Hideaki Ogura

Osteopontin mRNA expression during bone resorption : an in situ hybridization study of induced ectopic bone in the rat

To study the relationship between the expression of osteopontin mRNA and ectopic bone formation and resorption, in situ hybridization using 35S-labeled RNA probes was performed on ectopic bone that was induced in an experimental rat model. The expression of type I collagen and osteocalcin in this ectopic bone was also examined. After 6-week-old male Wistar rats were injected intravenously with colchicine at a dose of 1 mg/kg, trabecular bone-like ectopic calcified tissue had formed in the medial bone marrow cavity of the tibia on day 4, and then continued to increase progressing forward to the distal cavity. On day 8 peak growth was attained, after which it was resorbed within 4 days as a result of osteoclast recruitment. Subsequent in situ hybridization of sections of the ectopic bone at 4, 6, 8, and 10 days after this colchicine treatment revealed that high levels of the type I collagen mRNA were expressed in the osteoblasts of the mineralized ectopic bone surface, not only during the formation period but also during bone resorption. Although osteocalcin showed no specific signals throughout the experiments, osteopontin mRNA was expressed temporarily at day 10 during the initial phases of ectopic bone resorption, primarily in both osteoblasts and osteocytes and further in some osteoclasts. These results suggest that de novo synthesis of osteopontin is closely associated with bone resorption and could possibly be required to initiate and mediate this biological process.

Toxicity of Camphorated Phenol and Camphorated Parachlorophenol in Dental Pulp Cell Culture

The toxicity of phenol, parachlorophenol, camphorated phenol, camphorated parachlorophenol, and camphor was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay on established rat dental pulp cells (RPC-C2A). RPC-C2A cells at the confluent stage were incubated for 24 h in an experimental medium containing each compound at different concentrations. All tested drugs showed cytotoxicity in the MTT assay in a concentration-dependent manner. It is believed that camphor is a vehicle, and it reduces the toxicity of phenol and parachlorophenol. However, camphor itself showed cytotoxicity, and the addition of camphor increased the toxicity of phenol and parachlorophenol, reconfirming the cytotoxicity of these classical antiseptics.

The effect of sodium salicylate on the osteoclast-like cell formation and bone resorption in a mouse bone marrow culture.

Salicylates are reported to have an inhibitory effect on bone resorption in vivo and in vitro. The present study examined the effect of sodium salicylate on the formation of osteoclast-like cells in vitro. When mouse bone marrow cells were cultured for 8 days with 10-8 M 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), numerous clusters of mononuclear and multinucleated cells (MNCs) formed, which stained positive for tartrate-resistant acid phosphatase (TRAP-positive). In similar cultures using sodium salicylate, the number of both TRAP-positive mononuclear and TRAP-positive MNCs were found to diminish in proportion to the concentration of sodium salicylate. A time-course experimental model showed that the number of TRAP-positive MNCs decreased slightly when sodium salicylate was given early in the culture period, and decreased markedly when the drugs were given later in the culture period. Pit formation and bone-resorption area on the bone slices were also inhibited by adding sodium salicylate continuously with 1α,25(OH)2D3. The sodium salicylate showed no cytotoxic effect because the total number of adherent cells, including both TRAP-positive and TRAP-negative cells, was independent of the presence of sodium salicylate. These results suggest that sodium salicylate has an inhibitory effect on the recruitment of osteoclast-like MNCs and that this inhibition is greater during the later stage of mouse bone marrow culture.

Mechanism of induction of hypercalcemia and hyperphosphatemia by lead acetate in the rat.

SummaryThe present study is an investigation of the mechanism of hypercalcemia and hyperphosphatemia induced by the intravenous injection of lead acetate (Pb-Ac). A total of 118 male rats were injected with 30 mg/kg of Pb-Ac, or with 16.5 mg/kg of sodium acetate as the control. The levels of serum calcium, phosphorus and lead were then determined at various time periods after the injections. Serum calcium and phosphorus levels increased with time after Pb-Ac injection and the maximum values of calcium (17 mg%) were found after 1 h and of phosphorus (13.5 mg%) after 30 min. Both calcium and phosphorus levels reverted to the normal range after 12 h. The maximum net rates of increase of calcium and phosphorus were found immediately after Pb-Ac injection. At that time, deposition of lead at the calcifying sites of bone and incisor dentin was demonstrated by a histochemical examination. In other experiments the changes in the calcium and phosphorus contents in the medium after shaking bone powder in serum with Pb-Ac in an in vitro system were studied. It was confirmed that the calcium and phosphorus were displaced from the bone mineral, the extent of the displacement being correlated with the concentration of the Pb-Ac added to the medium, and that these displacements were very rapid reactions. These results suggest that hypercalcemia and hyperphosphatemia following Pb-Ac injection results from a direct action of lead on the bone mineral.

Scanning electron microscopical study on the fluorosis of enamel in rats

SummarySixteen 58-day-old male rats of Wistar strain, with a mean body weight of 179 g, were divided into two equal groups. Each group of eight animals was maintained for 70 days on drinking water, ad lib., containing no fluorine (control group) and 100 ppm of fluorine (experimental group). All specimens examined were obtained from the incisal portions of the incisors. The following types of enamel specimens were prepared for scanning electron microscopy: (1) acid-etched specimens; (2) acid-etched specimens followed by low temperature microincineration; and (3) fractured specimens. The enamel formed during high fluoride exposure showed marked hypocalcification, that is, the crystallite density in the prism core and interprismatic region was lower than that of control animals. The organic substances appeared to increase in these regions. These changes were prominent in the outer and middle enamel layers. Such changes following fluoride administration appear to indicate an inhibition of enamel maturation, that is, an inhibition of the mineral deposition and/or an inhibition of organic matrix withdrawal.

Aspirin-induced hypocalcemia in the rat.

Abstract Male rats of the Wistar strain (4 to 5 weeks) were used to study dose- and time-related effects of single doses of aspirin on plasma calcium in intact rats. In addition, aspirin was administered to bilaterally adrenalectomized, nephrectomized, or thyroparathyroidectomized rats to examine the mechanism of aspirin-induced hypocalcemia in rats. Rats were fasted overnight, and treatments were administered with a stomach tube. About 60 μl of blood was collected from the end of the tail for determination of plasma calcium. After the administration of aspirin (450 mg/kg), the level of plasma calcium decreased with time showing the minimum value (7.10 mg%) at 6 hr and reverted to normal range 12 hr after the administration. The decrease in plasma calcium 2 hr after the administration of aspirin was dose related. The administration of indomethacin (10 mg/kg) to rats, however, did not alter the values for plasma calcium. This finding suggests that prostaglandins may not be involved in the process of aspirin-induced hypocalcemia. The hypocalcemia due to treatment with aspirin (200 mg/kg) also occurred in bilaterally adrenalectomized rats. This finding suggests that the aspirin-induced hypocalcemia was not secondary to increased secretion of endogenous adrenocorticosteroids. When aspirin was administered immediately after the nephrectomy, the increment of plasma calcium due to the kidney damage was inhibited by the administration of aspirin. Since aspirin lowered the level of plasma calcium in the thyroparathyroidectomized rats, the thyroparathyroid apparatus and kidney seem to be dissociated from the mechanism of aspirin-induced hypocalcemia.

Low-temperature ashing of bovine dentine

The present studies were performed to obtain data on the low-temperature ashing (LTA of dentine. Observations of colour and acid solubility, measurement of weight loss, and chemical analyses of carbon, hydrogen, and nitrogen assessed the ashing efficiency. The LTA method was sufficient to deproteinize dentine powder below 300 mesh. The determinations of calcium, magnesium, and phosphorus in dentine showed no significant differences between LTA, muffle furnace ashing (MFA), and wet ashing. The carbonate content was not significantly different after drying and LTA, but approximately 48% carbonate was lost using MFA. X-ray diffraction patterns of dentine powder showed no detectable changes before and after LTA treatment compared to the patterns after treatment by MFA and/or ethylenediamine extraction.

The effects of colchicine or vinblastine on the blood calcium level in rats

In order to clarify how microtubule inhibitors induce hypocalcemia, rats were injected with intravenous colchicine (1 mg/kg) or vinblastine (2 mg/kg). The blood calcium levels decreased rapidly, and the minimum values, reached 4 h after the injection, were 7.55 +/- 0.70 mg/100 ml (mean +/- S.D., P < 0.001) for colchicine and 7.61 +/- 0.17 mg/100 ml (P < 0.001) for vinblastine. At 24 h, these values returned to the normal range (10.13 +/- 0.42 mg/100 ml). The blood calcium values in rats fed a low calcium diet and in thyroparathyroidectomized rats were also reduced by colchicine. The incorporation of blood 45Ca into bone was reduced by the injection of colchicine. Histologically, the bone cells of rats injected with either drug were severely damaged 8 h after the injection. These results indicate that hypocalcemia may be mediated by interference with the regulatory mechanisms of bone cell calcium homeostasis, and that the destruction of microtubules may be closely related to the development of the hypocalcemia.

Mineral phase in experimental ectopic calcification induced by lead acetate in the rat

SummaryExperimental ectopic calcification caused by intravenous injection of lead acetate (Pb−Ac) followed by subcutaneous injection of polymyxin B sulfate (PMX) in the rat was studied by the methods of quantitative chemical analysis and X-ray diffraction analysis using samples freed of organic matter by low temperature ashing (LTA). In all specimens, X-ray diffraction data showed the presence of an apatitic phase. In addition, several unknown peaks, the intensities of which weakened with time, were found. These peaks were established to be those of lead pyrophosphate (Pb2P2O7) when compared with the Joint Committee on Powder Diffraction Standards (JCPDS), formerly the ASTM index. Improvements in crystallinity of the apatitic phase was accompanied by the increases in the amounts of calcium, phosphorus and carbonate in the LTA ash. The amount of magnesium, on the contrary, decreased from 3 to days. The molar Ca/P ratio was near to 1.5 up to 10 days and then increased to 1.59 at 40 days. When the LTA ash was heated at 600° C, the major crystal phase wasβ-tricalcium phosphate and the minor phase was hydroxyapatite up to 10 days; after that the relationship between the two phases was reversed. It is suggested that the characteristics of calcium phosphate in the native state was transformed between 10 and 20 days after administration of Pb−Ac and PMX.

Studies on the Transport Mechanism of Iron in Rat Incisor Enamel

The enamel of the continuous growing incisors of adult rats is characterized by the presence of a orange-yellow pigmentation, which is caused by the content of large amounts of iron. Hi stochemical studies and electron microprobe analysis have shown that iron-containing pigment is also identified within the rat incisor ameloblasts during the enamel maturation stage [1–4]. Autoradiographic studies using 55Fe have demonstrated that the iron is incorporated from papillary layer to the ameloblasts and accumulated during the progress of maturation stage, and finally secreted onto the completely mineralized enamel surface at the end of maturation[5–7]. It is thought that iron is transported as the form of transferrin in the blood plasma and stored as the form of either ferritin or hemosiderin within the cells.