Tsuneyoshi Sano

Nrf2 deficiency causes tooth decolourization due to iron transport disorder in enamel organ

Rodents have brownish‐yellow incisors whose colour represents their iron content. Iron is deposited into the mature enamel by ameloblasts that outline enamel surface of the teeth. Nrf2 is a basic region‐leucine zipper type transcription factor that regulates expression of a range of cytoprotective genes in response to oxidative and xenobiotic stresses. We found that genetically engineered Nrf2‐deficient mice show decolourization of the incisors. While incisors of wild‐type mice were brownish yellow, incisors of Nrf2‐deficient mice were greyish white in colour. Micro X‐ray imaging analysis revealed that the iron content in Nrf2‐deficient mouse incisors were significantly decreased compared to that of wild‐type mice. We found that iron was aberrantly deposited in the papillary layer cells of enamel organ in Nrf2‐deficient mouse, suggesting that the iron transport from blood vessels to ameloblasts was disturbed. We also found that ameloblasts of Nrf2‐null mouse show degenerative atrophy at the late maturation stage, which gives rise to the loss of iron deposition to the surface of mature enamel. Our results thus demonstrate that the enamel organ of Nrf2‐deficient mouse has a reduced iron transport capacity, which results in both the enamel cell degeneration and disturbance of iron deposition on to the enamel surface.

Comparative Histology of the Laminar Bone between Young Calves and Foals

Laminar bone or primary plexiform tissue, not Haversian bone, shows an alternative concentric pattern of laminar-bone units or plates around the bone marrow periphery of long bones, although the laminar bone is gradually replaced by osteons during the growth period. One laminar-bone unit is constructed with a hypercalcified line in the center, woven bone on both sides of the line, and lamellar bone with laminated appositional lines. Such a laminar bone showing a homogeneous calcification has been reported in young calves and some young large animals, but it has not been reported in foals although a previous report proposed that the bone structure was distinguishable from plexiform tissue. In this study, we compared young calves with foals by backscattered electron imaging mainly of transverse ground sections of mid-diaphysis. Foals had many hypercalcified lines arranged concentrically around the bone marrow periphery, which were similar to those of young calves. However, rows of cylindrical osteon-like structures with Haversian canal-like canals running along the long-bone axis were arranged between the concentric hypercalcified lines. Each Haversian canal-like structure was enclosed with laminated appositional rings of lamellar bone deposited on the woven bone. In the developing period, the bone units containing the concentric hypercalcified lines were basically equal to the laminar-bone units. The osteon-like structures or ‘pseudo-osteons’ were gradually replaced by ‘true osteons’ during the growth period. The blood vessels in the Haversian canal-like canals of foals ran along the long-bone axis, whereas the blood vessels in the concentrically prolonged bone cavities of young calves ran transversely to obliquely against the long-bone axis. Thus, the long-bone cortex of foals showing an alternative concentric pattern of a row of the osteon-like structures arranged between the hypercalcified lines will be histologically classified into a variety of laminar bone caused by the different arrangement of blood vessels. Such a laminar bone may have a biomechanical structure against physical stress, especially the modified laminar bone of foals with osteon-like structures, when compared with the typical concentric laminar bone of young calves and also Haversian bone possessing variously calcified numerous osteons caused by bone remodeling.

Defects in mandibular bone area, enamel iron content and dentine formation following gastrectomy in rats

Fourteen 5-week-old male Sprague-Dawley rats were equally divided into two groups, sham-operated and gastrectomized. Tetracycline and calcein were given to label dentine. Four weeks after surgery, blood was collected for measurement of serum iron, calcium and parathyroid hormone (PTH) and the mandibles and maxillae were then removed. Sagittal sections of the maxilla or cross-sections of the mandible were prepared and examined. Backscattered electron images of the maxilla were taken and the iron content at the neck of incisors was measured by energy-dispersive X-ray. The dentine apposition rate in maxillary incisors was measured by fluorescence microscopy. Serum iron was significantly decreased, while PTH was significantly elevated without any change in the serum calcium in gastrectomized rats. Gastrectomy caused a gross loss of iron content in superficial enamel. The dentine apposition rate was significantly reduced by 30%. Both cortical and cancellous bone in the mandibula were significantly reduced. However, the total bone area in gastrectomized rats was similar to that in sham-operated rats. These results suggest that bone resorption was enhanced and dentine formation was reduced after gastrectomy.

Fructooligosaccharides prevent disorders of the femoral neck following gastrectomy in growing rats

Gastrectomy-evoked osteopenia in the femoral metaphysis of rats can be prevented by the consumption of fructooligosaccharides (FOS). We examined the effect of FOS on the femoral neck. Twenty-eight 5-week-old male Sprague-Dawley rats were divided into two groups, sham-operated (SH) and gastrectomized (GX). One week after each operation, the rats were fed diets containing 0.5% calcium with or without 7.5% FOS for 4 weeks. After dietary treatment, the middle of the femoral neck was cross sectioned. Backscattered electron images of the sections were then taken to calculate the following morphometric parameters: (1) percent trabecular bone volume (%TBV), (2) percent cortical bone volume (%CBV), and (3) percent bone marrow cavity (%MV); all were determined relative to the entire scan area (SC). Calcium, phosphorus, and magnesium (weight percent) were then measured on the cortical bone by energy-dispersive X-ray analysis. Total bone volume (%BV = %TBV + %CBV) and %CBV were almost identical among the groups, except in GX rats. In GX rats, these variables were significantly (approximately 20% and 30%, respectively; P ≪ 0.01) less than those in SH rats, whereas there were no changes in the other groups over the entire scan area. The calcium concentration close to the periosteal surface of cortical bone was markedly reduced by gastrectomy. This reduction was completely prevented by FOS consumption. These results suggest that FOS consumption prevents gastrectomy-evoked osteopenia regarding both volume and calcium concentration of the femoral neck.

Fine structure and mineral components of primary calculi in some human prostates

The fine structure of prostatic calculi has not been elucidated yet, although the chemical components were reported in detail. We studied the primary or endogenous calculi removed from eight human prostates by secondary scanning electron microscopy, backscattered electron imaging, energy-dispersive X-ray microanalysis and X-ray diffraction. The primary calculi containing Mg, Zn and S, besides Ca and P were basically classified into four stone groups (I-IV) by fine structure and mineral components. Stone I had the core deposits of calcospherites showing concentric rings and the laminated deposits concentrically around the core. Their deposits were identified as apatite. Stone II was occupied with the calcospherite deposits of apatite although the stone growth showed a rough concentric formation. Stone III contained the core of calcospherites and concentric laminated structures, similar to a smaller type of group I, whereas the wider peripheral region was deposited with needle-like structures, identified as calcium oxalates. Stone IV had the core deposits containing small hexahedral structures, identified as whitlockite, which were surrounded with several incompletely concentric laminated bands of apatite. Whitlockite crystals were also found between the fused large calculi. The initial and formative calculi were basically observed as the deposition of mineralizing spherical structures suggesting variously sized corpora amylaceous bodies. Thus, the primary prostatic calculi of stones I-III will begin from the mineralization of amylaceous bodies as a core, while the organic substances, which form stone IV, might be derived from the simple precipitation of prostatic secretion.

Structural and analytical comparison of gallbladder stones collected from a single patient : studies of five cases

We observed the gross and fine structure of gallbladder stones collected from five adult patients (cases I–V) by optical photography, radiography, scanning electron microscopy, and backscattered electron microscopy, and then measured the components by energy-dispersive X-ray microanalysis and infrared spectroscopy. From the stones, calcium (Ca) phosphate, Ca bilirubinate, and Ca palmitate or fatty acid Ca were identified. The 3 cholesterol stones (case I) and the 2 brown pigment stones (case II) showed macroscopic homogeneity, respectively. In addition, their fine structure and components were also similar to each other. The black pigment stones (case III) showed macroscopic homogeneity, but they were divided into radiopaque (∼30 stones) and radiolucent types (∼60 stones). The former had Ca phosphate in the center surrounded with Ca bilirubinate, and the latter was dotted with minute deposits of Ca bilirubinate. The 6 cholesterol stones (case IV) were divided into two types in size. The 5 large stones, of macroscopic homogeneity, had a core region of Ca palmitate and clear concentric rings of Ca phosphate, whereas the smaller stone was almost filled with Ca phosphate deposits in the center. From the different distributions of Ca phosphate, the smaller stone may have been formed later than the 5 large stones. Case V contained 4 stones. The 3 large cholesterol stones, of more or less macroscopic homogeneity, had a core region and concentric rings of Ca phosphate, but 1 smaller stone was dotted with minute deposits mainly containing iron (Fe) and/or silicon elements (rare type). Therefore, the stones of cases III, IV, and V showed considerable heterogeneity, respectively. In many stones, the initial precipitation of Ca salts will have become the nidus, and the concentric rings and dotted deposits of Ca salts may have accelerated cholesterol stone growth. In addition, the dotted deposits of Ca bilirubinate in the black pigment stones and the dotted deposits containing Fe in the rare stone may have become also the nidi.

Diclofenac Sodium Inhibits NFκB Transcription in Osteoclasts

A non-steroidal anti-inflammatory drug, diclofenac, acts efficiently against inflammation; however, down-regulation of diclofenac on bone remodeling has raised concerns. The inhibitory mechanisms of diclofenac are poorly understood. We hypothesized that diclofenac down-regulates osteoclast differentiation and activation via inhibition of the translocation of phosphorylated nuclear factor kappa B (NFκB). When osteoclasts prepared from mouse hematopoietic stem cells were treated with diclofenac, tartrateresistant acid phosphatase-positive multinucleated cells decreased in a concentration-dependent manner. Pit formation assay revealed the abolition of osteoclastic bone resorption; levels of cathepsin K transcripts, an osteoclastic resorption marker, were down-regulated time-dependently. Diclofenac induced the accumulation of the inhibitor of kappa B in cytosol, which led to suppression of the nuclear translocation of NFκB and phosphorylated NFκB. These results suggest that the novel mechanism of diclofenac for bone remodeling is associated with phosphorylated NFκB reduction, which regulates osteoclast differentiation and activation.

Fine structure and mineral components of fibrous stonelike masses obtained from the human mesenteries.

We investigated the fine structure and mineral components of 29 stonelike masses obtained from the mesenteries of four adult cadavers, using optical microscopy, backscattered electron imaging, scanning electron microscopy, energy-dispersive X-ray microanalysis, and X-ray diffraction. Although the overall appearance of the stonelike masses measuring about 5–20 mm in diameter and 0.06–3.1 g in dry weight was roughly grouped into smooth bulb- and uneven bulk-shaped types, all the calcified masses basically consisted of core and mantle regions. The smooth bulb-shaped masses had a broad mantle with many concentric rings, whereas the uneven bulk-shaped masses contained a large core. In their core regions, spherulitic and short bundle-shaped deposits composed of needle-shaped apatite crystals were mainly found among loose collagen fibers. Their mantle regions, on the other hand, showed the concentric structures of dense collagen fibers in the intra- and/or extrafibrous calcification with fine sandy grain-shaped deposits. The mineral elements were mainly Ca and P, and the major crystals were hydroxyapatite. Hexahedral whitlockite containing Mg was a minor component. The fiber-rich mantle regions showed lower calcification and lesser crystallization than the fiber-poor core region. When necrotic or some tumor adipose tissues and necrotic lymphoid tissues that might have been caused by some digestive diseases are recognized as foreign matter, their tissues occasionally will be calcified and grow into stonelike masses. These stonelike masses tend to occur more often in women than in men.

Inhibitory Mechanism of Non-steroidal Anti-inflammatory Drugs on Osteoclast Differentiation and Activation

Abstract Non-steroidal anti-inflammatory drugs (NSAIDs) act against not only inflammation but also down-regulation of bone remodeling. The inhibitory mechanisms of their action on bone remodeling are still unclear. We hypothesized that an NSAID, diclofenac, down-regulates osteoclast differentiation and activation via inhibition of the translocation of phosphorylated nuclear factor kappa B (NFκB). When osteoclasts prepared from mouse hematopoietic stem cells were treated with diclofenac, tartrate-resistant acid phosphatase-positive multinucleated cells decreased in a concentration-dependent manner, leading to the abolition of osteoclastic bone resorption. Levels of cathepsin K transcripts, an osteoclastic resorption marker, were down regulated. Diclofenac induced the accumulation of the inhibitor of kappa B in cytosol, which led to suppression of the nuclear translocation of NFκB and phosphorylated NFκB. These results suggest that the novel mechanism of diclofenac for bone remodeling is associated with phosphorylated NFκB reduction, which regulates osteoclast differentiation and activation.

Fructooligosaccharides consumption inhibits the loss of iron from the incisor enamel surface in gastrectomized rat

We examine the effects of fructooligosaccharides (FOS) on the reduction in the incisor iron content in gastrectomized rat. Twenty-eight 5-wk-old male Sprague-Dawley rats were divided into two groups: sham operated (bSH) and gastrectomized (bGX). After 4 wk each group was divided into two subgroups according to the presence or absence of 7.5% FOS in the synthetic diet (SH, SH+FOS, GX, and GX+FOS). At 10 wk wafter surgery, the maxilla was prepared to examine the iron content of the incisor enamel surface at four points. These points corresponded to the iron content at 6,7,8, and 10 wk, respectively. Blood was collected to determine serum iron levels at 4 and 10 wk. The serum iron level significantly decreased at 4 and 10 wk the GX group. At 10 wk, the level in the GX+FOS group significantly increased but did not reaach that in the SH group. The iron content of the enamel surface time-dependently increased and no significant differences were seen between SH and GX+FOS at 8 and 10 wk. These results suggest that FOS consumption impaired the loss of enamel content following gastrectomy, and this effect preceded the effect on the serum iron level.