Implanted octacalcium phosphate is more resorbable than beta-tricalcium phosphate and hydroxyapatite.

Our previous studies have suggested that synthetic octacalcium phosphate (OCP) could be resorbed and replaced by newly formed bone if implanted in rat skull defects. We hypothesized that the implanted OCP is more resorbable than other commonly used bone graft substitutes of calcium phosphate compounds, such as hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP). To test the hypothesis, the present study was designed to compare histomorphometrically resorption of the implanted OCP, HA, and beta-TCP, which were kept in the experimental cranial defect of rats for a long term. A full thickness of standardized trephine defect was made in the rat parietal bone, and the same volume of granules of OCP, HA, and beta-TCP were implanted into the defect. Five specimens of each group were fixed 6 months after implantation. The percentage of remaining implants (r-Imp%) and newly formed bone (n-Bone%) in the defect was analyzed histomorphometrically. The statistical analysis showed that the r-Imp% of OCP was significantly lower than that of HA and beta-TCP. In contrast, the n-Bone% of OCP was significantly higher than that of HA and beta-TCP. The present study has shown that the implanted OCP in the rat cranial defect is more resorbable than the implanted beta-TCP and HA, whereas the implanted OCP enhances bone formation more than the implanted beta-TCP and HA.     

Age-related changes in gene expression patterns of matrix metalloproteinases and their collagenous substrates in mandibular condylar cartilage in rats

Matrix metalloproteinases (MMPs) have been implicated in physiological cartilage matrix remodelling as well as in pathological and invasive extracellular matrix remodelling of tissue. Age-related changes in the gene expression patterns of MMPs in mandibular condylar cartilages (MCCs) were analysed. We examined the gene expression patterns of Mmp-8 and -13 and their substrates, Col1a1, Col2a1 and Col10a1, in MCC of growing and ageing rats. Temporomandibular joints of male Wistar rats aged 4, 8, 16 and 32 weeks were subjected to in situ hybridization analysis. Histologically, MMCs showed characteristics of growth plate cartilage at ages 4 and 8 weeks, and more closely resembled articular cartilage thereafter. Mmp-8 was expressed in the cells in all cartilaginous cell layers at ages 4 and 8 weeks, and then was localized only in the mature cells at ages 16 and 32 weeks. Whereas Mmp-13 expression was limited to the lowermost hypertrophic chondrocytes in the growth stage, mature chondrocytes instead of hypertrophic chondrocytes expressed Mmp-13 in adult non-hypertrophic MCC. Because Mmp-8 and -13 expression overlapped with Col2a1 and Col10a1, chondrocytes could play a pivotal role in degradation as well as production of the cartilaginous matrix in MCC.     

Cementum formation in rat molar roots

Cementum is the calcified tissue covering roots of teeth and serves as attachment sites of the periodontal ligament. Although recent studies have suggested that extracellular matrix of cementum is very similar to that of bone, cementogenesis on a biological basis is still poorly understood. There are variations in the distribution and mineral contents of cementum depending on animal age, tooth species and position within the tooth roots. This paper reviews the formation and age-related changes of cellular and acellular cementum in rat molar roots, and discusses the effect of mechanical stress to the cementum formation.     

Interindividual variability in 2-hydroxylation, 3-sulfation, and 3-glucuronidation of ethynylestradiol in human liver

In the current study, we investigated interindividual variability of the 2-hydroxylation, 3-glucuronidation, and 3-sulfation of ethynylestradiol (EE2) using human liver microsomes and cytosol. Km values for the 2-hydroxylation and 3-glucuronidation in pooled liver microsomes and for the 3-sulfation in pooled liver cytosol were 3.34, 23.3, and 2.85 microM, respectively. Vmax/Km (ml/min/g liver) was highest for the 3-sulfation, followed by 2-hydroxylation, suggesting that 3-sulfation is the major metabolic pathway of EE2 in human liver. All further studies were performed at a substrate concentration of 0.1 microM. Microsomal 2-hydroxylation and 3-glucuronidation activities ranged from 0.21 to 5.02 (2.04+/-1.34, mean+/-S.D., n=35) and 0.20 to 4.84 (1.20+/-1.00, n=35) pmol/min/mg protein, respectively. Cytosolic 3-sulfation activity ranged from 4.2 to 24.3 (11.8+/-4.4, n=21) pmol/min/mg protein. All the measured enzyme activities were neither gender-related nor age-dependent, except that 2-hydroxylation was significantly higher in females than in males (p<0.05). The relative contribution of CYP3A to the 2-hydroxylation in liver microsomes was estimated from the degree of inhibition by 1 microM ketoconazole. The degrees of inhibition were between 17.8 and 78.0% (51.6+/-16.0%, n=27). These results indicate that there are large interindividual differences in the enzyme activities towards the respective metabolic pathways of EE2 and the relative contribution of CYP3A to the 2-hydroxylation of EE2 in human liver.     

Tissue distribution after intravenous dosing of micafungin, an antifungal drug, to rats

The tissue distribution after an intravenous dose of micafungin (1 mg/kg), a new echinocandin-like lipopeptide antifungal agent, to male rats was investigated. Micafungin in plasma disappeared biexponentially with a terminal half-life of 5.03 h. Micafungin concentrations in liver, kidney, and lung at the first sampling time (5 min) after dosing were 1.15, 1.64, and 2.58-fold higher than the plasma concentration, and the AUC(0- infinity ) were 1.61, 3.42, and 2.89-fold higher than that for plasma. The terminal half-lives for these tissues were 5.14, 4.87, and 5.31 h, respectively, which were comparable to those for plasma. These results suggest that micafungin distributes rapidly and moderately into tissues such as the liver, kidney, and lungs, and that the concentrations in tissues decreased in parallel with the unchanged drug in plasma.     

Confocal microscopy of Tomes’ granular layer in dog premolar teeth

Tomes’ granular layer is the hypomineralized area of radicular dentin, but knowledge concerning it is limited. The present study was designed to investigate the structural characteristics of Tomes’ granular layer in the dog’s teeth by confocal microscopy. Permanent premolars of four beagles, two at 7 months and the other two at 14 months of age, were used for observation. During premolar root formation, the 7-month-old dogs were injected with calcien and alizarin red S for vital staining of dentin, and ground sections of the teeth were prepared. Both ground and decalcified-paraffin sections were made from the teeth of the 14-month-old dogs and stained with basic fuchsin or with hematoxylin and eosin. All sections were examined by fluorescence and confocal microscopy. In the ground sections, granules of Tomes’ layer and dentinal tubules were stained with basic fuchsin and with calcein. The granules of Tomes’ layer stained with calcein were seen only near the labeling lines by calcein. The granules of Tomes’ layer appeared as bright spots in cross sections, and as lines in longitudinal sections. When the sections were cut tangentially through the surface of dentin, the granules of Tomes’ layer showed a reticular structure. Most of the dentinal tubules were seen to pass between the granules and terminated in the dentin-cementum junction. Looped tubules were not found in this area. In the paraffin sections stained with hematoxylin and eosin, extracellular matrix of dentin showed fluorescence of various intensities and dentinal tubules appeared dark. At the surface of the radicular dentin, the granules of Tomes’ layer appeared as fluorescent fibers running parallel to the surface of dentin in the longitudinal sections. The fibers appeared as bright spots in the cross sections and as a mesh in the tangential sections. In the periodontal ligament, collagen fibers showed intense fluorescence, whereas most cells were negative. From these results we conclude that Tomes’ granular layer of dog’s teeth may be the collagen fiber bundles that remained uncalcified or hypocalcified within the radicular dentin.     

A histochemical study on lectin binding in the immature enamel and secretory ameloblasts of rat incisors

Four lectin-fluorescent marker conjugates (Con A-F, MPA-F, PNA-F, WGA-R) were used for visualizing their binding sites on tissue sections by fluorescent microscopy. The immature enamel, Tomes' processes and distal cytoplasm of ameloblasts were stained with MPA-F and WGA-R on paraformaldehyde-fixed and paraffin-embedded tissue sections. The MPA- and WGA-binding glycoconjugates seemed to be main components of the organic enamel matrix because of the intense fluorescence from the tissue sections. The central portion of distal cytoplasm of ameloblasts was stained with PNA-F but the immature enamel and Tomes' processes were not. This suggests the PNA-binding glycoconjugates to be intermediates during the process of modifying their oligosaccharide chains or to be elements of Golgi area. Tomes' processes, the distal cytoplasm and periphery of nuclei of ameloblasts were stained with Con A-F. The Con A-binding glycoconjugates except those localizing on Tomes' processes may also be intermediates and/or intracellular elements like the PNA-binding glycoconjugates. Cells of stratum intermedium were stained with Con A-F, MPA-F and WGA-R. Cells of stellate reticulum and outer enamel epithelium were stained with Con A-F and WGA-R. It was indicated that enamel proteins, major organic components of immature enamel, are highly possible candidates for the MPA- and WGA-binding glycoconjugates.