Mitsuo Kakei

ASPECTS OF CARBONIC ANHYDRASE AND CARBONATE CONTENT DURING MINERALIZATION OF THE RAT ENAMEL

We examined carbonic anhydrase and carbonate content in developing dental enamel to clarify some problems in connection with the initial mineralization. Two-dimensional electrophoretic analysis showed that the type of carbonic anhydrase isoenzyme in developing enamel tissue was a low-activity form. A relatively high activity of carbonic anhydrase was detected in the developing enamel of rat lower incisors. We also determined that the content rate of carbonic anhydrase accounts for about 4% of the total matrix proteins of early developing enamel. A remarkable reduction of carbonic anhydrase activity occurred at the maturation stage, and then disappeared in the fully matured enamel. The carbonate content also decreased gradually, showing a parallel profile to that of carbonic anhydrase activity, from 3.5% to 1.9% in mineral phase. From the present study, we proposed that carbonic anhydrase in the initial site of mineralization may act as a trigger for the crystal nucleation by supplying the carbonate ions into the precursor mineral at the early stage of crystal development.

Behavior of carbonate and magnesium ions in the initial crystallites at the early developmental stages of the rat calvaria

Analysis of the contents of calcium (Ca), magnesium (Mg), phosphate, and carbonate ions in the mineral phase of rat calvaria specimens obtained at different developmental stages indicated that the mineral at the newborn stage contained a negligible amount of carbonate, but a high content of Mg. There was no significant difference in Ca and phosphate (as PO4) contents between the newborn material and that from later stages. A relatively large amount of carbonate was detected in the calvaria from 6-day-old rats, in which only immature crystals were observed, thus indicating the beginning of apatite formation. Furthermore, using laser Raman microprobe analysis we confirmed that the Raman peak at 1120 cm-1 band, indicative of a Mg-CO3 compound, appeared at the 6-day stage. We also observed that the Raman peak at 988 cm-1 found in the samples from the newborn seemed to have shifted to 963-962 cm-1 in the case of those obtained from 6-day-old rats, a shift which suggests the conversion from the non-apatitic to the apatitic form. These results indicate that carbonate ions might facilitate the initiation of crystal development by converting the inhibitory Mg ion into its inactive form (Mg-carbonate compound).

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.).

Ultrastructural and Protein Aspects of Apatite Formation in Vertebrate Hard Tissues

The central dark line (CDL) which is one of the 100 planes of hydroxyapatite and represents the site of initiation of crystal growth, was observed not only in the crystallites of enamel but in dentin, bone and baleen as well. The area of CDL probably contains high concentration of carbonate ions. It was demonstrated in all the hard tissues tested that the earliest appearance of mineral structure is composed of a ribbon- or disc-shaped precursor mineral plate intimately surrounded by an organic envelope. The CDL appears at the center of the mineral portion as the first step in crystal growth.

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.

High-resolution Electron Microscopy of the Crystallites of Fossil Enamels Obtained from Various Geological Ages

To elucidate the stability of the central dark line (CDL) in biologically induced hydroxyapatite crystals, we examined the diagenetic changes on the microstructures of the crystallites during the course of fossilization. Using transmission electron microscopy, we investigated the enamel crystallites of fossil animals of various geological ages ranging from Pleistocene to Cretaceous. Electron micrographs indicated that the microstructures and lattice images of each crystallite in fossil enamels were well-preserved regardless of the thickness of the enamel layer, and the presence of CDLs in fossil enamel crystals was also confirmed. The results indicated that the microstructure of hydroxyapatite crystals containing lattice images of CDLs appear stable during long geological periods. In addition, we conclude that the existence of lattice images in apatite with CDLs may be an indicator for the assessment of the evolution of dental enamel from fossil remains.

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.