Ultrastructural study of the effects of cytochalasin D administration on the structure and acid trimetaphosphatase activity of osteoclast.

To determine the role of the cytoskeleton consisting of the microfilaments in osteoclasts, cytochalasin D (CD) conjugated with PBS was administered intravenously to rats at a dose of 1 mg/100 g b.w. Control rats were given only PBS. Then the osteoclasts in the metaphyses of the humeri were examined ultrastructurally, as well as acid trimetaphosphatase (TMPase) cytochemistry. The plasma calcium (Ca) concentrations before and after CD and calcitonin administration were also measured. CD administration first caused a prominent reduction of ruffled borders and spreading of clear zones associated with the occurrence of large pale vacuoles in adjacent cytoplasm. At 1 hr after CD administration, the osteoclasts mostly lacked both ruffled borders and clear zones, but still maintained a normal intracellular organization of cytoplasmic organelles. Morphometric analysis confirmed that CD administration resulted in about 70% reduction of both ruffled border and clear zone areas. TMPase secretion from osteoclasts towards the resorbing bone surfaces was strongly inhibited by CD administration. At 1 hr after administration, although CD caused a decrease of plasma Ca concentrations in 4 of 7 examined rats similar to that caused by calcitonin treatment, there was a slight increase of plasma Ca concentrations in the other 3 rats. These results suggest that the structure and bone-resorbing function of the ruffled border-clear zone complex of the osteoclast is highly regulated by cytoskeleton consisting of microfilaments containing F-actin.     

Osteoclast differentiation at growth plate cartilage-trabecular bone junction in newborn rat femur

Using 3-day-old newborn rats, we examined the differentiation processes of osteoclasts associated with the destruction of the femoral growth plate cartilage and primary trabecular bone. In the growth plate cartilage, thin mineralized areas were detected solely in the longitudinal septal cartilage matrix in the hypertrophic zone, but the transverse septal cartilage matrix between adjacent chondrocytic lacunae within a row of chondrocytes remained unmineralized. The longitudinal septal cartilage between adjacent rows of chondrocytes appeared to persist, forming the walls of opened lacunar canals. Consistent with the removal of the transverse septal cartilage matrix, the longitudinal canals of opened chondrocytic lacunae were deeply invaded by capillary vessels, mononuclear cells and multinucleated pre-osteoclasts lacking a ruffled border. CD34-positive endothelial cells of capillary vessels deeply penetrated into the transverse septal cartilage matrix facing the medullary cavity and the opened chondrocytic lacunae. ED1-positive monocytes/macrophages were distributed at the chondro-osseous junction, but they were distant from the erosive front of the transverse septa. Tartrate-resistant acid phosphatase-positive multinucleated pre-osteoclasts lacking a ruffled border and differentiated osteoclasts with a ruffled border were localized mainly at two locations: the chondro-osseous junction and the growth front of primary bone trabeculae. Osteoclasts were located on the type-I collagen-positive bone trabeculae close to the growth plate, but they appeared to be distant from the type-II collagen-positive cartilage matrix. Even within opened chondrocytic lacunae, when osteoclasts were distant from the cartilage and bone matrix, they lacked polarized cytoplasmic organization and a ruffled border. The osteoclasts located in the remaining septal cartilage also exhibited neither a ruffled border nor a clear zone. Osteoclasts with a prominent ruffled border and clear zone were located in bone matrix covering the remaining septal cartilage. These results suggest that osteoclasts require hydroxyapatite crystals and bone matrix constituents for ruffled border formation and are not involved in resorption of the unmineralized transverse and mineralized longitudinal septal cartilage without covering bone matrix at the chondro-osseous junction.     

The ruffled border and attachment regions of the apposing membrane of resorbing osteoclasts as visualized from the cytoplasmic face of the membrane

The aim of our present research was to visualize how the plasma membrane is modified and how the cytoskeleton interacts with the attachment and ruffled border regions of resorbing osteoclasts. In order to view the surface modification of membranes and associated cytoskeleton, we employed the method of cell-shearing combined with quick-freezing and rotary replication to expose and replicate an extensive area of the cytoplasmic face of the surface membrane of osteoclasts in contact with synthetic apatite as a substratum. The membrane apposed to the apatite was composed of three different domains: the attachment zone, ruffled border and the remainder. In the attachment zone, a highly organized actin filament network formed dot-shaped, F-actin rich adhesion sites, so-called podosomes, and the actin ring. The cytoskeletal filament of podosomes and actin ring appeared to be in direct contact with the cytoplasmic surface of the underlying membrane. Within the actin ring, individually recognizable podosomes were well preserved, which indicates that the actin ring was probably derived from the fusion of podosomes. After shearing at the ruffled border region, the ruffled border projections and membrane regions among the projections were left behind. These ruffled border projections contained the cytoskeletal network. These actin networks also appeared to be in direct contact with the inner side of the ruffled border membrane or in contact with it via membrane-associated particles. At the basal portion of the ruffled border, numerous clathrin-coated patches or pits were well preserved. Deeper clathrin-coated pits and vesicles were also found, which indicates an active site for receptor-mediated endocytotic events. Clathrin sheets were also observed in the cell periphery outside of the actin ring. This type of clathrin sheets adhered to the apatite substrate, but was not anchored to the actin microfilaments. Our study thus clearly visualized the interaction between the cytoskeletal filaments and the underlying membrane at the ruffled border, attachment zone and podosome in osteoclasts cultured on apatitepellets.     

Alkaline phosphatase activity in the osteoclasts induced by experimental tooth movement.

The localization of alkaline phosphatase (Alpase) activity in the osteoclasts was examined cytochemically. Alpase activity was located in the basolateral membrane in mature osteoclasts having ruffled borders and clear zones, and also in the basolateral membrane in the osteoclasts lacking a ruffled border or a clear zone on the bone surface. But in the preosteoclasts situated away from the bone surface the enzyme activity was noted in the whole plasma membrane. These results suggest that the localization of Alpase activity may be altered in relation to the changes in morphology associated with the functional activity of the osteoclasts.     

The three-dimensional structure of the clear zone of a cultured osteoclast

Osteoclasts collected from long bones of newborn mice were cultured on dentine slices. Then, osteoclasts were sectioned by alternating semithin and ultrathin sections, and the three-dimensional reconstruction was made by the serial semithin sections. By this method, the present study showed first the three-dimensional structure of an osteoclast, especially the clear zone. A reconstructed osteoclast with complicated contours shifted from the lacuna, and its clear zone was a ring-shaped structure. By TEM, a new small lacuna was formed under the ruffled border, and the clear zone could be further classified into three types. The present study suggested that the synthetic observations of both three-dimensional structure and ultrastructure by transmission electron microscopy were necessary to determine whether the osteoclast was resorbing or migrating. According to these observations, the reconstructed osteoclast seemed to be migrating.     

Differentiation and function of osteoclasts cultured on bone and cartilage

We examined the differentiation and resorptive function of osteoclasts (OC) cultured on the slices of calcified bone, decalcified bone and hyaline cartilage, and found that OC differentiation depends on the co-cultured substratum, as well as osteoblast-derived factors. Bone marrow-derived macrophages (BMM) were formed from marrow cells of 5 week old ddY mice and cultured for 3 days on freeze-dried slices of calcified bone, decalcified bone or cartilage, all prepared from rabbit costal bone. BMM cultured on calcified bone slices exhibited tartrate-resistant acid phosphatase (TRAP) activity and were structurally characterized by multinucleation and ruffled border development. However, on decalcified bone slices, BMM seldom became multinucleated and exhibited weak TRAP activity. BMM cultured on cartilage slices were mononuclear, devoid of TRAP activity and structurally resembled mononuclear phagocytes. In SEM observations of co-cultured slices, resorption lacunae were formed only on calcified bone slices, and not on slices of decalcified bone and cartilage. Our results, therefore, indicated that BMM could differentiate into functional OC only on calcified bone slices, suggesting a key role of calcified components in the bone matrix for the terminal OC differentiation. Then, we cultured BMM on the same slices with yeast particles. In cultures with yeast particles, BMM exhibited intense TRAP activity, developed a ruffled border-like structure and formed resorption lacunae even on decalcified bone and cartilage slices. Vacuolar-type H+-ATPase was strongly expressed along the ruffled border membranes of these OC. Only the BMM that had not incorporated yeast particles developed a ruffled border, whereas the BMM that had incorporated yeast particles did not become multinucleated and lacked a ruffled border structure. Thus, our results further suggest that, even on uncalcified substrata, the terminal differentiation of BMM into functional OC is induced by an unidentified external stimulus, which may be contained in the cell membrane of yeast particles.     

Ultrastructural and cytochemical study of the odontoclasts in physiologic root resorption of human deciduous teeth.

Using extracted human deciduous teeth undergoing physiologic root resorption, this author studied the ultrastructural and cytochemical features of odontoclasts. The scanning electron microscopic observation of trypsin-treated dentin and cementum surfaces of resorption lacunae showed the exposure of collagen fibrils and prominent loss of the peritubular matrices around the dentinal tubules. In the resorption lacunae formed in enamel, there was dissolution of either the rod or the interrod regions. The odontoclasts developed extensive ruffled borders apposed to these resorbing matrices and had round phagosomes containing tannic acid-stainable fine amorphous inclusions, which were identical to those in the extracellular canals of the ruffled borders. The odontoclasts did not phagocytose the collagen fibrils. The odontoclasts showed the enzymatic activities of the acid trimetaphosphatase and acid p-nitrophenyl phosphatase (p-NPPase) in the Golgi-lysosome system, the ruffled border region, and along the resorbing dentin surfaces. The p-NPPase activity was inhibited by sodium tartrate. Also, the odontoclasts showed H(+)-K(+)-ATPase activity in the cytoplasm along the plasma membranes including those of ruffled border and the limiting membranes of the lysosomes. These results suggest that: 1) the odontoclasts are associated with resorption of non-collagenous organic matrices and/or extracellularly-degraded collagenous fragments rather than the incorporation of intact collagen fibrils; 2) the odontoclasts release the hydrolytic enzymes onto the lacunal surfaces and/or the lysosomes for the extra/intracellular degradation of the organic matrices; and 3) they also have H(+)-K(+)-ATPase for extracellular demineralization of the inorganic crystals.     

Localization of receptor activator of NFkappaB ligand, RANKL, in periodontal tissues during experimental movement of rat molars

The receptor activator of NFkappaB ligand, RANKL, is one of the key regulatory molecules in osteoclast formation and function. We examined RANKL localization in the periodontal tissues during experimental movement of rat molars. To produce orthodontic force, an elastic band was inserted between the upper first and second molars for 4 days, and the dissected maxillae were subjected to light and electron microscopic immunocytochemical examination for RANKL. Expression of RANKL protein was detected in osteoblasts, osteocytes, fibroblasts, and osteoclasts mostly located in resorption lacunae. In osteoblasts, osteocytes, and fibroblasts, RANKL localization was mainly observed in the cytoplasm, the cisternae of rough endoplasmic reticulum and along plasma membranes. In osteoclasts, RANKL was expressed along the ruffled, border membranes and in the cytoplasm, including the clear zone. These results suggest that during tooth movement, osteoclast differentiation and activation are regulated, at least in part, by RANKL, possibly produced by osteoblasts/stromal cells and osteoclasts themselves in the periodontal tissues.     

The electrical characteristics of InP implanted with the column IV elements

A study of the electrical characteristics of InP implanted with C, Si, Ge and Sn demonstrates that all of these column IV elements are donors, although the net electrical activation achieved with the light ion C was only about 5%. Samples implanted at temperatures of 150–200°C generally had lower sheet resistivities, higher mobilities and except for high doses, higher sheet carrier concentrations than those done at room temperatures. Implants at 150–200°C with 1 × 10¹⁴cm^?2 of the heavier ions, Si, Ge or Sn, resulted in layers with sheet carrier concentrations of 7.8 × 10¹³, 5.6 × 10¹³ and 4.7 × 10¹³cm^?2, respectively. Carrier concentration profiles of samples implanted at 200°C with 1 × 10¹⁴cm^?2 of Si agreed reasonably well with LSS theory. Higher doses gave rise to substantial diffusion of the implanted Si, whereas room temperature implants showed poor activation near the surface.     

Effects of osteoprotegerin administration on osteoclast differentiation and trabecular bone structure in osteoprotegerin-deficient mice

Osteoprotegerin (OPG)-deficient mice exhibit severe bone loss including the destruction of growth plate cartilage. Using OPG-deficient mice, we attempted to clarify the differentiation and ultrastructure of osteoclasts located on the destroyed growth plate cartilage and trabecular bone matrix in long bones. In (-/-) homozygous OPG knockout mice, adjacent to the growth plate cartilage, the formation of bone trabeculae without a calcified cartilaginous core resulted in an irregular chondrocyte distribution in the growth plate cartilage. At the metaphyseal ossification center, TRAP-positive osteoclasts showed unusual localization on both type-II collagen-positive cartilage and type-I collagen-positive bone matrix. Osteoclasts located on cartilage matrix lacked a typical ruffled border structure, but formed resorption lacunae. During growth plate cartilage destruction, osteoclasts formed ruffled border structures on bone matrix deposited on the remaining cartilage surfaces. These findings suggest that, in OPG (-/-) mice, osteoclast structure differs, depending on the matrix of either cartilage or bone. Then, we examined the effects of OPG administration on the internal trabecular bone structure and osteoclast differentiation in OPG (-/-) mice. OPG administration to OPG (-/-) mice significantly inhibited trabecular bone loss and maintained the internal trabecular bone structure, but did not reduce the osteoclast number on bone trabeculae. For most osteoclasts, OPG administration caused disappearance or reduction of the ruffled border, but induced neither necrotic nor apoptotic damages. These results suggest that OPG administration is an effective means of maintaining the internal structure and volume of trabecular bone in metabolic bone diseases by inhibition of osteoclastic bone resorption.     

In situ SEM imaging at temperatures as high as 1450 degrees C

Modifications to a scanning electron microscope (SEM) and a commercially available heating stage permits in situ imaging at temperatures as high as 1450 degrees C. Here we report on the technical modifications necessary to allow such high-temperature in situ imaging. In addition, in order to underline the potential of this technique in the field of materials science, three heating-stage experiments are presented, which reveal microstructural changes occurring at high temperature. The respective in situ experiments are: (i) surface crystallization of a cordierite glass at 1050 degrees C; (ii) thermal recovery of asbestos (chrysotile) fibers at 1250 degrees C; and (iii) residual pore-structure evolution of tricalcium phosphate during sintering at 1450 degrees C.     

Osteoclast differentiation and characteristic trabecular bone formation during growth plate destruction in osteoprotegerin-deficient mice

Osteoprotegerin (OPG) is an osteoblast-derived secreted member of the tumour necrosis factor receptor superfamily that inhibits osteoclastogenesis. Mice that are OPG-deficient have severe bone loss, including growth plate cartilage destruction. Using OPG-deficient mice as a useful animal model, we attempted to clarify differentiation and ultrastructural features of osteoclasts located on destructed growth plate cartilage and trabecular bone matrix. In the humerus and femur of OPG homozygous (-/-) mice, adjacent to the growth plate cartilage, bone trabeculae without a calcified cartilage core were characteristically formed at the metaphyseal side of the medullary cavity, which resulted in an irregular chondrocyte distribution and arrangement in growth plate cartilage. During growth plate cartilage destruction, osteoclasts positive for tartrate-resistant acid phosphatase showed unusual localization on both type-II collagen-positive cartilage and type-I collagen-positive trabecular bone matrix at the ossification centre of the epiphyseal/metaphyseal border. Although multinucleated osteoclasts were distributed within open lacunar canals in the growth plate, those on uncalcified cartilage matrix lacked a ruffled border. Facing the calcified cartilage matrix within lacunar canals, osteoclasts showed irregularly formed ruffled borders. After growth plate destruction, a thin bone layer was deposited on the remaining cartilage surfaces by invading osteoblasts. Osteoclasts formed prominent ruffled border structures on bone matrix, deposited on the remaining growth plate cartilage. These results suggest that, in OPG (-/-) mice, terminal osteoclast differentiation requires the presence of newly produced bone matrix, as the coupled phenomenon of bone formation and resorption, as well as osteoblast-derived cytokines.     

Radiation damage in N+-implanted ZnSe

The radiation damage in 200 keV N⁺ -ion implanted (111) single crystalline ZnSe has been studied as a function of dosage and annealing treatment using cross-sectional transmission electron microscope techniques. For dosages less than 10¹⁴ /cm² no observable damage is present at room temperature or after annealing at 700°C for one hour. For a dosage 10¹⁵/cm² the radiation damage is observable at room temperature and on annealing the initial high density of small black dot defects form Frank loops which on growing still further form prismatic vacancy type loops. For dosages of 10¹⁷/cm² a very high concentration of defects form at room temperature although the material remains single crystalline. On annealing at 700°C dodecahedron voids form in the damage region and cause swelling of ? 1%. The stresses induced by this swelling is accommodated by dislocations of the a/2 <110> type which glide in from the surface on the {111} slip planes. The distribution of the radiation damage for all the dosages and on annealing up to 700°C is Gaussian with a peak coinciding with the projected range of N⁺ -ions in ZnSe as predicted by the Lindhard, Scharff and Schiot (LSS) theory. Auger depth profiling on 10¹⁷ N -ions/cm² implanted ZnSe revealed no nitrogen in the damage zone of either the as implanted (R.T.) ZnSe or those annealed at 700°C.     

Exfoliation and blistering of Cd<Subscript>0.96</Subscript>Zn<Subscript>0.04</Subscript>Te substrates by ion implantation

As part of a series of wafer bonding experiments, the exfoliation/blistering of ion-implanted Cd_0.96Zn_0.04Te substrates was investigated as a function of postimplantation annealing conditions. (211) Cd_0.96Zn_0.04Te samples were implanted either with hydrogen (5×10¹⁶ cm⁻²; 40–200 keV) or co-implanted with boron (1×10¹⁵ cm⁻²; 147 keV) and hydrogen (1–5×10¹⁶ cm⁻²; 40 keV) at intended implant temperatures of 253 K or 77 K. Silicon reference samples were simultaneously co-implanted. The change in the implant profile after annealing at low temperatures (<300°C) was monitored using high-resolution x-ray diffraction, atomic force microscopy (AFM), and optical microscopy. The samples implanted at the higher temperature did not show any evidence of blistering after annealing, although there was evidence of sample heating above 253 K during the implant. The samples implanted at 77 K blistered at temperatures ranging from 150°C to 300°C, depending on the hydrogen implant dose and the presence of the boron co-implant. The production of blisters under different implant and annealing conditions is consistent with nucleation of subsurface defects at lower temperature, followed by blistering/exfoliation at higher temperature. The surface roughness remained comparable to that of the as-implanted sample after the lower temperature anneal sequence, so this defect nucleation step is consistent with a wafer bond annealing step prior to exfoliation. Higher temperature anneals lead to exfoliation of all samples implanted at 77 K, although the blistering temperature (150–300°C) was a strong function of the implant conditions. The exfoliated layer thickness was 330 nm, in good agreement with the projected range. The “optimum” conditions based on our experimental data showed that implanting CdZnTe with H⁺ at 77 K and a dose of 5×10¹⁶/cm² is compatible with developing high interfacial energy at the bonded interface during a low-temperature (150°C) anneal followed by layer exfoliation at higher (300°C) temperature.     

Ion implantation damage in GaAs: A TEM study of the variation with ion species and stoichiometry

GaAs samples have been implanted with a dose of 2 × 10¹⁴ cm^?2 of each ion in the following combinations: Ga, As, Ga + As, Se, Ga + Se and As + Se. Implantation was at 200°C, and post implantation annealing at 700°C. Subsequent examination by transmission electron microscopy (TEM) showed clear and reproducible differences in the dislocation loop size and density, depending on the ion species implanted. The simplest results were obtained with the single implants, particularly Ga and As. These observed variations could be explained in terms of point defect populations, and hence rates of annealing at a given anneal temperature, being affected significantly by the stoichiometric effect of the implant. These simpler aspects were also seen to be incorporated in the more complex “dual” implants.     

Microwave irradiation and ambient temperature interact to alter rat behavior following overnight exposure.

Each of twelve male hooded rats was trained to insert its head into a food cup for food pellets on a random-interval schedule of reinforcement. After performances stabilized, the rats were assorted into 3 groups of 4 animals. Groups were matched for response rates. Animals were exposed in groups of 4 for 15.5 h to CW 2450-MHz microwaves once every 6 nights. Animals of each group were exposed to microwaves at only one power density, either 5, 10, or 15 mW/cm2; they were exposed three times at an ambient temperature of 22 degrees C, then three times at 28 degrees C, and then once more at 22 degrees C. The relative humidity was 50% during all exposures. Rats were sham irradiated (at 0 mW/cm2) the night before each microwave exposure. Behavior was tested daily after termination of microwave irradiation or after sham exposures. None of the exposures to microwaves at 22 degrees C altered rates or durations of responding. Exposures at 28 degrees C reduced response rates and increased response durations in direct relation to the power density. The results are interpreted as the transient debilitation of behavior produced by the interaction of a mild elevation of ambient temperature and microwave irradiation.     

Thiamine pyrophosphatase activity in the Golgi apparatus of calcitonin-treated osteoclasts.

The thiamine pyrophosphatase (TPPase) activity described by Novikoff and Goldfisher was examined in osteoclasts affected by calcitonin in order to elucidate whether the morphological and functional changes of the osteoclasts have an influence over the secretion function of their Golgi apparatus. The Golgi apparatus of osteoclasts of which the ruffled border had disappeared and bone resorption discontinued as the result of treatment with calcitonin showed a slight TPPase activity. The reaction products of the enzyme in these inactive osteoclasts were distinctly fewer than that of control osteoclasts, which were not affected by calcitonin. From these results, it is suggested that there may be a connection between the morphological and functional changes of osteoclasts and the secretion function of the Golgi apparatus.     

Unusual absorption “band” in the infrared spectrum of silicon annealed at high temperature and then rapidly cooled

An unusual absorption “band” with a giant half-width has been detected in the infrared absorption spectra of silicon obtained by zone melting and subjected to cyclic heat treatment at 1250 °C with rapid cooling after each anneal and with partial removal of the thermal oxide in each cycle. A model explaining the observed features of the spectrum in terms of the transformation, occurring during heat treatment of impurity nanoprecipitates contained in the initial silicon and in terms of the microblock structure of the material is proposed. Fiz. Tekh. Poluprovodn. 31, 247–249 (February 1997)     

Sub-Nanowires Boost Superior Capacitive Energy Storage Performance of Polymer Composites at High Temperatures

Polymer dielectrics with high breakdown strength (E_b) and high efficiency are urgently demanded in advanced electrical and electronic systems, yet their energy density (U_e) is limited due to low dielectric constant (ε_r) and high loss at elevated temperatures. Conventional inorganic fillers with diameters from nano to micrometers can only increase ε_r at the cost of compromised E_b and U_e due to their poor compatibility with polymer matrix. Herein, hydroxyapatite (HAP) sub-nanowires with a diameter of ≈0.9 nm are incorporated in polyetherimide (PEI) matrix to form HAP/PEI sub-nanocomposites. ε_r and E_b of the composites are concomitantly enhanced with only 0.5 wt.% of HAP sub-nanowires, leading to high U_e of 5.14 (@150 °C) and 3.1 J cm⁻³ (@200 °C) with efficiency of 90% and high-temperature stability up to 3 × 10⁵ charge-discharge cycles at 200 °C. Microstructural analysis and molecular dynamics simulations indicate that the sub-nanowires with comparable diameter as polymer chains induce enormous interfacial area, substantially increase mobility of polymer chains and form dense traps for charge carriers. This work extends the current research scope of polymer-inorganics composite dielectrics to the sub-nano-level incorporation and provides a novel strategy for fabricating high performance polymer dielectrics at elevated temperatures.     

Performance of a Multiple HAP System Employing Multiple Polarization

In this paper we address the potential gain of using compact MIMO antenna array configurations in conjunction with HAP (High Altitude Platforms) diversity techniques in order to increase the data rates in HAP communication systems. We will also investigate the effects of spatial correlation and mutual coupling between the separate antenna elements on system performance. Simulation results show that although the capacity is degraded by correlation and mutual coupling, we still achieve significant capacity gain compared to the single HAP case. In addition, we evaluate the performance of the system for different separation angles between HAPs, and determine the optimal separation angle that maximizes the total capacity of the system.