma membrane but has no effect on single channel conductance or the calcium/voltage sensitivity. Rather, S0-S1 linker palmitoylation is a critical determinant of cell surface expression of BK channels, as steady state surface expression levels are reduced by ∼55% in the C53:54:56A mutant. STREX variant channels that could not be palmitoylated in the S0-S1 linker also displayed significantly reduced cell surface expression even though STREX insert palmitoylation was unaffected. Thus our work reveals the functional independence of two distinct palmitoylation-dependent membrane interaction domains within the same channel protein and demonstrates the critical role of S0-S1 linker palmitoylation in the control of BK channel cell surface expression.

Chemical durability and microstructural analysis of glasses soaked in water and in biological fluids

A new glass, obtained from Bioglass ® BG45S5 original composition by substituting CaO with MgO, was produced and its chemical durability and microstructural characteristics were compared with that of Bioglass ®. The two glasses (labelled as BG45 and MG45) were soaked up to 4 weeks at physiological temperature in different solutions, i.e. bi-distilled water, Hank's Buffered Salt Solution 61200 (labelled as HBSS+), Hank's Buffered Salt Solution 14170 (labelled as HBSS−), and Kokubo's SBF. Moreover, the influence of either flat or flake surfaces was analysed for both glasses. Results showed that the chemical durability of a glass in saline at 37 °C, evaluated through pH and ICP-AES chemical analysis of the leached components, depended mainly on the chemical composition of the soaking solution. Moreover, the MG45 glass never exhibited hydroxyapatite crystal formation on its surface also after soaking in calcium-containing solutions. The apatite crystallisation and deposition mechanism, typical of a bioactive glass, was induced only if the glass itself contained calcium. The contemporaneous presence of calcium in the glass and in the soaking solution improved the reactivity of the glass, as apatite crystals nucleated in a shorter time and grew more quickly. As regards the morphology of the glass surface, rougher surfaces favoured the formation of hydroxyapatite crystals on glasses containing calcium.

Angiopoietin-1 Induces Krüppel-like Factor 2 Expression through a Phosphoinositide 3-Kinase/AKT-dependent Activation of Myocyte Enhancer Factor 2

Angiopoietin-1 (Ang1) regulates both vascular quiescence and angiogenesis through the receptor tyrosine kinase Tie2. We and another group have recently shown that Ang1 and Tie2 form distinct signaling complexes at cell-cell and cell-matrix contacts and further demonstrated that the former selectively induces expression of Krüppel-like factor 2 (KLF2), a transcription factor involved in vascular quiescence. Here, we investigated the mechanism of how Ang1/Tie2 signal induces KLF2 expression to clarify the role of KLF2 in Ang1/Tie2 signal-mediated vascular quiescence. Ang1 stimulated KLF2 promoter-driven reporter gene expression in endothelial cells, whereas it failed when a myocyte enhancer factor 2 (MEF2)-binding site of KLF2 promoter was mutated. Depletion of MEF2 by siRNAs abolished Ang1-induced KLF2 expression, indicating the requirement of MEF2 in KLF2 induction by Ang1. Constitutive active phosphoinositide 3-kinase (PI3K) and AKT increased the MEF2-dependent reporter gene expression by enhancing its transcriptional activity and stimulated the KLF2 promoter activity cooperatively with MEF2. Consistently, inhibition of either PI3K or AKT and depletion of AKT abrogated Ang1-induced KLF2 expression. In addition, we confirmed the dispensability of extracellular signal-regulated kinase 5 (ERK5) for Ang1-induced KLF2 expression. Furthermore, depletion of KLF2 resulted in the loss of the inhibitory effect of Ang1 on vascular endothelial growth factor (VEGF)-mediated expression of vascular cell adhesion molecule-1 in endothelial cells and VEGF-mediated monocyte adhesion to endothelial cells. Collectively, these findings indicate that Ang1/Tie2 signal stimulates transcriptional activity of MEF2 through a PI3K/AKT pathway to induce KLF2 expression, which may counteract VEGF-mediated inflammatory responses.