Thyroid hormone influence upon lung surfactant metabolism.

Thyroid administration and thyroidectomy in the rat profoundly affect the morphological characteristics of the type II pneumonocyte and the quantitative harvest of lung surfactant obtained from alveolar washings. The correlation of the ultrastructural changes with quantitative alterations in lung surfactant is evidence that the lamellar bodies within this cell are the source of the surface-active phospholipids utilized at the air-liquid interphase of lung alveoli. Our findings suggest that in the rat, L-thyroxine may be a potent regulator of lung surfactant metabolism.

Bioenergetic pattern of isolated type II pneumocytes in air and during hypoxia.

The bioenergetic pattern of a cell clone derived from rat lung with ultrastructural and biochemical characteristics like those of type II pneumocytes (T-II-P), has been studied in a tissue culture system. During air cultivation, these cells have a high rate of aerobic and anaerobic glycolysis associated with high activities of two rate-limiting enzymes in glycolysis (pyruvate kinase [PyKi] and phosphofructokinase [PFK]). This is present despite the rates of oxygen consumption and activities of cytochrome oxidase (CyOx) similar to other lung cells. Presumably the high rate of aerobic glycolysis explains the substantial lactate production previously described in lung slices and in the intact perfused lung. Hypoxic cultivation results in a decrease in CyOx. Acute re-exposure to air does not restore the oxygen consumption to normal, presumably as a result of decreased mitochondrial O(2) utilization associated with decreased CyOx activity. As a result, hypoxically cultivated T-II-P cells have a decreased capacity for mitochondrial ATP generation in air as compared to air-cultivated cells. During hypoxia, aerobic and anaerobic glycolysis are further increased as well as the activities of PyKi and PFK. The high rate of glycolysis and high activities of PyKi and PFK in cultivated T-II-P appear to reflect intrinsic genetic regulation. The decreased CyOx activity and increased PyKi and PFK activities in hypoxic T-II-P appear to reflect alterations in enzyme biosynthesis/biodegradation regulated by O(2) availability.

Involvement of cell surface heparin sulfate in the binding of lipoprotein lipase to cultured bovine endothelial cells.

It has been postulated that lipoprotein lipase, an enzyme important in the uptake of fatty acids into tissues, is bound to the vascular endothelial cell surface and that this binding occurs through attachment to heparinlike glycosaminoglycans. Furthermore, it is thought that heparin releases the enzyme from its attachment to the endothelium into the circulation. These hypotheses have never been tested directly in cell systems in vitro. In the present study we have directly evaluated the interaction of lipoprotein lipase, purified from bovine skim milk with monolayer cultures of endothelial cells, isolated from bovine pulmonary artery. Endothelial cells in primary culture had no intrinsic lipoprotein lipase activity but were able to bind lipoprotein lipase quantitatively. The binding reached equilibrium and was saturable at 0.24 nmol of lipoprotein lipase/mg of cell protein. The concentration of lipoprotein lipase at half-maximal binding was 0.52 microM. Bound lipoprotein lipase could be detached from cultured cells by increasing concentrations of heparin, and at and above 0.6 microgram/ml of heparin, 90% of the cell-bound lipoprotein lipase activity was released. Heparan sulfate and dermatan sulfate released the enzyme to a lesser extent and chondroitin sulfate caused little, if any, release of lipoprotein lipase. The release of lipoprotein lipase with heparin was not associated with a release of [3S]glycosaminoglycans from 35S-prelabeled cells. Reductions of lipoprotein lipase binding to endothelial cells and of cell surface-associated [3S]glycosaminoglycans in 35S-prelabeled cells occurred in parallel both when cells were pretreated with crude Flavobacterium heparinum enzyme before lipoprotein lipase binding and when cells were treated with this enzyme after lipoprotein lipase binding. The removal of heparan sulfate from the cell surface by purified heparinase totally inhibited the binding of lipoprotein lipase by endothelial cells, but the removal of chondroitin sulfate by chondroitin ABC lyase had no effect on this binding. These results provide direct evidence for lipoprotein lipase attachment to endothelial cells through heparan sulfate on the cell surface, and provide evidence for the release of lipoprotein lipase by heparin through a detachment from this binding site.

Prostaglandin production by type II alveolar epithelial cells.

Prostaglandin production was studied in fetal and adult type II alveolar epithelial cells. Two culture systems were employed, fetal rat lung organotypic cultures consisting of fetal type II cells and monolayer cultures of adult lung type II cells. Dexamethasone, thyroxine, prolactin and insulin, hormones which influence lung development, each reduced the production of prostaglandin E and F alpha by the organotypic cultures. The fetal cultures produced relatively large quantities of prostaglandin E and F alpha and smaller quantities of 6-keto-prostaglandin F1 alpha and thromboxane B2. However, prostaglandin E2 production was predominant. In contrast, the adult type II cells in monolayer culture produced predominantly prostacyclin (6-keto-prostaglandin F1 alpha) along with smaller quantities of prostaglandin E2 and F2 alpha. The type II cells were relatively unresponsive to prostaglandins. Exogenously added prostaglandin E, had no effect on cell growth, and only a minimal effect on cyclic AMP levels in the monolayer cultures.

Effect of dexamethasone upon surfactant phosphatidylcholine and phosphatidylglycerol synthesis in organotypic cultures of type II cells.

Organotypic cultures of pulmonary type II epithelial cells were treated with dexamethasone at concentrations between 10(-10) and 10(-5) M for 48 h followed by a 3 h incubation in 5.6 mM [U-14C]glucose. A surfactant and a residual fraction was isolated from the cultures by discontinuous sucrose gradient centrifugation. Phosphatidylcholine and phosphatidylglycerol were purified from each fraction and analyzed for total content. The specific activity of each phospholipid was measured as an index of the rate of synthesis. Dexamethasone treatment produced a dose-dependent increase in synthesis and content of surfactant phosphatidylcholine, with a maximum response occurring at 10(-6) M dexamethasone. At concentrations of 10(-5) M, dexamethasone ceased to produce a significant stimulation. Dexamethasone produced an increase in surfactant phosphatidylglycerol synthesis only at a concentration of 10(-8) M and higher. There was not a significant effect upon the content or rate of synthesis of phosphatidylcholine or phosphatidylglycerol in the residual fraction at any of the dexamethasone concentrations tested.

Stabilization of lipoprotein lipase by endothelial cells.

Lipoprotein lipase, purified from bovine milk, lost 90% of its activity when incubated in Hanks' balanced salt solution for 5 min at 37 degrees C. Bovine pulmonary artery endothelial cells, maintained in culture, markedly stabilized this enzyme. The stabilizing factor of endothelial cells was non-dialyzable, resistant to heating at 100 degrees C and to changes in pH, and unaffected by treatments of cells with proteolytic enzymes or with heparinase (Flavobacterium heparinum enzyme). However, the stabilizing effect on lipoprotein lipase was reduced by 60-70% by the extraction of cells with chloroform/methanol (2:1). The lipid extract of the cells stabilized the enzyme, suggesting that lipid component(s) of the endothelial cells account for their stabilizing effect. Since the endothelial cell is thought to be the site of action of lipoprotein lipase, stabilization of the enzyme by this cell may play a role in its preservation and function in vivo.

The frictional coefficients and associated wear resistance of novel low-shrink resin-based composites.

OBJECTIVES: Frictional forces play a major role in the oral wear process of dental resin-based composites (RBCs) and it would be of interest to consider how the energy from friction is dissipated at the material surface. Consequently, the micromechanical wear properties of conventional methacrylate compared with novel oxirane RBCs were assessed. METHOD: The frictional coefficient (mu), volume loss and Vickers hardness number (VHN) of oxirane (EXL596 and H1) and methacrylate RBCs (Z100 and Filtek Z250) were evaluated. Archard's wear equation was implemented to obtain the wear coefficient (K) and expressed as a 'fraction of friction' (K/micro) to indicate the dissipation of frictional energy that resulted in wear. Scanning electron microscopy (SEM) was used to qualitatively asses the wear facets of each RBC following 50000-cycles. RESULTS: The mean frictional coefficients observed between the oxirane and methacrylate RBCs were not significantly different (P > 0.05). However, the volume loss of EXL596 and H1 (5.9 +/- 0.4 and 4.7 +/- 0.3 x 10(-2) mm(3)) was significantly increased compared with Z100 and Filtek Z250 (1.7 +/- 0.2 and 2.3 +/- 0.3 x 10(-2) mm(3)). The VHN of EXL596 and H1 was either significantly greater (P = 0.021) or similar (P = 0.089) to Filtek Z250, respectively. An increase in K/micro was reported for EXL596 and H1 (34.7 +/- 4.1 and 22.8+ /- 2.4 x 10(-4)) compared with Z100 and Filtek Z250 (8.50 +/- 0.7 x 10(-4) and 8.62 +/- 1.0 x 10(-4)) (P < 0.05). SEM images of the oxirane RBCs exhibited increased surface fatigue and delamination of the surface layers compared with the methacrylate RBC specimens following 50,000-cycles. CONCLUSION: The significant decrease in wear resistance of the oxirane compared with methacrylate RBCs was unexpected since frictional coefficients and/or surface hardness were statistically similar. The decreased wear resistance of EXL596 and H1 compared with Z100 and Filtek Z250 was further explained by the increase in K/micro from wear theory and the associated increase in surface fatigue identified from SEM. The simplistic testing procedure combined with SEM utilized in the current investigation provided a greater insight into the wear mechanism by considering the effect of frictional energy at the specimen surface which may benefit the development of improved wear resistance for experimental RBC materials.

Glucocorticoid binding by isolated lung cells.

Synthesis of surfactant in the lung of fetal, and perhaps adult, animals responds to glucorticoids, and glucocorticoid receptor activity has been identified in this tissue of several sepecies. To determine whether receptor is present in the alveolar type II cell, which is the site of surfactant production, we studied glucocorticoid binding by various populations of lung cells. Specific binding was demonstrated in freshly isolated populations of rat lung cells containing primarily alveolar type II cells, in organotypic cultures derived from fetal rat lung containing 90% type II cells, in cultured A549, L-2, and F-42 cell lines which apparently originated from type II cells, and in human lung fibroblastic cells. The equilibrium dissociation constants for nuclear binding of dexamethasone by intact cells at 37 C ranged from 5.0--10.8 nM, and the number of binding sites per cell ranged from 5,700--57,000. In cytosol preparations from L-2 and A549 cells, there was equivalent specific binding of both natural and synthetic corticosteroids, and binding activity had the expected specificity for steroids with glucocorticoid activity. These findings indicate that glucocorticoid receptor is present in both fetal and adult pulmonary type II cells and in cell lines which apparently originated from these cells. The presence of receptor in type II cells in consistent with direct action of glucocorticoids on these cells in vivo.

Organotypic cultures of diploid type II alveolar pneumonocytes: surfactant associated esterase activity.

Organotypic cultures, established from enzymatically dispersed day 19 fetal rat lung, are comprised primarily of cells which are morphologically similar to type II alveolar pneumonocytes, the cells involved in surfactant synthesis. To further characterize these cultures, the nonspecific esterase pool was examined to determine if these cultures contained certain nonspecific esterases previously shown to be enzyme markers for the surfactant system. The results of biochemical, electrophoretic and cytochemical studies indicate that these organotypic cultures contain the same nonspecific esterases already demonstrated in surface active fractions derived from rat and mouse lung homogenates and pulmonary lavage fluid. As in whole lung, the major site of esterase activity in the organotypic cultures is the type II cell lamellar body, the putative site of surfactant synthesis and storage. These findings support the concept that the organotypic cultures derived from fetal rat lung are comprised predominantly of type II cells which retain surfactant associated functions in vitro.

Scanning electron microscopy of type I collagen at the dentin-enamel junction of human teeth.

The dentin-enamel junction constitutes a unique boundary between two highly mineralized tissues with very different matrix composition and physical properties. The nature of the boundary between the ectoderm-derived enamel and mesoderm-derived dentin is not known. This study was undertaken to identify the presence, type, and distribution of collagen at the dentin-enamel junction as an initial step in understanding its structural-functional role in dental occlusion. Sections of human teeth were demineralized with 0.1 M neutral EDTA and examined by high-resolution field-emission scanning electron microscopy at low accelerating voltage. Enamel and dentin were observed to be linked by many parallel 80-120-nm diameter fibrils, which were inserted directly into the enamel mineral and also merged with the interwoven fibrillar network of the dentin matrix. Immunogold labeling for collagen was visualized by secondary electron imaging and backscatter electron imaging at low accelerating voltage. The collagen fibrils at the junctional zone as well as in the dentin matrix were identified as Type I collagen. Collagenase digestion led to loss of the fibrillar structures and prevented immunogold labeling with antibody specific to Type I collagen. Consequently, the dentin-enamel junction can be regarded as a fibril-reinforced bond which is mineralized to a moderate degree.

Isolation of cells that retain differentiated functions in vitro: properties of clonally isolated type II alveolar pneymonocytes.

We have isolated byclonal culture techniques a diploid cell strain (L-2) from adult rat lung. These cells appear to retain differentiated functions that are present in type II alveolar epithelial cells of intact lung. The L-2 cells are diploid, epithelial cells, they contain osmiophilic lamellar bodies in their cytoplasm and they synthesize lecithin by the same de novo pathways as whole lung.

Comparison of surface layer properties of composite resins by ESCA, SEM and X-ray diffractometry.

Hyperfine surface layer properties of three types of dental composite resins, highly-filled, conventional, and micro-filled resins, were studied using X-ray photoelectron spectroscopy (ESCA) by a combination of X-ray diffractometry and scanning electron microscopy (SEM). X-ray diffraction and SEM analysis showed clearly that each resin has different characteristics of SiO2 particle size and distribution. ESCA depth resolution with argon ion etching indicated that, in contrast to conventional and microfilled resins, carbon due to polymers of highly-filled resin decreases dramatically with increasing depth in the nanometer range of the resin-rich surface layer.

Comparison of the porosity of hand-mixed and capsulated glass-ionomer luting cements.

The strength of dental glass-ionomer cements will be influenced by defects present within its structure. This study measured the surface area porosity, percentage surface area porosity, and mean surface area of small bubbles (<0.01 mm2) and the surface area porosity, percentage surface area porosity and diameter of large bubbles within 40-microm-thick layers of four cements, using image analysis software. Two hand-mixed cements (Fuji I and KetacCem) and two capsulated cements (Fuji Cap I and KetacCem Maxicap) were viewed under transmitted light at x117.6 magnification. For each selected area (64.75 mm2) of each cement sample, five independent measurements were made of each of these parameters. Analysis of variance (ANOVA) indicated that there were no significant differences between the four cements in the small bubble parameters measured, whilst there were significant differences in the surface area porosity, percentage surface area porosity and diameter of the large bubbles. It was concluded that the hand-mixed cements tested had a greater number of larger diameter bubbles compared with the capsulated cements.

The metal oxide/eugenol cements. II. A diffuse reflectance spectrophotometric study of the setting of zinc oxide and magnesium oxide cements.

In a continuing study, spectrophotometry has been used to study the diffuse reflectance of the setting of ZnO and MgO cements.

The metal oxide/eugenol cements. I. The chelating power of the eugenol type molecule.

The chelating power of the orthomethoxy phenolic (guaiacyl) group is fundamental to the accepted setting reaction of the metal oxide/eugenol cements. In this report, the chelating power is quantitatively described by the sequence of beta2 for the chelates of the relevant metal ions.

Tear strength of non-aqueous impression materials.

Examples from each of the four major groups of dental impression materials (polysulfides, polyethers, condensation polysiloxanes, and addition polysiloxanes) were subjected to a trouser-leg tear test. Tear energies were determined, and materials were ranked with respect to each other. Polysulfides displayed the highest tear energy, while condensation polysiloxanes were the lowest, with the addition polysiloxanes occupying an intermediate position.

Structure-property relations and crack resistance at the bovine dentin-enamel junction.

The present report is a study of the fracture behavior of the dentin-enamel complex, involving enamel, dentin, and the dentin-enamel junction (DEJ), that combines experimental design, computational finite element analysis, and fractography. Seven chevron-notched short-bar bovine DEJ specimens were utilized in this study. The general plane of the DEJ was approximately perpendicular to the fracture plane. All specimens were stored at 37 degrees C and 100% relative humidity for 24 h prior to being tested. A fracture test set-up was designed for application of tensile load on the DEJ specimens to initiate a crack at the vertex of the chevron in the enamel, across the DEJ zone and into the bulk dentin. During fracture testing, a water chamber was used to avoid dehydration of the specimen. The results showed that the lower boundary value of the fracture toughness of the DEJ perpendicular to its own plane was 3.38 +/- 0.40 MN/m1.5 and 988.42 +/- 231.39 J/m2, in terms of KIC and GKC, respectively. In addition, there was an extensive plastic deformation (83 +/- 12%) collateral to the fracture process at the DEJ zone. The fractography revealed that the deviation of the crak path involved an area which was approximately 50-100 microns deep. The parallel-oriented coarse collagen bundles with diameters of 1-5 microns at the DEJ zone may play a significant role in resisting the enamel crack. This reflects the fact, that in the intact tooth, the multiple full thickness cracks commonly found in enamel do not typically cause total failure of the tooth by crack extension into the dentin.

Development of an artificial oral environment for the testing of dental restoratives: bi-axial force and movement control.

The integration of two closed mechanical loops was used to produce a force-movement cycle, using servo-hydraulics. Several of the parameters were of interest in clinically-simulated laboratory studies. The system represented the first phase in developing an artificial oral environment.

Why do shear bond tests pull out dentin?

It is widely accepted that a dentin shear bond test which pulls out dentin must mean that the adhesive strength is superior to the cohesive strength of the dentin. Using numerical modeling techniques, Van Noort et al. (1988, 1989) and DeHoff et al. (1995) alerted the scientific community that there were massive stress concentrations in the familiar dentin bond test. It is not inconceivable that these localized high tensile stresses could initiate cracks which diverge monolithically into dentin, leaving the interface unchallenged. To test this hypothesis, we developed a failure accumulation simulation program which determined localized failure interactively "on the fly" with a finite element solver, and also included brittle behavior, adhesive and cohesive failure, stochastic response, and dynamic remeshing. All of the familiar dentin bond variables were included in the simulation. A parallel experimental dentin bond test validation was run, and the fractography was examined in the scanning electron microscope for mode of failure. The simulation confirmed the tensile monolithic fracture hypothesis. It is also confirmed that dentin pull-out was partly due to the biomechanics of the test and did not necessarily mean superior adhesive strength or even that the cohesive strength of the dentin was reduced. There is clear need for a new technology for the evaluation of biological interfaces, and the present work has shown the vital role of numerical modeling in the interpretation of such experimental procedures.