Masayoshi Yoshikawa

Electron microscopy of octacalcium phosphate in the dental calculus.

The purpose of this study was to morphologically demonstrate the presence of octacalcium phosphate in the dental calculus by judging from the crystal lattice image and its rapid transformation into apatite crystal, as part of our serial studies on biomineral products. We also aimed to confirm whether the physical properties of octacalcium phosphate are identical with those of the central dark lines observed in crystals of ordinary calcifying hard tissues. Electron micrographs showed that crystals of various sizes form in the dental calculus. The formation of each crystal seemed to be closely associated with the organic substance, possibly originating from degenerated microorganisms at the calcification front. Many crystals had an 8.2-A lattice interval, similar to that of an apatite crystal. Furthermore, some crystals clearly revealed an 18.7-A lattice interval and were vulnerable to electron bombardment. After electron beam exposure, this lattice interval was quickly altered to about half (i.e. 8.2 A), indicating structural conversion. Consequently, a number of apatite crystals in the dental calculus are possibly created by a conversion mechanism involving an octacalcium phosphate intermediate. However, we also concluded that the calcification process in the dental calculus is not similar to that of ordinary calcifying hard tissues.

Demonstration of the central dark line in crystals of dental calculus

Using an electron microscope and Fourier transform infrared (FTIR) microspectroscopy, we studied the lattice images of crystallites of dental calculus to demonstrate the presence of the central dark line (CDL) in its crystallite and to compare this CDL with that of bone and synthetic hydroxyapatite crystals. Ultrastructural observations revealed clearly a number of crystallites, which displayed a proper lattice image and CDL similar to that of bone, in the dental calculus. FTIR microspectroscopy revealed that the dental calculus displayed a set of major spectra analogous to that of bone. These results suggest that the formation process of hydroxyapatite crystals with CDL in dental calculus, which is considered to be an unusual type of calcified structure in association with microorganisms, is basically similar to that of the ordinary calcifying hard tissues (bone, enamel, etc.).

Mechanism of cadmium induced crystal defects in developing rat tooth enamel

It is well known that exposure to environmental cadmium causes itai-itai (ouch-ouch) disease. However, the exact mechanism underlying this bone disease remains unresolved. By focusing on the calcification mechanism, we examined developing tooth enamel in rats exposed to cadmium to test the hypothesis that cadmium exposure may cause defects in crystal formation. Electron microscopy revealed the presence of perforated crystals in developing tooth enamel, indicating that the process of crystal nucleation may have been interrupted by cadmium exposure. Furthermore, biochemical analyses revealed that the catalytic activity of carbonic anhydrase in the immature enamel matrix declined remarkably despite the fact that quantitative reduction of this enzyme was insignificant, suggesting that the decline of catalytic activity may have resulted from the replacement of zinc with cadmium ions. Therefore, we concluded that the poor catalytic activity of cadmium-binding carbonic anhydrase might hinder the nucleation process, leading to an impairment in mineralization that causes itai-itai disease.

Fluoride Exposure May Accelerate the Osteoporotic Change in Postmenopausal Women: Animal Model of Fluoride-induced Osteoporosis

Carbonic anhydrase is a key enzyme for initiating the crystal nucleation, seen as “the central dark line” in the crystal structure in calcified hard tissues such as tooth enamel, dentin and bone. Both estrogen deficiency and fluoride exposure adversely affected the synthesis of this enzyme in the calcifying hard tissues. This led to the notion that fluoride exposure might increase the risk of developing osteoporosis in postmenopausal women. Using ovariectomized rats, which represent an estrogen (Es)-deficient state, as an animal model of postmenopausal women, we examined the causal relationship between fluoride (F) exposure and risk of developing osteoporosis. Two groups of rats, an Es-deficient group and a non-Es-deficient group, were administered free drinking water containing F ions (1.0 mg/L). Two other groups, an Es-deficient group and a control-group, were administered tap water. Soft X-ray radiography demonstrated a significant increase of radiolucent areas in the calvaria of the combined Esdeficient plus F group compared to that in the other experimental groups. Electron microscopy revealed an increase of amorphous minerals in the radiolucent areas. Light microscopy demonstrated that combined effects evidently of Es-deficiency and administration of F caused deterioration of the rat tibia with a coarse pattern of trabecular architecture, suggesting that a decline in bone formation might be the primary cause of osteoporosis. Consequently, F exposure might accelerate osteoporotic changes in postmenopausal women even at a low dose.

Combined effects of estrogen deficiency and cadmium exposure on calcified hard tissues: Animal model relating to itai-itai disease in postmenopausal women

Using ovariectomized rats as a model of postmenopausal women, we studied the effects of estrogen (Es) deficiency and in combination with cadmium (Cd) exposure on the calcified hard tissues related to the development of itai-itai disease. Es deficiency suppressed the synthesis of carbonic anhydrase required for the crystal nucleation process, causing the crystal structure defects in the tooth enamel. Regarding the combined effects of Es deficiency and Cd exposure on the bone, in which rats were given drinking water containing Cd ions, soft X-ray radiography revealed a development of labyrinthine pattern in the calvaria, and micro-computed tomography demonstrated the declining trabecular architecture of the tibia, suggesting Cd–induced osteoporotic change. Further, electron microscopy showed the increase of amorphous minerals in the calvaria. In conclusion, the combined effects of Es deficiency and Cd exposure can be responsible for accelerating the declining bone strength together with the crystal structure defects resulting in the preferential occurrence of itai-itai disease in postmenopausal women.

TEM Study of the Radular Teeth of the Chiton Acanthopleura japonica

The radula chiton teeth, Acanthopleura japonica, were examined using transmission electron microscopy (TEM). After cutting into segments corresponding roughly to three developmental stages from the onset of tooth development, the middle and the fully matured stages, toluidine blue staining has given the posterior side three different color patterns, colorless, reddish-brown, and black colors, respectively. At the colorless stage, the microvilli attached along the surface of the tooth cusp appeared to be dissembled and convert into the lamellar structure in the tooth interior. At the reddish-brown stage, the electron density between fibrous layers increased. A complex of tiny clusters of grains appeared along the fibrous layers. They seemed to aggregate each other to become larger. At the black stage, multiple layers consisting of irregular-shaped and various size of iron minerals were formed. After treating with an aqua regia solution, organic substances have remained between iron minerals, suggesting the abrasion-resistant role at the posterior side of chiton teeth during feeding. In addition, these minerals were randomly arranged. The lattice intervals of the ion minerals varied at an approximate range from 4.8 to 10.2 A. Also, we have confirmed clearly the lattice fringe of apatite crystal in the core region.

A statistical analysis of outpatients in the Meikai University Hospital Clinic of Orthodontics during the past ten years

approximately one-half and one-third those of stainless steel wire and cobalt–chromium–nickel alloy wire, respectively. For a cyclic bending test, stainless steel wire, cobalt–chromium– nickel alloy wire, and beta-titanium alloy-2 wire did not fracture even after 10,000 times of bending cycles, although beta-titanium alloy-1 wire fractured after 4500 bending cycles. Orthodontic forces delivered by a quad helix made from both betatitanium alloy wires were lower than those for stainless steel wire and cobalt–chromium–nickel alloy wire.

Fluoride exposure accelerates the development of postmenopausal osteoporosis: animal model

From the viewpoint of the crystal formation mechanism (Fig. 1), we demonstrated recently that the combined effects of estrogen (Es) deficiency together with cadmium (Cd) exposure adversely affected the supply of carbonate ions during the crystal nucleation process, resulting in the increase of amorphous minerals in the bone associating with itai-itai disease, Cd-induced osteoporosis. We also reported that fluoride (F) exposure was much greater than that of Cd exposure in terms of the detrimental effect on the crystal nucleation process. This led to the assumption that F schemes such as water fluoridation for public health may accelerate the risk of developing postmenopausal osteoporosis. Therefore, the present study was conducted to raise awareness that water fluoridation for public health is the misconception. Furthermore, it is suggested that the primary cause of osteoporosis may result from the declining bone formation. (No conflict of interest)