Confocal Laser Microscopy Images Research Articles

Capillary force-driven formation of native starch granule oleogels for 3D printing

3D printing has extremely high requirements for the design of food matrices with suitable rheological properties. Starch exhibits considerable potential as a material for 3D printing. However, there is limited research available on the direct fabrication of oleogels using starch granules as building blocks. In this study, we developed a simple approach to fabricate plant-based oleogels as edible 3D printable materials by exploiting capillary forces between starch granules. When a small amount of water (less than 5 wt%) was added to starch granules dispersed in an oil phase, a pendular bridging network was formed between the starch granules. The rheological properties of the starch-oil suspension were significantly changed, transitioning it from a fluid-like to a gel-like state. The rheological properties of the oleogel could be altered by varying the starch granule volume fraction, the amount of water added, the structure of starch granules, and the oil-water interfacial tension. Confocal laser microscopy imaging demonstrated that starch granule oleogels exhibit three distinct structural states: pendular state, pendular bridge network, and capillary aggregation. Furthermore, the presence of water was identified as a liquid bridge between starch granules. Additionally, starch granule oleogels exhibited excellent thermal stability and freeze-thaw stability. Overall, this approach enhances the feasibility of preparing starch oleogels and provides valuable insights for the personalized 3D printing of oleogels.

Hair regrowth boosting via minoxidil cubosomes: Formulation development, in vivo hair regrowth evaluation, histopathological examination and confocal laser microscopy imaging

Minoxidil has been used as an effective and cost-efficient topical treatment for androgenic alopecia. However, due to its poor water solubility, commercially available formulations contain alcohol and propylene glycol in a concentration that causes skin reactions such as irritation and dryness. Therefore, nanotechnology-based formulations can offer an alternative that might increase penetration and deposition of the drug in the skin while minimizing its adverse reactions.Minoxidil cubosomes (MXD-CUB) were prepared by melt dispersion emulsification technique according to full 23 factorial design. Three independent variables, namely, the dispersed phase concentration, glyceryl monooleate: Poloxamer 407 ratio and Tween 80 concentration were tested. Particle size, polydispersity index and the zeta potential were the dependent variables. The optimized formula was investigated by transmission electron microscopy, X-ray diffractometry and in vitro release test. In vivo study included Draize test, histopathological examination, hair regrowth efficacy and confocal laser scanning microscopy (CLSM).Particle size, zeta potential and polydispersity index of the optimal MXD-CUB were measured to be 131.10 ± 1.41 nm, −23.5 ± 0.42 mV and 0.185 ± 0.0, respectively, and its entrapment efficiency was 80.4 ± 4.04 %. Draize test and histopathological testing proved safety and tolerability of MXD-CUB. In vivo hair regrowth study revealed greater hair growth boosting effect of the prepared cubosomes compared to minoxidil solution. CLSM proved superior penetration and retention of rhodamine B-loaded cubosomes in the skin compared to rhodamine B solution. Therefore, MXD-CUB can be a safe and effective dosage form for minoxidil that overcome the drawbacks of the commercial formulations.

Photodynamic toluidine blue-gold nanoconjugates as a novel therapeutic for Staphylococcal biofilms.

Staphylococci are among the most frequent bacteria known to cause biofilm-related infections. Pathogenic biofilms represent a global healthcare challenge due to their high tolerance to antimicrobials. In this study, water soluble polyethylene glycol (PEG)-coated gold nanospheres (28 ppm) and nanostars (15 ppm) with electrostatically adsorbed photosensitizer (PS) Toluidine Blue O (TBO) ∼4 μM were successfully synthesized and characterized as PEG-GNPs@TBO and PEG-GNSs@TBO. Both nanoconjugates and the TBO 4 μM solution showed remarkable, if similar, antimicrobial photodynamic inactivation (aPDI) effects at 638 nm, inhibiting the formation of biofilms by two Staphylococcal strains: a clinical methicillin-resistant Staphylococcus aureus (MRSA) isolate and Staphylococcus epidermidis (S. epidermidis) RP62A. Alternatively in biofilm eradication treatments, the aPDI effects of PEG-GNSs@TBO were more effective and yielded a 75% and 50% reduction in viable count of MRSA and S. epidermidis RP62A preformed biofilms, respectively and when compared with untreated samples. This reduction in viable count was even greater than that obtained through aPDI treatment using a 40 μM TBO solution. Confocal laser microscopy (CLSM) and scanning electron microscope (SEM) images of PEG-GNSs@TBO's aPDI treatments revealed significant changes in the integrity and morphology of biofilms, with fewer colony masses. The generation of reactive oxygen species (ROS) upon PEG-GNSs@TBO's aPDI treatment was detected by CLSM using a specific ROS fluorescent probe, demonstrating bright fluorescence red spots across the surfaces of the treated biofilms. Our findings shine a light on the potential synergism between gold nanoparticles (AuNPs) and photosensitizers in developing novel nanoplatforms to target Staphylococcal biofilm related infections.

Synthesis of luminescent chitosan-based carbon dots for Candida albicans bioimaging

In this work, we used chitosan as a raw material to synthesize carbon dots using fast microwave carbonization. We studied the influence of the synthesis time, doping agent, and the molar ratio between the reactants on the quantum yield of carbon dots. Chitosan-based carbon dots displayed stable blue fluorescence emission with excitation-dependent behavior and quantum yield values ranging from 1.16 to 7.07 %. ANOVA results showed that the interaction factor between the doping agent and the molar ratio of the reactants was a significant combination to produce carbon dots with higher quantum yield. The presence of the doping agent improved the carbon dots optical properties by obtaining higher fluorescence intensity values. Confocal laser microscope images showed that the carbon dots internalized in the Candida albicans cellular membrane, exhibiting blue, green, and red emissions, acting as a promising agent for bioimaging.

Formation of protein oleogels via capillary attraction of engineered protein particles

Edible oleogels have gained popularity as an approach for formulating food products, profiting from improving nutritional profiles related to health-related concerns, and playing an essential role in food processing. Protein oleogels provide an additional opportunity to load and deliver a high proportion of natural macronutrient in lipid-based food products. However, structuring oleogels with proteins directly were rarely reported, which has greatly limited their practical applications. Herein, we proposed a simple and robust method for structuring protein oleogels. By taking advantage of the improved interparticle cohesion through adding a much small amount of water (e.g., 2%) dispersed by ball milling, a space-spanning fractal network of engineered colloidal protein nanoparticles was formed, allowing for physically trapping the liquid oil into a self-standing gel-like system. Rheology and confocal laser microscopy imaging were adopted to investigate the viscoelasticity and microstructure of the resulting oleogels, showing that these capillary protein oleogels can be designed and fabricated with tunable properties upon water addition. Meanwhile, protein oleogels can be successfully formed by this approach, varying from proteins with different wettability, as well as oils differing in polarity. Overall, our findings raise the possibility of structuring protein oleogels with high operability and easy scale-up in practical applications.

Calcium-induced skim milk gels: Effect of milk powder concentration and pH on tribo-rheological characteristics and gel physico-chemical properties

The physico-chemical and tribo-rheological characteristics of calcium-induced skim milk gels obtained by heating of milk dispersions prepared with different concentrations of skim milk powder (10, 20 and 30% w/w) and calcium chloride (30, 60 and 90 mmol kg−1) were studied. The effect of pH on the gelation and on the final gel structure was also analyzed. For this purpose, the pH of selected samples was readjusted to the natural pH of milk of 6.66 after the calcium salt addition. The gelation process was analyzed by rheometry throughout temperature and time sweeps. Gelation at temperatures lower than 70 °C was observed in samples with 10% w/w of skim milk powder (SM) and 30 mmol kg−1 of calcium chloride or similar ratio, when the amount of calcium remaining in the serum phase is enough to induce gel formation. The results obtained from temperature sweeps also suggest that the pH strongly affects the temperature at which gelation initiates. Structuring parameters confirmed these results. From time sweeps, it was observed that the kinetics of gelation depended on both composition and pH. Gels obtained using higher SM concentrations (20 and 30% w/w) showed better physical properties (low syneresis and high water holding capacity). Confocal laser microscopy images also showed a more homogeneous structure in those samples. Higher SM and calcium chloride concentrations improved the lubrication characteristics analyzed by tribology. Friction factors at 10 mm s−1 (typical speed in oral processing) decreased as the SM concentration increased. It is concluded that calcium-induced skim milk gels with different microstructure can be obtained by varying the concentration of skim milk powder and calcium salt added, and pH adjustment.

An anticancer drug loading and release study to ternary GO-Fe3O4-PPy and Fe3O4 @PPy-NGQDs nanocomposites for photothermal chemotherapy

Photothermal therapy (PTT) is a new treatment method being developed against traditional cancer therapy methods. In this study, graphene oxide-iron oxide-polypyrrole (GO-Fe3O4-PPy) and iron oxide@polypyrrole-nitrogen doped graphene quantum dots (Fe3O4 @PPy-NGQDs) ternary nanocomposites having superior photothermal and magnetic properties were loaded with doxorubicin (DOX) anticancer drug. The loading and release of the DOX was investigated by UV–visible spectroscopy. At the end of 48 h, the cumulative release of DOX from GO-Fe3O4-PPy/DOX and Fe3O4 @PPy-NGQDs/DOX at pH 5.5 and pH 7.4 was obtained as 99.55% ± 0.20, 48.87% ± 0.20% and 97.88% ± 0.45, 38.82% ± 0.26, respectively. After a short period of time like 4 h and 8 h, NIR-triggered release of DOX was increased significantly compared to drug release without NIR. The cytotoxic effects on the 4T1 mouse breast cancer cells of GO-Fe3O4-PPy/DOX and Fe3O4 @PPy-NGQDs/DOX drug loaded nanocomposites were determined by MTT test. The increase of DOX release with NIR effect was demonstrated by both in-vitro cytotoxicity studies and confocal laser microscopy images. In summary, multifunctional GO-Fe3O4-PPy/DOX and Fe3O4 @PPy-NGQDs/DOX nanocomposites have great potential for photothermal chemotherapy.

Ganoderic Acid A Promotes Amyloid-β Clearance (In Vitro) and Ameliorates Cognitive Deficiency in Alzheimer's Disease (Mouse Model) through Autophagy Induced by Activating Axl.

Alzheimer’s disease (AD) is thought to be caused by amyloid-β (Aβ) accumulation in the central nervous system due to deficient clearance. The aim of the present study was to investigate the effect of ganoderic acid A (GAA) on Aβ clearance in microglia and its anti-AD activity. Aβ degradation in BV2 microglial cells was determined using an intracellular Aβ clearance assay. GAA stimulated autophagosome formation via the Axl receptor tyrosine kinase (Axl)/RAC/CDC42-activated kinase 1 (Pak1) pathway was determined by Western blot analyses, and fluorescence-labeled Aβ42 was localized in lysosomes in confocal laser microscopy images. The in vivo anti-AD activity of GAA was evaluated by object recognition and Morris water maze (MWM) tests in an AD mouse model following intracerebroventricular injection of aggregated Aβ42. The autophagy level in the hippocampus was assayed by immunohistochemical assessment against microtubule-associated proteins 1A/1B light-chain 3B (LC3B). Intracellular Aβ42 levels were significantly reduced by GAA treatment in microglial cells. Additionally, GAA activated autophagy according to increased LC3B-II levels, with this increased autophagy stimulated by upregulating Axl and Pak1 phosphorylation. The effect of eliminating Aβ by GAA through autophagy was reversed by R428, an Axl inhibitor, or IPA-3, a Pak1 inhibitor. Consistent with the cell-based assay, GAA ameliorated cognitive deficiency and reduced Aβ42 levels in an AD mouse model. Furthermore, LC3B expression in the hippocampus was up-regulated by GAA treatment, with these GAA-specific effects abolished by R428. GAA promoted Aβ clearance by enhancing autophagy via the Axl/Pak1 signaling pathway in microglial cells and ameliorated cognitive deficiency in an AD mouse model.

Torque Requirements and the Influence of Pilot Holes on Orthodontic Miniscrew Microdamage

This study aimed to quantify the microdamage to cortical bone of different thickness and the maximum insertion torque during orthodontic miniscrew implant (OMI) placement with and without a pilot hole. Forty-five porcine bone specimens were prepared with thicknesses of 1.5, 2 and 2.5 mm. Ten bone specimens per thickness had a pilot hole drilled prior to the insertion of an OMI, and the remaining 15 bone specimens had an OMI without a pilot hole inserted. Sequential staining was used to identify damage caused by bone preparation and surface microdamage from OMI insertion and confocal laser microscopy images were used to quantify damage characteristics. Of the five damage characteristics, only one decreased when a pilot hole was used for all bone specimens (p = 0.025), while two increased as cortical bone thickness increased (p = 0.0064, p = 0.0003). There was no evidence that maximum insertion torque differed according to pilot hole status (p = 0.1144) and increased as cortical bone thickness increased (p = 0.0001). The presence of a pilot hole had minimal effect on microdamage characteristics and no effect on maximum insertion torque. As cortical bone thickness increased, an increase in microdamage and in maximum insertion torque was observed.

Novel rhein integrate transphytosomes as non-invasive local therapy for osteoarthritis to ameliorate cartilage deterioration in MIA-arthritic rats

Rhein (RH), a natural chondroprotective agent, suffers from poor systemic availability (20–25%) after oral administration concomitant to side effects on the gastrointestinal tract and liver. We present a new approach for non-invasive local targeted delivery of rhein to ameliorate cartilage deterioration employing cartilage-homing phospholipids nanocarriers. This is the first work to elaborate RH loaded transphytosome (RH-T-PHY) as novel nanovesicular systems for transdermal drug delivery based on an advantageous hybrid between phytosomes and transfersomes or bilosomes. Here, we developed transphytosomes through incorporating various edge activators (EAs) such as Tween 80, Span 80 and sodium deoxycholate into the lipid bilayer of RH phytosomes to affix the flexibility. RH-T-PHY with high flexibility and entrapment efficacy showed the highest significant skin permeation compared to conventional phytosomes. Additionally, RH-T-PHY have a magnificent potential in maintaining high chondroprotective activity as demonstrated by enhanced repair, regeneration of chondrocytes and GAG formation in MIA-induced osteoarthritis (OA) rat model. Besides, histological examination of vital organs revealed the formulation safety. Confocal laser microscopy images revealed the highest drug availability in the articular cartilage of RH-T-PHY treated group. Conclusively, novel RH-T-PHY can serve as a promising alternative means for delivery of chondroprotective drugs for effective non-invasive local therapy of OA.

Comparison between Allogenic and Xenogenic Bone Blocks on the Osteogenic Potential of Cultured Human Periodontal Ligament Stem Cells: Confocal Laser and Scanning Electron Microscopy Study.

Aim:The aim of this study was to compare between equine and human bone blocks in the osteogenic differentiation of cultured human periodontal ligament stem cells (hPDLSCs) at 14 and 21 days of culture, using confocal laser microscopy and scanning electron microscopy.Materials and Methods:In vitro cultures of commercially obtained hPDLSCs were seeded onto equine and human bone blocks. At 14 days and 21 days of culture, confocal laser microscope images were obtained to assess cellular differentiation and adhesion, and scanning electron microscope images were obtained to validate the osteogenic differentiation by showing the morphological characteristics of the new bone cells.Results:Both equine and human bone blocks showed positive staining for newly formed bone cells through the confocal laser microscope analysis, however, a higher signal intensity was expressed at 21 days of culture. These findings indicate the biocompatibility of hPDLSC with both types of bone blocks, cellular differentiation, and adhesion. Scanning electron microscopy images validated the osteogenic differentiation by showing the common characteristics of bone cells as flattened, polygonal morphology with multiple extending cytoplasmic processes.Conclusion:Both equine and human bone blocks were able to confirm the osteogenic capability of seeded human PDLSC. There was no significant difference between equine and human bone blocks on the human PDLSC differentiation. Superior osteogenic differentiation of cultured hPDLSCs was evident at 21 days in comparison to 14 days.

Liquid droplet imbibition into a thin coating layer: Direct pore-scale modeling and experimental observations

In order to control ink droplet movement into the printing-paper layer, a set of pore-scale two-phase flow simulations were performed. The high-resolution three-dimensional pore space of the paper was obtained using focused ion beam scanning electron microscopy (FIB-SEM). Solving Navier-Stokes equations yielded details about dynamic movement of a droplet into the layer. To evaluate simulation results, for the first time, confocal laser microscopy imaging technique was integrated into a FIB-SEM chamber. Doing so, high resolution imaging of the droplet penetration inside paper was conducted and computed volume of penetrated ink at final stage was compared to the imaged volume. The ink penetration and spreading extent showed a good agreement with simulation results. Therefore, the developed simulation case was further investigated to study impact of liquid contact angle, real ink properties, and droplet arrival velocity on paper surface on final print quality. A faster penetration into the paper coating was observed for smaller equilibrium contact angles; meanwhile, more radial wicking was observed. In case of velocity of impact, higher velocity caused creation of irregular shapes of the ink footprint on paper surface. In addition to that, higher velocity caused ink splash which consequently created satellite droplets and lowered the print quality. Comparing ink-like liquid (representing real ink liquid properties) with water, water moves faster than ink-like liquid into the paper. This is mainly due to the higher viscosity and lower surface tension of the ink-like liquid.

Three-dimensional structure analysis of melanocytes and keratinocytes in senile lentigo.

Senile lentigo or age spots are hyperpigmented macules of skin that commonly develop following long-term exposure to ultraviolet radiation. This condition is caused by accumulation of large numbers of melanosomes (melanin granules) produced by melanocytes within neighboring keratinocytes. However, there is still no consensus regarding the melanosome transfer mechanism in senile lentigo. To date, most pathohistological studies of skin have been two-dimensional and do not provide detailed data on the complex interactions of the melanocyte-keratinocyte network involved in melanosome transfer. We performed a three-dimensional reconstruction of the epidermal microstructure in senile lentigo using three different microscopic modalities to visualize the topological melanocyte-keratinocyte relationship and melanosome distribution. Confocal laser microscopy images showed that melanocyte dendritic processes are more frequently branched and elongated in senile lentigo skin than in normal skin. Serial transmission electron micrographs showed that dendritic processes extend into intercellular spaces between keratinocytes. Focused ion beam-scanning electron micrographs showed that dendritic processes in senile lentigo encircle adjacent keratinocytes and accumulate large numbers of melanosomes. Moreover, melanosomes transferred to keratinocytes are present not only in the supranuclear area but throughout the perinuclear area except on the basal side. The use of these different microscopic methods helped to elucidate the three-dimensional morphology and topology of melanocytes and keratinocytes in senile lentigo. We show that the localization of melanosomes in dendritic processes to the region encircling recipient keratinocytes contributes to efficient melanosome transfer in senile lentigo.

Ozonation as non-thermal option for bacterial load reduction of Chlorella biomass cultivated in airlift photobioreactor

The present study proposes the selective reduction of bacterial load in the microalgal cultures without affecting the microalgae. Bacteria was found to be symbiotically associated with microalgae and exponentially increased in the range of 103–1011 CFU mL-1 during Chlorella cultivation in the airlift photobioreactor. The bacterial load needs to be reduced to meet USFDA standards (∼103 CFU mL-1) for the use of microalgal biomass in food and feed applications. Ozone was evaluated as a non-thermal option for bacterial load reduction of Chlorella cultivated in the airlift photobioreactor. The results of ozonation w.r.t to time (5, 10, 15, 20, 25, and 30 min) and dissolved ozone concentrations (0.25, 0.49, 0.75, 0.99, and 1.25 mg of O3 L-1) showed a significant reduction of bacterial load in the range of 3 – 6.5 log. The bacterial disinfection kinetics of ozonation showed an overall second-order reaction with a rate constant (K) of 0.93 mg−1 min−1 L. Further, confocal laser microscopy imaging of Chlorella and its associated bacterial cells confirmed the selective and significant reduction of associated bacteria. The viability of the Chlorella cells were confirmed with trypan blue viability assay (∼99%) under a fluorescent microscope. The biomass quality with respect to metabolite integrity was also validated. In conclusion, ozonation can be a potential solution for selective reduction of bacterial load associated with Chlorella without affecting Chlorella cells viability.

CATIONIC NANOSTRUCTURED LIPID CARRIERS: OPTIMIZATION OF ZETA POTENTIAL AND EVALUATION

Objective: To fabricate, optimize and evaluate, Lipid-based cationic nanoparticulate dispersed system to improve the bio-adhesion property for ophthalmic use.
 Methods: Lipid-based cationic nanoparticulate dispersed system was fabricated by melt emulsification ultrasonication method and Box-Behnken design was utilized for optimization of formulation through the Design-Expert® program. The concentration of stabilizer, liquid lipid and Cetyltrimethylammonium Bromide (CTAB) was selected as variables (factors) while particle size, zeta potential and polydispersity index (PDI) were selected as a response for optimization purpose. Characterization of particle properties was performed using Transmission Electron Microscopy (TEM), Photon correlation spectroscopy (PCS). Fourier-transform infrared spectroscopy (FT-IR) was performed to study chemical interaction among ingredients. Rhodamine B entrapped CNLC formulation was used to study the interaction of NLC and CNLC on a three-dimensional and two-dimensional ocular tissue model for cell uptake and penetration properties.
 Results: Cationic nanostructured lipid carrier system was successfully fabricated by melt emulsification ultrasonication method. Characterization of nanoparticulate system using PCS revealed particle size in the range of 113.1 to 274.2 nm, PDI in a range of 0.147 to 0.280, while zeta potential in the range of 7.2 to 49.8 mV. The validation of statistical design suggested that it was suitable for navigation of design space. TEM imaging confirmed the results of PCS characterization. FT-IR study suggested a minimal chemical interaction among ingredients in CNLC. Confocal laser microscopic imaging for the interaction of NLC and CNLC on a three-dimensional and two-dimensional ocular tissue model revealed good penetration and bio-adhesion properties.
 Conclusion: The fabricated Cationic nanostructured lipid carrier system was found to be the potential ophthalmic novel drug delivery system with good bio-adhesion and penetration properties.

Osteogenic Potential of Periodontal Ligament Stem Cells Cultured in Osteogenic and Regular Growth Media: Confocal and Scanning Electron Microscope Study

To evaluate the ability of osteogenic culture media in comparison with regular growth culture media in enhancing the osteoblastic cell differentiation of human periodontal ligament stem cells (hPDLSCs). In vitro cultures of commercially obtained hPDLSCs were seeded onto xenograft bone blocks in both regular and osteogenic media. Confocal laser microscope images were obtained for cellular differentiation and adhesion, and scanning electron microscopy (SEM) images were obtained to validate the osteogenic differentiation by showing the morphological characteristics of the newly formed cells. Confocal laser microscope analysis showed positive staining for new bone cells with an increased signal intensity when samples were cultured in osteogenic culture media compared with regular culture media. These findings indicate the effect of the active ingredients of the osteogenic culture media in enhancing the osteogenic differentiation hPDLSC. Scanning electron microscopy images validated the osteogenic differentiation showing a flattened, polygonal morphology with multiple extending cytoplasmic processes of new cells. Xenograft bone blocks are biocompatible scaffold for the osteogenic differentiation of seeded hPDLSCs. Osteogenic culture media enhances and increases the osteogenic differentiation of hPDLSCs into new bone cells more than regular growth culture media. Periodontal ligament stem cells are a predictable biological input as a cell-based tissue-engineered construct and biologically acceptable when it is cultured in a suitable growth media that mimics the intended environment. Consideration of the clinical use of equine bone blocks and periodontal ligament stem cells in a suitable biological environment as a potential new option for bone regeneration techniques.

Deep transfer learning methods for colon cancer classification in confocal laser microscopy images.

The gold standard for colorectal cancer metastases detection in the peritoneum is histological evaluation of a removed tissue sample. For feedback during interventions, real-time in vivo imaging with confocal laser microscopy has been proposed for differentiation of benign and malignant tissue by manual expert evaluation. Automatic image classification could improve the surgical workflow further by providing immediate feedback. We analyze the feasibility of classifying tissue from confocal laser microscopy in the colon and peritoneum. For this purpose, we adopt both classical and state-of-the-art convolutional neural networks to directly learn from the images. As the available dataset is small, we investigate several transfer learning strategies including partial freezing variants and full fine-tuning. We address the distinction of different tissue types, as well as benign and malignant tissue. We present a thorough analysis of transfer learning strategies for colorectal cancer with confocal laser microscopy. In the peritoneum, metastases are classified with an AUC of 97.1, and in the colon the primarius is classified with an AUC of 73.1. In general, transfer learning substantially improves performance over training from scratch. We find that the optimal transfer learning strategy differs for models and classification tasks. We demonstrate that convolutional neural networks and transfer learning can be used to identify cancer tissue with confocal laser microscopy. We show that there is no generally optimal transfer learning strategy and model as well as task-specific engineering is required. Given the high performance for the peritoneum, even with a small dataset, application for intraoperative decision support could be feasible.

Percutaneous immunization with 40-nm antigen-encapsulated elastic liposomes

In this study, we aimed to verify the influence of the particle diameter of elastic liposomes for transdermal delivery on immune responsiveness by evaluating the amount of antibody. Albumin from chicken egg white (OVA) solution and elastic liposomes with mean volume diameter of 40 nm and 130 nm were percutaneously administrated to mice. We prepared 40-nm elastic liposomes using mechanochemical process and 130-nm elastic liposomes using extrusion process. Subcutaneous injection of OVA was also as a positive control. Forty-nanometer elastic liposomes significantly promoted antibody production compared with the OVA solution and 130-nm elastic liposomes. Furthermore, this result was nearly equivalent to that of the subcutaneous injection of OVA solution. Besides, skin permeability study shows that coumarin-6 used as a fluorescent label encapsulated in 40-nm elastic liposomes were 2.2 times more permeated than 130-nm elastic liposomes in the skin. From the observation of confocal laser microscopy images, 40-nm elastic liposomes accumulated more in the stratum corneum and deeper follicle compared to 130-nm elastic liposomes. It has been reported that particles with a diameter of 40 nm are more captured by antigen presenting cells. Thus, we considered that the 40-nm elastic liposomes are useful for percutaneous immunization.

BIAXIAL FLEXURAL STRENGTH OF UN-SHADED AND SHADEDMONOLITHIC TRANSLUCENT ZIRCONIA

INTRODUCTION: The natural white color of zirconia together with the veneer chipping problem has led to the development of tooth colored monolithic (full-anatomic) zirconia. In order to optimize esthetics shading of monolithic zirconia were done by two main approaches, either by powder mixing method or by infiltration technique. The influence of infiltration technique on mechanical properties of zirconia has been reviewed previously, while the influence of powder mixing method on the mechanical properties of shaded zirconia, specially the biaxial flexural strength has not been widely studied before. OBJECTIVES: To evaluate and compare the biaxial flexural strength and the crystal structure of shaded CAD/CAM monolithic translucent zirconia by powder mixing method with un-shaded ones. MATERIALS AND METHODS: Twenty fully sintered discs of un-shaded [T 0] and shaded [T S] monolithic translucent zirconia of diameter 12 mm and thickness 1 mm has been divided into 2 parallel groups of 10 discs each. Their biaxial flexural strength was measured using a ball-on-ring test fixture and universal testing machine at room temperature. Statistical significance was measured using One-Way ANOVA test. Then crystallographic analysis was done for both groups using X-ray diffraction. Finally the nature of failures of the tested specimens was examined using confocal laser microscopy. RESULTS: Biaxial flexural strength test showed no significant difference between un-shaded and shaded specimens. X-ray diffraction spectrum only showed crystals of tetragonal zirconia with no evidence of neither monoclinic zirconia crystals nor coloring oxides crystal phases. Confocal laser microscopy images of both groups showed the classic fracture patterns of monolithic zirconia materials. CONCLUSIONS: shading of zirconia blocks by powder mixing methods does not significantly (P≥0.05) affect either biaxial flexural strength or crystallographic structure of monolithic translucent zirconia

Renin Production and Involvement of ß1 Integrin in Blood Pressure Regulation in Diabetic Nephropathy

Diabetic nephropathy (DN) and hypertension mutually exacerbate each other, but many aspects of the mechanisms of these two pathologies remain unclear. The juxtaglomerular cells (JGC) secrete renin and trigger the renin-angiotensin-aldosterone system involved in blood pressure regulation. Integrins (Itgs) are cell surface proteins that interact with the extracellular matrix and transduce intracellular signals in response to extracellular stimuli. There are several phosphorylation sites in the β chain, and phosphorylation plays an important role in the cell-binding ability and intracellular signaling functions of Itgs. Our previous immunohistochemical analysis of renal tissue sections of diabetic patients showed that β1 Itg, a cellular matrix adhesion factor, undergoes JGC-specific phosphorylation. To characterize the relationship between the phosphorylated β1 (pβ1) Itg and renin expression in DN, the following studies were conducted in rats with streptozotocin (STZ)-induced diabetes. Immunohistochemical analysis of the prepared sections showed JGC-specific phosphorylation of β1 Itg in the kidneys of diabetic rats. Immunofluorescent staining of these kidney tissue specimens confirmed the coexpression of pβ1 Itg and renin in JGC. The mRNA levels of pβ1 Itg decreased continuously with time in diabetic rats after the onset of diabetes, till month 2. However, the mRNA levels of pβ1 Itg increased subsequently, but ultimately showed recovery to the baseline level. In contrast, the renin mRNA ratio exhibited an inverse correlation with pβ1 Itg. Quantification by image analysis of confocal laser microscopic images confirmed the negative correlation between pβ1 Itg and renin. Our results showed a negative correlation between pβ1 Itg and renin expression in renal JGC, suggesting a link between β1 Itg and the regulatory system that controls renin production and secretion. Disclosure M. Toyoda: None. N. Saito: None. M. Kimura: None. H. Moriya: None. K. Sawada: None. M. Fukagawa: None.