Regulation of RAW264.7 macrophage polarization on smooth and rough surface topographies by galectin-3.

Recognition of topographical features induces phenotypic changes in macrophages although the receptors and signaling pathways are not completely characterized. As integrin molecules in focal adhesions/podosomes are in intimate contact with topography and topography modulates the NFkB pathway through cholesterol enriched raft-associated adhesive signaling structures we hypothesized that a cell-surface signaling complex comprised of galectin-3 together with its ligand CD98 and integrinß1 is important for topography-directed lineage determination. This study used polished, sand blasted and acid etched (SLA) surfaces and two novel grooved topographies (G1 and G2) produced by anisotropic etching of Si <1 1 0> to evaluate the role of galectin-3 in macrophage polarization in RAW 264.7 macrophages, as determined by gene expression and morphology. In the presence of the galectin-3 inhibitor, lactose, the M2 marker (mannose receptor) was down-regulated while the M1 marker (iNOS) was up-regulated on smooth and rough surfaces. This skewing of phenotype suggests a role for galectin-3 in macrophage polarization towards the M2 phenotype. Additionally, we evaluated the role of PI3K on polarization using PI3K inhibitor LY294002. We found that the M2 marker was down-regulated on both PO (surface polished) and G1 surfaces implicating PI3K in lineage determination. We also found that surface topography altered cell morphology; macrophages had a larger area on G2 surfaces. Lactose treatment significantly reduced the cell area on all topographies suggesting that the galectin-3 is also involved in signaling complexes triggering the rearrangement of the actin cytoskeleton. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2499-2509, 2017.

Synergistic interaction of topographic features in the production of bone-like nodules on Ti surfaces by rat osteoblasts.

The objective of this study was to study the responses of osteoblast-like cells to rough Titanium (Ti)-coated epoxy surfaces of differing topographic complexity. Four topographies were studied: polished (PO), coarse-blasted (CB), acid-etched (AE) and coarse-blasted+acid-etched (SLA). Rat osteoblasts were cultured on these surfaces and their morphology, thickness as well as the number and size of bone-like nodules measured. To determine cell shape and cell thickness, fluorescein-5-thiosemicarbazide was used to stain the cell components including the cell membrane, the stained cells were optically sectioned using epifluorescent microscopy and the optical sections were computationally reconstructed to obtain three-dimensional images in which cell volume and cell thickness could be determined. Similarly optical sections of bone-like nodules labeled with tetracycline were also reconstructed to determine their size. The different surface topographies were found to alter the thickness and morphology of osteoblasts cultured on these surfaces. Osteoblasts produced significantly more and larger nodules on SLA compared to other surfaces. Nevertheless and perhaps surprisingly, given the evidence in various cell populations that cell shape can affect cell differentiation, cell thickness was not directly correlated with an increase in bone-like nodule formation. Data were analyzed by factorial analysis of variance. In this way the primary effect of each surface treatment ( i.e. blasting and acid etching) could be assessed as well as their interaction. Both the acid etching and blasting processes significantly affected the number and size of bone-like nodules cultured on Ti surfaces. Moreover there were significant interaction effects indicating that surface topographic features can act synergistically to enhance bone formation. This result suggests that a useful approach to the optimization of surfaces for bone production could involve systematic investigation of combinations of processes each of which produces distinct surface topographical features.

A PLGA membrane controlling cell behaviour for promoting tissue regeneration.

Barrier membranes are used in periodontal applications with the aim of supporting bone regeneration by physically blocking migrating epithelial cells. We report a membrane design that has a surface topography that can inhibit epithelial cell migration and proliferation on one side and a topography that guides osteoblast migration to a desired area. A PLGA copolymer (85:15) blended with MePEG, was cast to have surfaces with smooth, grooved or sandblasted-acid-etched topographies. Epithelial cells spread on smooth surfaces after 24 h, and cell numbers increased after 5 days. Cells on the smooth surface exhibited no preferred direction of migration. On the sandblasted-acid-etched topography epithelial cells spread but the cell number did not significantly increase after 5 days. Cell migration was inhibited on this surface. Osteoblasts spread on both grooved and smooth surfaces and cell number increased after 5 days on all surfaces. The cells that adhered in the grooves migrated preferentially in the direction of the grooves. Positive alkaline phosphatase staining was seen on all surfaces within 4 weeks and positive Von Kossa nodule staining within 6 weeks. These results suggest that surface topographies replicated on opposite sides of a biodegradable polymers membrane can inhibit proliferation and migration of the epithelial cells, and promote proliferation and directional migration of osteoblasts. Addition of appropriate surface topographies to membranes used in guided tissue regeneration has the possibility of improving clinical performance in periodontal tissue regeneration procedures.

Substratum roughness alters the growth, area, and focal adhesions of epithelial cells, and their proximity to titanium surfaces.

Epithelial (E) cells were cultured on smooth tissue culture plastic (TCP), TCP-Ti, polished Ti (P), and rough grit-blasted Ti (B), acid-etched Ti (AE), and grit-blasted and acid-etchedTi (SLA) surfaces and their growth, area, adhesion, and membrane-Ti proximity assessed. Rough surfaces decreased the growth of E cells compared to smooth surfaces in cultures up to 28 days. In general rough surfaces decreased the spreading of E cells as assessed by their area with the most pronounced affect for the SLA surface. On the other hand, the strength of E cells adhesion as inferred by immunofluorescence staining of vinculin in focal adhesions indicated that E cells formed more and larger focal adhesions on the smooth P surface compared to the rougher AE surface. As this finding indicates a stronger adhesion to smooth surfaces, it is likely that E cells on rough surfaces are more susceptible to mechanical removal. An immunogold labeling method was developed to visualize focal adhesions using back-scattered electron imaging with a scanning electron microscope (SEM). On rough surfaces focal adhesions were primarily localized on to the ridges rather than the valleys and the cells tended to bridge over the valleys. Transmission electron microscopy (TEM) measurements of membrane proximity to the Ti surface indicated that average distance of cell to the Ti increased as the Ti surface roughness increased. Therefore, the size and shape of surface features are important determinants of epithelial adhesive behavior and epithelial coverage of rough surfaces would be difficult to attain if such surfaces become exposed.

Parathyroid hormone- and prostaglandin E1-response in a selected population of bone cells after repeated subculture and storage at -80C.

Five different cell populations, designated I to V, were isolated from minced newborn rat calveria by 5-sequential 20 min incubations with an enzyme mixture containing collagenase, elastase and DNAse. In primary culture, all five populations responded to parathyroid hormone (PTH) to a different degree, population IV giving the highest increase in cyclic-3'5'-adenosine monophosphate (cAMP) level. None of the five populations gave any response to calcitonin. Upon subsequent subcultures, all populations, except population IV, either lost or considerably decreased their response to PTH. Population IV gave a two to three-fold increase in cAMP concentration in response to PTH up to the third subculture. No morphological differences could be observed among the five populations. The third subculture of population IV cells that had been stored in 10% glycerol at -80C for four months was subsequently thawed and subcultured to the sixth subculture. These cells still responded to PTH with an increase in cAMP level. In a second experiment, 5 different cell populations designated I to V were isolated in a similar way by incubation with collagenase and DNAse. The maximum response to PTH was found in population 3. The preservation of the PTH-responsiveness of this population, after subculturing, freezing, storing in 10% glycerol at -80 C and subsequent subculturing, was likewise demonstrated. The hormone-responsiveness of cells from the sixth subculture of previously frozen and thawed population IV cells was further analyzed. These cells responded to PTH at a concentration of 0.1 U/ml to 5U/ml and to prostaglandin E1 (PGE1) at a concentration of 0.1 microng/ml to 10 microng/ml. The time course of action on population IV of PTH was found to be different from that of PGE1, suggesting a possible difference in the regulation of intracellular cAMP levels by these hormones.

Observation of fibronectin distribution on the cell undersurface using immunogold scanning electron microscopy.

Immunogold staining followed by observation with scanning electron microscopy (SEM) has been quite effective in showing the distribution of proteins on dorsal cell surfaces. However, observation of proteins on the ventral cell surface using SEM has not been developed to the same extent. In this study, human gingival fibroblasts cultured on titanium-coated wafers were embedded in resin. After fracturing the wafers off the embedded cells, the undersurface of the cell was exposed by argon gas glow discharge etching. After 15 min of glow discharge etching, the resin covering the cell undersurface was completely removed. The distribution of fibronectin (FN) on the cell undersurface was demonstrated using an anti-FN antibody and colloidal gold (30 nm) conjugated with IgG. The undersurface was then coated with carbon or gold-palladium and observed by SEM. Using backscattered electron detection, gold beads could be identified in high contrast. On cells cultured for 5 hr, gold beads were distributed randomly on the entire cell undersurface. However, a line of gold beads was sometimes observed close to the edge of the cell. These results indicated that this immunogold/SEM etching method provides a powerful means for studying cell adhesion molecules on the cell undersurface. (J Histochem Cytochem 47:1487-1493, 1999)

Impact of the electrocardiogram on the delivery of thrombolytic therapy for acute myocardial infarction.

The initial electrocardiogram is crucial in accurately selecting patients with chest pain for thrombolytic therapy. An electrocardiogram with a large amount of ST-segment elevation and depression is "visually alarming," and therefore, may influence the efficiency of patient treatment with thrombolytic therapy. It was hypothesized that the amount of ST-segment deviation present on the initial electrocardiogram was an important variable in determining the time to initiation of thrombolysis in the emergency department. The time from arrival at the emergency department to thrombolysis was measured in 93 consecutive patients with suspected acute myocardial infarction (AMI) who were treated with intravenous thrombolytic therapy by emergency department physicians. This was correlated with the sum of ST-segment elevation and depression present on the initial electrocardiogram. AMI was proved in 83 patients (89%). In patients with proved AMI, the average time to thrombolysis was 50.8 +/- 25.6 minutes. Treatment began within the goal of < or = 30 minutes in 18 patients (22%) and was excessively delayed at > or = 60 minutes in 24 (29%). Regression analysis of multiple clinical variables revealed that ST-segment sum was the only variable that significantly influenced the time to thrombolysis (r = -0.42; p < 0.001). For patients treated in < or = 30 minutes, the average ST-segment sum was 21.1 +/- 13.5 vs 11.5 +/- 11.4 mm for those treated in > or = 60 minutes (p = 0.01). In 10 patients mistakenly treated with thrombolytic therapy, the electrocardiographic processes responsible for ST-segment elevation included the early repolarization variant, left ventricular hypertrophy, old anterior AMI with persistent ST-segment elevation, and conduction delay.(ABSTRACT TRUNCATED AT 250 WORDS)

Impact of a thrombolysis research trial on time to treatment for acute myocardial infarction in the emergency department.

Because time to treatment in AMI is a critical factor in long-term outcome, it is important that complex trials designed to improve reperfusion therapy do not delay the time to treatment. Participation in the TIMI 5 trial did not significantly prolong our door-to-needle time. These results indicate that, if done carefully, complex, labor-intensive studies can be performed within a reasonable time limit. Care should be taken to design protocols incorporating easy drug preparation, informed consent by the ED, and efficiency of trial initiation.

Chemically patterned, metal oxide based surfaces produced by photolithographic techniques for studying protein- and cell-surface interactions I: Microfabrication and surface characterization.

Chemical patterns on smooth wafer substrates comprising areas with two different metals have been produced by vacuum metal deposition and photolithographic techniques. The combination of metals has been chosen from the series titanium (Ti), aluminium (Al), vanadium (V) and niobium (Nb), producing patterns (dots and stripes with dimensions of 50, 100 and 150 micrometer) with one of the metals as the background and with the second metal (foreground pattern) deposited on the background metal. The structure and chemical composition of the patterned surfaces were evaluated by scanning electron microscopy, X-ray photoelectron spectroscopy and imaging time-of-flight secondary-ion mass spectrometry. The surfaces proved to be geometrically well defined with the expected surface-chemical composition, i.e. a surface oxide (passive) film essentially composed of TiO(2),Al(2)O(3),V(2)O(5), or Nb(2)O(5). Ti/Ti patterned surfaces were produced as controls and found to show no chemical composition contrast. The surface roughness of the pattern was greater than that of the background by a factor of 2-3, but was still extremely smooth with Ra<2nm. The patterns serve as model surfaces for studying in vitro the behaviour of cells as well as the adsorption of serum proteins on different metal oxides, which will be reported in a companion paper. These surfaces can be used to compare and contrast the response of osteoblasts to Ti and other alloy components, such as Al, V, or Nb, which are used in load-bearing medical implants.

Chemically patterned, metal-oxide-based surfaces produced by photolithographic techniques for studying protein- and cell-interactions. II: Protein adsorption and early cell interactions.

Protein adsorption and adhesion of primary human osteoblasts on chemically patterned, metal-oxide-based surfaces comprising combinations of titanium, aluminium, vanadium and niobium were investigated. Single metal samples with a homogeneous surface and bimetal samples with a surface pattern produced by photolithographic techniques were used. The physical and chemical properties of the samples have been extensively characterised and are presented in a companion paper. Here, we describe their properties in terms of cell responses during the initial 24h of cell culture. Regarding the cell number and activity there was no significant difference between any of the single metal surfaces. However the morphology of cells on vanadium surfaces became spindle-like. In contrast to the behaviour on single metal samples, cells exhibited a pronounced reaction on bimetallic surfaces that contained aluminium. Cells tended to stay away from aluminium, which was the least favoured metal in all two-metal combinations. An initial cell alignment relative to the pattern geometry was detectable after 2h and was fully developed after 18h of incubation. The organisation of f-actin and microtubules as well as the localisation of vinculin were all more pronounced on non-aluminium regions. We hypothesised that the differences in cell response could be associated with differences in the adsorption of serum proteins onto the various metal oxides. Protein adsorption experiments were performed using microscopy in conjunction with immunofluorescent stains. They indicated that both fibronectin and albumin adsorption were significantly greater on the non-aluminium regions, suggesting that differences in cellular response correlate with a modulation of the concentration of serum proteins on the surface.

Expression of fibronectin and integrins in cultured periodontal ligament epithelial cells.

The process of attachment of epithelial cells obtained from the porcine periodontal ligament (cell rests of Malassez) to different extracellular matrix proteins and their expression of fibronectin and integrin receptors were studied by means of immunocytochemistry, in situ hybridization, and time-lapse cinemicrography techniques. The cell lines of periodontal ligament epithelial cells (PLE cells) attached to and spread rapidly on fibronectin, vitronectin, and type I collagen. One of the cell lines also attached to laminin, while the other cell line showed poor attachment to both laminin and Matrigel, a basement membrane material. By use of the in situ hybridization technique, some PLE cells were found to express the fibronectin gene strongly. Immunocytochemical staining localized fibronectin in extracellular fibrils and intracellular granules. Fibronectin was also found in the tracks left behind by the cells migrating on the substratum. Arg-gly-asp-ser peptide inhibited the attachment of the PLE cells to fibronectin, laminin, type I collagen, and vitronectin by 47%, 43%, 83%, and 94%, respectively, suggesting that the cell-matrix interactions were partly mediated by receptors related to the integrin family. Antibodies against the beta 1-integrin subunit stained the cell bodies and the plasma membrane projections of spreading cells. After 24 h or longer in culture, beta 1-integrins were localized to the regions of cell-cell contact. Cinemicrography of the arg-gly-asp-ser-peptide-treated cells demonstrated that the spreading and migration of isolated cells were prevented by the peptide. The peptide did not appear to dissociate the cell-cell contacts or interfere with migration of spread-cell colonies.(ABSTRACT TRUNCATED AT 250 WORDS)

Grooved titanium surfaces orient growth and migration of cells from human gingival explants.

A silicon mask-etching technique was used to prepare grooved surfaces that control the direction of outgrowths of human gingival explants. The method used to produce the grooves is excellent in terms of both the uniformity of the grooves and the control with which surfaces of the desired specifications can be obtained.

Cerebral resuscitation after cardiac arrest using hetastarch hemodilution, hyperbaric oxygenation and magnesium ion.

This study was done to investigate the effects of hemodilution, hyperbaric oxygenation, and magnesium sulfate on cerebral resuscitation. Sixteen mongrel dogs were anesthetized, and monitored via pulmonary artery catheter, arterial catheter and electrocardiogram. A left lateral thoracotomy was done. Ventricular fibrillation was obtained by application of a 6-volt AC current. Mechanical ventilation was stopped. Total arrest time was 12 min. All dogs were cardiac resuscitated within 6 min using internal massage, ventilation, bicarbonate, epinephrine and internal defibrillation. The animals were then randomized into three groups. Group I represented controls, and were not treated. Group II dogs received normvolemic hemodilution using hetastarch (Hespan) containing magnesium sulfate (2000 mg/l), resulting in a hematocrit of 20%-30%. Group III dogs received the above hemodilution plus compression in a hyperbaric oxygen chamber to 2 atmospheres absolute. Critical care management and hourly neurologic scoring was performed for 7 days by blinded observers. All dogs at the time of death underwent autopsies for gross study. Data analysis revealed no statistical difference among the three groups with respect to survival time, cardiac function or neurologic scoring.

Effects of titanium-coated micromachined grooved substrata on orienting layers of osteoblast-like cells and collagen fibers in culture.

Osteogenic cells from newborn rat calvariae were cultured on titanium surfaces on which cell orientation could be manipulated. Substrata included smooth surfaces and substrata with smooth regions (gaps) flanked by grooves of 47-microm pitch and 3-, 10-, or 30-microm depth. Orientation angles of the cells were measured over time using propidium-iodide staining and confocal laser scanning microscopy. In addition, collagen fibers were identified using picro-sirius staining and reflected light polarization microscopy. Grooves proved effective in orienting cells, but their orienting ability decreased above the ridge level. Cells on the smooth surface showed no preferred orientation. Cells in the gaps became oriented as a result of cell-cell interactions with the cells on the flanking grooves. Cells in grooves produced oriented collagen fibers, but in the gaps, fibers could be parallel, perpendicular, or diagonal to the grooves. Collagen fibers on the smooth surfaces formed arrays of parallel fibers in a crisscross pattern. In long-term cultures, bone-like nodules were formed, but mostly above the ridge level. These data demonstrate that grooved surfaces can influence cell orientation both in cell populations above the cells in contact with the grooves and in cell populations adjacent to the grooves.

The interactions of benzo(a)pyrene with cell membranes: uptake into Chinese hamster ovary (CHO) cells and fluorescence studies with isolated membranes.

The interactions of benzo(a)pyrene (B(a)P) with the cell surface membrane were studied by measuring B(a)P uptake into intact mammalian cells and by determining B(a)P fluorescence in the presence of isolated cell surface membranes. It was found that 0.19 mu-g B(a)P were taken up by 10-6 Chinese hamster ovary (CHO) cells after 30 min exposure to a solution containing 0.59 mu-g/ml. Culture conditions were found to markedly alter B(a)P uptake. Low cell culture densities resulted in a four-fold increase in rate of B(a)P uptake per cell relative to confluent monolayer cultures. The uptake rate of B(a)P was reduced in the presence of bovine serum (BS) and, under some conditions, perylene. This information should be considered in the design of experiments on the biological effects of B(a)P. Another aspect of B(a)P membrane interaction was that the binding of B(a)P to cell surface membranes could be measured by fluorescence. The additional B(a)P fluorescence, found in the presence of cell surface membranes, was sufficiently large that the methods of data treatment used in the study of fluorescent probe-membrane interactions could be applied to get quantitative information on B(a)P-membrane interactions. It was found that 0.6 x 10-8 moles B(a)P were bound per mg membrane protein and that the apparent statistical dissociation constant for the complex was 3.8 x 10-7 M. The data suggest that the mechanism of uptake of B(a)P is probably passive diffusion.

Cholera toxin and dibutyryl cyclic-AMP stimulate the growth of epithelial cells derived from epithelial rests from porcine periodontal ligament.

The epithelial cells (E-cells) grew best at high (greater than 5 per cent) concentrations of fetal bovine serum (FBS). Growth could be obtained at low concentrations of dialysed FBS (DFBS) if the medium (alpha-MEM) was modified so that the levels of Ca2+ and K+ were reduced to 0.1 and 1.0 mM, respectively (beta-MEM). The addition of 0.5 per cent DFBS to the beta-MEM did not initiate good growth but was sufficiently supportive to enable the effects of various growth promoters to be tested. Cholera toxin and dibutyryl cyclic-3'5'-adenosine monophosphate (Bt2cAMP), which are known to increase intracellular cAMP levels, at concentrations of 1 ng/ml and 0.5 mM, respectively increased cell number. Cholera toxin caused the E-cells to be more flattened when viewed by phase-contrast; this appeared to be due to spread of the cells. No difference in cell-size distributions obtained between the trypsinized E-cells grown in the presence or absence of cholera toxin was observed. Epithelial proliferation that occurs in dental cysts could result from a rise in intracellular cAMP levels in epithelial cell rests.

Wavelength-dependent roughness: a quantitative approach to characterizing the topography of rough titanium surfaces.

Topographies of grit-blasted, etched, grit-blasted and etched, and microfabricated and etched surfaces of commercially pure titanium have been investigated. Such surface topographies vary across the scale range of interest for dental implants, extending from nanometers to millimeters. The complete characterization of topography requires the use of complementary methods. This study compared the topographic characterization methods of non-contact laser profilometry, interference microscopy, stereo-scanning electron microscopy (stereo-SEM), and atomic force microscopy. Non-contact laser profilometry was shown to be a useful method to characterize topographic features in the micron to millimeter range, whereas interference microscopy and stereo-SEM can be employed down to the submicron range. Stereo-SEM is particularly useful for quantifying topographies with complex, strongly corrugated ("sharp"), and high-aspect-ratio features and was shown to be complementary to non-contact laser profilometry and interference microscopy. Because of tip-related envelope problems, atomic force microscopy was not found to be suitable for the type of surfaces investigated in this study. Independent of the method used, the commonly used "integral" amplitude roughness parameters, such as Ra, Rq, or Rt, were often of limited value in the description of actual implant surfaces. The application of the wavelength-dependent roughness approach was shown to be an effective method for the description of surface topographies in the complete range of characteristic roughness and is also a useful means of examining the effects of surface treatment processes.

Neuro-ophthalmologic emergencies.

This article is designed to provide the emergency physician with the knowledge necessary for the diagnosis and management of the more common neuro-ophthalmologic emergencies. Emphasis is placed on the recognition and initial evaluation of neuro-ophthalmologic disease.

The effects of dentifrice systems on oral malodor.

Chronic oral malodor is a serious concern for about one-fifth of the North American population, and a field of emerging research interest. The present three studies, one involving gas chromatography and two employing odor judge assessment, examined the efficacy of baking soda and other toothpastes in reducing breath odor. The most common cause of oral malodor is elevated levels of volatile sulfur compounds (VSC's), primarily hydrogen sulfide (H2S) and methyl mercaptan (CH3SH), in the breath. Gas chromatography, an accurate means of measuring breath VSC, was employed to evaluate the breath levels of VSC in 11 men after brushing with baking soda-containing dentifrices with or without the addition of Zn++. Dentifrices with either Zn++ or a concentration of baking soda 20% or greater significantly reduced VSC levels. The addition of Zn++ to baking soda dentifrices enhanced the anti-odor effects. In the first organoleptic study, dentifrices containing 20% baking soda and 30% baking soda demonstrated significantly greater ability to reduce breath odor than a standard sodium fluoride/silica dentifrice. The subjects' baseline mouth odor evaluations, initially rated as strong, declined after brushing with the baking soda toothpastes to a barely detectable level at one hour, then rising to a faint level at two hours and moderate levels at three hours. In the second organoleptic study, a dentifrice containing 65% baking soda demonstrated significantly greater ability to reduce breath odor than a standard sodium fluoride/silica tartar control dentifrice, but did not differ significantly from a standard dentifrice containing 0.76% sodium monofluorophosphate in a dicalcium phosphate dihydrate base. The results of these studies indicate that dentifrices containing 20% or more baking soda can confer a significant odor-reducing benefit for time periods up to three hours.