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PubMed2006

Stem cell attachment to layer-by-layer assembled TiO2 nanoparticle thin films.

Surface topography is one of the most important factors influencing the attachment and spreading of cells. In the present study, layer-by-layer assembled titanium dioxide (TiO2) nanoparticle thin films were chosen for attachment, proliferation and spreading studies on mouse mesenchymal stem cells (MSC). Increasing surface roughness was observed with increasing number of layer-by-layer assembled TiO2 thin films. Four layer TiO2 thin film showed higher number of attached cells than a one layer thin film and control surfaces. MSCs experienced no cytotoxic effects after culture on the TiO2 coated substrates as observed from the cytotoxicity tests. Cell spreading, visualized with scanning electron microscopy, showed a faster rate of spreading on a rougher surface. Cells on a four-layer substrate, at 12 h showed complete spreading, where as most of the cells on a control surface and a one-layer surface, at 24 h, retained a rounded morphology. In conclusion, TiO2 nanoparticle thin films were successfully assembled in alternation with polyelectrolytes and in-vitro studies with MSC showed an increase in the attachment and faster spreading of cells on rougher surfaces.

Cell Adhesion↗

PubMed2005

Focal prostate basal cell layer disruptions and leukocyte infiltration are correlated events: A potential mechanism for basal cell layer disruptions and tumor invasion.

To assess the potential correlation between basal cell layer disruptions and leukocyte infiltration, consecutive sections of normal (n=5) and tumor (n=50) prostate tissues were double immunostained for cytokeratin 34betaE12 (CK 34betaE12) plus leukocyte common antigen, Ki-67, or proliferating cell nuclear antigen (PCNA). Of 2047 acini and ducts examined, 201 contained focal basal cell layer disruptions. Of those, 183 (91%) showed leukocyte infiltration, compared to 67 (33.3%) in 201 morphologically comparable structures with an intact basal cell layer (P<0.01). Basal cell layers adjacent to or surrounded by leukocytes were often attenuated or fragmented, and leukocytes were generally located at or near disruptions. Disrupted basal cell layers showed a markedly reduced proliferation rate, compared to their non-disrupted counterparts. Cells overlying focal basal cell layer disruptions often displayed distinct changes in the size, nuclear shape, density, and polarity, compared to those away from disruptions. A vast majority of proliferating tumor cells were located at or near basal cell layer disruptions. These findings suggest that focal basal cell layer disruptions and leukocyte infiltration are correlated events, representing a potential trigger factor for prostate tumor invasion.

Cell Aggregation↗

PubMed2006

Templated synthesis of silver nanowires based on the layer-by-layer assembly of silver with dithiodipropionic acid molecules as spacers.

The layer-by-layer assembly of silver nanoclusters with 3,3'-dithiodipropionic acid (DTDPA) as spacers was prepared through self-assembly on a gold foil and has been characterized by cyclic voltammetric and AFM techniques. The DTDPA molecules acting as spacers between the layers of silver serve as molecular interconnects for the four layers prepared in this work. The organization of layers was found to decrease with an increase in the number of layers. The layer-by-layer assembly of silver clusters motivated us to prepare silver nanowires stabilized by the bifunctional molecules DTDPA through template synthesis using cellulose nitrate membranes. The nanostructures formed by this method were characterized by SEM, TEM, AFM, FTIR, CV, and photoluminescence studies. It is observed that the DTDPA molecules, instead of forming molecular interconnects, protect the structures by self-assembling themselves along the edges of the nanostructures. The concept of self-assembly protecting the nanostructures is demonstrated in this work.

Journal Article↗

PubMed2003

Biocompatibility of layer-by-layer self-assembled nanofilm on silicone rubber for neurons.

Electrostatic layer-by-layer (LbL) self-assembly, a novel method for ultrathin film coating has been applied to silicone rubber to encourage nerve cell adhesion. The surfaces studied consisted of precursor layers, with alternating cationic poly(ethyleneimine) (PEI) and anionic sodium poly(styrenesulfonate) (PSS) followed by alternating laminin and poly-D-lysine (PDL) layers or fibronectin and PDL layers. Film growth increased linearly with the number of layers. Every fibronectin/PDL and laminin/PDL bilayer was 4.4 and 3.5 nm thick, respectively. All layers were more hydrophilic than the unmodified silicone rubber surface, as determined from contact angle measurements. Of the coatings studied, a PDL layer was the most hydrophilic. A multilayer film with composition [PSS/PEI]3+[fibronectin/PDL]4 or [PSS/PEI]3+[laminin/PDL]4 was highly favorable for neuron adhesion, in contrast to bare silicone rubber substrate. The film coated on silicone rubber is biocompatible for cerebellar neurons with active viability, as shown by lactate dehydrogenase (LDH) assay and fluorescence cellular metabolism observations. These results demonstrate that LbL self-assembly provides an effective approach to apply films with nanometer thickness to silicone rubber. Such only few nanometer thick films are biocompatible with neurons, and may be used to coat devises for long-term implant in the central nervous system.

Animals↗

PubMed2001

Cholesterol biosensors prepared by layer-by-layer technique.

The analysis of formation, deposition and characterization of cholesterol oxidase (COX) layer-by-layer films were performed. Initially, a layer of polyanion, poly(styrene sulfonate) (PSS) was adsorbed followed by a layer of polycation, poly(ethylene imine) (PEI) on each solid substrate from aqueous solutions. The alternating layers were formed by consecutive adsorption of polycations (PEI) and negatively charged proteins (COX) and cholesterol esterase (CE). A strong interaction between protein and polyelectrolyte improves the stability of the alternating multilayer; however, it can change a native protein conformation and impair the protein activity. The PSS/PEI/COX, PSS/PEI/COX/PEI/CE, PSS/PEI/COX-CE/PEI etc. layered structures were prepared on the surface of a platinum electrode, ITO coated glass plate, quartz crystal microbalance, quartz plates, mica and silicon substrates. Optical and gravimetric measurements based on an ultraviolet-visible absorption spectroscopy and a quartz crystal microbalance revealed that the enzyme multilayers thus prepared consist of molecular layered of the proteins. The surface morphology of such bilayer films was investigated by using atomic force microscopy. The electrochemical redox processes of the enzyme-layered films deposited either on platinum or ITO coated glass plate were investigated. The response current of cholesterol oxidase electrode with concentration of cholesterol was investigated at length.

Biosensing Techniques↗

PubMed2004

Structure of beta-casein layers at the air/solution interface: atomic force microscopy studies of transferred layers.

We report the nanoscale structural changes associated with the interfacial gelation of adsorbed beta-casein layers as a function of aging time. Adsorbed layers were transferred to solid supports and imaged by atomic force microscopy. The aging of the layer was accompanied by the formation of distinct disk-shaped protein nanoparticles ( approximately 20 nm in diameter). Under conditions where a gelled layer was expected (from previous interfacial rheology experiments), we observed ordering of the particles and the formation of elongated aggregates or linear rows. Brewster angle microscopy images were also obtained during the adsorption and gelation processes and during the degradation of the protein layer following addition of the surfactant sodium dodecyl sulfate (SDS). If SDS was added prior to interfacial protein gelation, the layer developed a foamlike morphology consistent with a fluid interfacial protein layer. However, if SDS was added after gelation, the protein layer was observed to fracture, consistent with the behavior of a solid phase.

Air↗

PubMed2002

Haemoglobin oxygenation of a two-layer tissue-simulating phantom from time-resolved reflectance: effect of top layer thickness.

A dual wavelength time-resolved reflectance system was developed for monitoring haemoglobin saturation noninvasively. At each wavelength, the time-resolved reflectance data were fitted to a diffusion model of light propagation in a homogeneous, semi-infinite medium to yield the absolute scattering and absorption coefficients. The absorption coefficients were then used to calculate haemoglobin saturation. A two-layer phantom containing human erythrocytes in a scattering solution in the bottom layer was used to study system performance under more realistic conditions. The top layer was chosen to simulate either skin or fat and the oxygenation of the bottom layer, which corresponded to muscle, was controlled. The thickness of the fat layer was varied from 1.5 to 10 mm to investigate the effects of increasing the top layer thickness. These results, obtained with the simple diffusion model, were compared with simultaneous measurements of oxygenation made directly in the bottom layer. Errors in estimating haemoglobin saturation with this method ranged from 5-11% depending on the thickness of the top layer and its optical properties.

Hemoglobins↗

PubMed1994

Plasminogen activator production by the granulosa layer is stimulated by factor(s) produced by the theca layer and inhibited by the luteinizing hormone surge in the chicken.

The stages of follicular maturation of a preovulatory follicle in the hen can be divided into an extended proliferative phase (prior to LH surge) and a brief ovulatory phase (after LH surge). Previous studies suggest involvement of plasminogen activator (PA) in both the proliferative and ovulatory phases. The goals of the present study were 1) to determine whether PA production by granulosa and theca is dependent upon interaction of the two cell layers; 2) to investigate whether the structural difference of the stigma (site of follicular rupture) and nonstigma regions of the theca layer affect PA production; 3) to determine whether there is a change in the ability of the granulosa layer and stigma or nonstigma regions of the theca layer to produce PA as the follicle makes the transition from the proliferative to the ovulatory phase; and 4) to characterize the type(s) of PA produced by the hen follicle. Equal proportions of the granulosa layer (10-mm diameter) and stigma or nonstigma regions of the theca layer (10 mg) obtained from the F1 preovulatory follicle 8 h before ovulation (before LH surge) or 2 h before ovulation (after LH surge) were incubated alone or in combination for 24 h. PA was measured in tissue homogenates and medium by use of the chromogenic substrate S-2251. The granulosa layer or stigma or nonstigma regions of the theca layer incubated alone and obtained either 8 h or 2 h before ovulation had very low amounts of PA activity in the medium and tissue homogenates.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Immunohistochemical study of human eccrine sweat ducts with anti-keratin antibodies. Presence of a layer between luminal and peripheral cell layers.

It is known that human eccrine sweat ducts are composed of luminal cells and peripheral cells. In this study, the immunohistochemical staining properties of human eccrine sweat ducts were investigated using seven different anti-keratin antibodies by light microscopy. Anti-keratin antibody MA904, which reacts with 68-kDa keratin peptide specifically stained an intermediate cell layer between the luminal cell layer and the peripheral cell layer in the ducts. Anti-keratin antibody CK8,60 stained both the luminal cell layer and the intermediate cell layer. Anti-keratin antibody MA903 stained all of the layers. Anti-keratin antibodies CK4.26, PKK1, and MAK-6 weakly to faintly stained the luminal cells. Anti-keratin antibody PKK3 stained no cells in the ducts. These results suggest that each cell layer has its own characteristic staining pattern with anti-keratin antibodies. Moreover, the presence of an intermediate cell layer was confirmed by immunoelectron microscopy using anti-keratin antibody MA904.

Eccrine Glands↗

PubMed2004

4 layer versus 1 layer small intestinal submucosa for correction of penile chordee: experimental study in a rabbit model.

PURPOSE: We evaluated the use of 4 vs 1 layer small intestinal submucosa (SIS) for covering defects in the ventral surface of the tunica albuginea to correct severe chordee. MATERIALS AND METHODS: A total of 18 New Zealand white rabbits underwent implantation of a 10 x 5 mm SIS graft following excision of a rectangular area in the ventral surface of the tunica albuginea. In 9 rabbits 4 layer SIS was used to cover the defect and in the remaining animals 1 layer SIS was used. The animals were sacrificed at 2, 6 and 12-week intervals postoperatively, respectively. The surface area of the grafts was measured and the percent of contracture was calculated. Transverse sections of the penis at the graft site were stained with hematoxylin and eosin, and Masson's trichrome, and examined microscopically. RESULTS: : None of the animals had hematoma or bleeding. At autopsy contracture was not seen in any of the rabbits with 1 layer SIS. On the contrary, there was 21% and 25% contracture at 6 and 12 weeks, respectively, in the 4 layer SIS group. At 12 weeks the 1 layer SIS graft was completely replaced by well collagenized tissue similar to that of normal tunica albuginea without inflammatory infiltrate, while the multilayer SIS graft was replaced by dense fibrous tissue with areas of chronic inflammation and other focal areas of calcification. CONCLUSIONS: Four layer SIS undergoes contracture and calcification when used to cover defects in the tunica albuginea. On the other hand, 1 layer SIS can be safely and reliably used for corporeal grafting.

Animals↗

PubMed2006

Comparison of one-layer (continuous Lembert) versus two-layer (simple continuous/Cushing) hand-sewn end-to-end anastomosis in equine jejunum.

OBJECTIVE: To evaluate single and double layer end-to-end anastomosis in equine jejunum. STUDY DESIGN: Experimental in vitro study. ANIMALS: Mid-jejunal sections from 12 adult horses without gastrointestinal disease. METHODS: Jejunal end-to-end anastomoses were performed by a continuous Lembert pattern or a simple continuous pattern oversewn with a Cushing pattern. Jejunal segments were distended with fluid at 1 L/min, and intraluminal pressure at failure, and mode of failure were recorded. Bursting pressure and bursting wall tension were calculated. Anastomosis construction time and degree of luminal reduction were recorded. Results- Single layer anastomoses were constructed in less time than 2-layer anastomoses. Both anastomotic techniques resulted in luminal reduction compared with control tissue; however, the reduction was smaller with a 1-layer continuous Lembert anastomosis. No differences were noted in bursting pressure or bursting wall tension between groups. CONCLUSIONS: Anastomosis using a 1-layer continuous Lembert pattern resulted in a larger stoma, was faster to perform, and as strong as a 2-layer anastomosis. CLINICAL RELEVANCE: Use of a 1-layer continuous Lembert pattern for jejunojejunosotomy may be beneficial by decreasing anastomosis time and produce a larger stoma than a 2-layer anastomosis.

Anastomosis, Surgical↗

PubMed1987

Nerve fibre layer loss in diseases of the outer retinal layer.

We present examples of nerve fibre layer changes in diseases thought to affect primarily the outer retinal layers. These disease processes include cone-rod dystrophies, rod-cone dystrophies, juvenile macular degeneration (Stargardt's disease) and fundus flavimaculatis, vitelliform macular dystrophy, and Leber's congenital amaurosis. All were associated with alterations in the retinal nerve fibre layer, either diffuse or focal. The presence of these nerve fibre layer changes raises the question of transsynaptic degeneration and of possible primary or associated disease of other retinal cells than the receptors-namely, bipolar, amacrine, Mueller, or ganglion cells--in these disease of the outer retinal layer. Involvement of the nerve fibre layer also indicates the need to examine patients with defects in the nerve fibre layer by electroretinograms and other tests for outer retinal layer disease when no obvious optic nerve disease is present.

Adult↗

PubMed2006

Thermal stability of poly(o-methoxyaniline) layer-by-layer films investigated by neutron reflectivity and UV-VIS spectroscopy.

Neutron reflectivity measurements were used to investigate the thermal stability of layer-by-layer (LBL) films of poly(o-methoxyaniline) (POMA), which was probed by increasing the temperature up to 80 degrees C of a D2O solution in contact with the LBL films. The study was made possible by adsorbing POMA layers on a PEI/(PSS/PAH)5/PSS LBL film template, leading to less rough POMA layers in comparison with the POMA/poly(vinylsulfonic acid sodium salt) (PVS) LBL films adsorbed directly on glass and silicon substrates. While the latter yielded almost fringeless neutron reflectivity curves due to the large roughness, the fitting of the data for POMA films adsorbed onto the template film and UV-vis measurements indicated that the topmost layer is affected for films heated in solution up to 80 degrees C. This is essentially the same thermal stability of LBL films from the template films made with conventional polyelectrolytes. A decrease in thickness of approximately 10 A was inferred when the solution temperature increased from 25 degrees C to 80 degrees C, which was maintained when the sample was cooled back to 25 degrees C. This decrease, observed for solutions of pH 3 and pH 8, is consistent with thermally-stimulated desorption and was corroborated by UV-VIS absorption experiments. The unexpected stability of the POMA layer at pH 8 is attributed to the layer-by-layer structure of the films that allows POMA to remain doped, in its salt emeraldine form, even at high pH.

Aniline Compounds↗

PubMed2005

Activity and lifetime of urease immobilized using layer-by-layer nano self-assembly on silicon microchannels.

Urease has been immobilized and layered onto the walls of manufactured silicon microchannels. Enzyme immobilization was performed using layer-by-layer nano self-assembly. Alternating layers of oppositely charged polyelectrolytes, with enzyme layers "encased" between them, were deposited onto the walls of the silicon microchannels. The polycations used were polyethylenimine (PEI), polydiallyldimethylammonium (PDDA), and polyallylamine (PAH). The polyanions used were polystyrenesulfonate (PSS) and polyvinylsulfate (PVS). The activity of the immobilized enzyme was tested by pumping a 1 g/L urea solution through the microchannels at various flow rates. Effluent concentration was measured using an ultraviolet/visible spectrometer by monitoring the absorbance of a pH sensitive dye. The architecture of PEI/PSS/PEI/urease/PEI with single and multiple layers of enzyme demonstrated superior performance over the PDDA and PAH architectures. The precursor layer of PEI/PSS demonstrably improved the performance of the reactor. Conversion rates of 70% were achieved at a residence time of 26 s, on d 1 of operation, and >50% at 51 s, on d 15 with a six-layer PEI/urease architecture.

Adsorption↗

PubMed1999

Developmental switch in the short-term modification of unitary EPSPs evoked in layer 2/3 and layer 5 pyramidal neurons of rat neocortex.

Amplitudes of EPSPs evoked by repetitive presynaptic action potentials can either decrease (synaptic depression) or increase (synaptic facilitation). To determine whether facilitation and depression in the connections between neocortical pyramidal cells varied with the identity of the pre- or the postsynaptic cell and whether they changed during postnatal development, whole-cell voltage recordings were made simultaneously from two or three pyramidal cells in layers 2/3 and 5 of the rat sensorimotor cortex. Unitary EPSPs were evoked when pre- and postsynaptic neurons were in the same and in different layers. In young [postnatal day 14 (P14)] cortex, EPSPs evoked in all connected neurons depressed. The degree of depression was layer specific and was determined by the identity of the presynaptic cell. EPSPs evoked by stimulation of presynaptic layer 5 neurons depressed significantly more than did those evoked by stimulation of layer 2/3 neurons. In mature cortex (P28), however, the EPSPs evoked in these connected neurons facilitated to a comparable degree regardless of the layer in which pre- and postsynaptic neurons were located. The results suggest that in young cortex the degree of synaptic depression in connected pyramidal cells is determined primarily by whether the presynaptic cell was in layer 2/3 or 5 and that maturation of the cortex involves a developmental switch from depression to facilitation between P14 and P28 that eliminates the layer-specific differences. A functional consequence of this switch is that in mature cortex the spread of excitation between neocortical pyramidal neurons is enhanced when action potentials occur in bursts.

Animals↗

PubMed1989

[Intestinal metaplasia of the stomach--the significance of single-layer and double-layer metaplasia].

Resected early gastric carcinomas (1,690 cases) and atypical epithelial proliferations (adenomatous lesions, 310 cases) were investigated by the 5 mm-wide step sections. There were two types of intestinal metaplasia; double-layer type, where intestinal metaplasia was in the superficial area with the remaining pyloric or pseudopyloric glands in the deep region of the propria mucosae, and single-layer type, where intestinal metaplasia was usually present in a single layer without remaining deep non-metaplastic glands. These two types of intestinal metaplasia were usually found in the same specimen, however, most of the atypical epithelial proliferations (adenomatous lesions) arose from the area of intestinal metaplasia showing a double-layer type. The mitotic activity was usually found in the transitional zone showing these double-layer intestinal metaplasia. Atypical epithelial cells arose in the transitional zone of the double-layer intestinal metaplasia and spread into the luminal side by budding or replacement of existing epithelial cells. However, the author suspected that the cells in the under area of the transitional zone reproduce non-atypical cells to supplement the cells in the existing pyloric or pseudopyloric glands. On the other hand, atypical epithelial proliferations of single-layer type were rarely found showing a concaved appearance. Some of them may arise from the intestinal metaplasia of single-layer type, where the mitotic region moved towards the lower 1/3 of the glands. It seemed likely that most of the well-differentiated adenocarcinomas arise with intimate relation to these two types of intestinal metaplasia, where the incidence of malignant change of each type has been unknown.

Adenocarcinoma↗

PubMed2001

Layer-by-Layer Construction of Novel Biofunctional Fluorescent Microparticles for Immunoassay Applications.

A novel class of biofunctional fluorescent microparticles for application in immunoassays was constructed by using the layer-by-layer self-assembly method to deposit multiple layers of fluorescently labeled polyelectrolytes onto colloidal particles, followed by deposition of a protein (immunoglobulin G, IgG) layer. Microelectrophoresis experiments revealed alternating negative and positive zeta-potentials with deposition of each successive polyelectrolyte layer, indicating that the alternate electrostatic adsorption of polyelectrolytes of opposite charge was successfully achieved. Transmission electron microscopy images showed a change of the particle surface texture after polyelectrolyte multilayer deposition. Fluorescence microscopy image (FMI) analysis provided direct measurement of the fluorescence intensity of single microparticles. The observed systematic increase of the fluorescence intensity of individual microparticles with increasing polyelectrolyte layer number from FMI analysis further demonstrated the controlled regular adsorption of polyelectrolyte layers onto the polystyrene (PS) particles. Protein immobilization onto the polyelectrolyte multilayer-coated particles was verified by the different surface properties of the microparticles with respect to surface charge under pH conditions above and below the isoelectric point of the proteins. The assembly of IgG and fluorescein isothiocyanate-labeled IgG onto polyelectrolyte multilayer-coated PS microparticles and their potential use was ultimately confirmed by a solid phase immunotest. Copyright 2001 Academic Press.

Journal Article↗

PubMed1978

[Comparative study on single layer and two layer anastomoses of small intestine (author's transl)].

The one layer anastomosis after Gambee is compared with the two layer anastomosis after Dick at the small bowel of 20 minipigs. The end-to-end anastomosis after Gambee consists of an interrupted, inverting suturing of the bowel wall. Dick suggested a closed method, applying bronchial clamps before resecting the bowel. Seromuscular sutures and inner all layer sutures are inserted interrupted. Experimental studies have been done after 4, 7, 14 and 21 days. In Histology two layer anastomoses caused delayed wound healing after 4 days. Twenty-one days after the operation histological findings in both groups were equal. Microangiographic studies showed, that in single layer anastomoses revascularization began 3 days earlier than in two layer anastomoses. Measuring of the bursting strength showed no significant differences. All parameters suggest equal value of both techniques. In our opinion the two layer anastomosis after Dick combines the advantages of a closed method with a simple, easy performable technique. We therfore apply it all over the gastrointestinal tract with exception of esophagel and rectal anastomoses.

Animals↗