Decrease in fluorescence lifetime by glycation of collagen and its application in determining advanced glycation end-products in human dentin.

Advanced Glycation End-products (AGEs) are produced by the Maillard reaction, which causes cross-linking of collagen and results in changes in the mechanical properties of collagen tissues. Several types of AGE fluoresce, and measurement of this fluorescence is effective for determining the presence of AGEs. Because fluorescence intensity by steady-state fluorometry is affected by sample surface condition and light source, we focused on fluorescence lifetime measurement (FLM). We found that fluorescence lifetime of collagen gel decreased with glycation progress. In vivo application of FLM for determination of AGEs was confirmed in human dentin.

Accumulation of advanced glycation end-products in human dentine.

Cross-linking of collagen by Advanced Glycation End-products (AGEs) occurs by non-enzymatic glycation (Maillard reaction). The purpose of this study was to examine whether AGEs are formed in human dentinal collagen, and to consider any possible influence of AGEs on dentinal physiology. Mechanical characteristics, fluorescence spectra and immunohistochemical analyses of demineralized dentine sections from young subjects were compared with those of aged ones. The same investigations were performed with young dentine artificially glycated by incubation in 0.1M ribose solution. Indentation measurement indicated that the sections from aged dentine were mechanically harder than those from young dentine. The hardness of young dentine increased after incubation in ribose solution. Fluorescence peak wavelength of the young dentine was shorter than that of the aged one, but shifted towards the peak wavelength of the aged one after incubation in ribose solution. These changes were considered to be due to accumulation of AGEs. Existence of AGEs in dentinal collagen was confirmed by immunohistochemical analysis. The obtained results suggest that AGEs accumulation occurs in dentinal collagen and is affected by both human age and physiological conditions such as glucose level in blood because dentinal collagen receives nourishment via dental pulp and tubules.

Structural modifications of dentinal microcracks with human aging.

Several studies have reported the mechanism of crack propagation with aging. Although structural modifications of dentinal microcracks with aging have been evaluated by observing the cracked surface using scanning electron microscope (SEM), very few attempts have been made at sectional observation of the microcracks inside dentine using transmission electron microscopy (TEM). The objectives of this study were to: (1) examine the process of dentinal microcrack formation using TEM and (2) to morphologically evaluate the relation between dentinal microcrack propagation and human aging. Molars from 'young' (16-28 years) and 'aged' (62-76 years) subjects were evaluated. Dentine blocks were cracked with an indenter and sectioned using a diamond knife and ultramicrotome after embedding in epoxy resin. Microcracks were observed by TEM and ultra-high-voltage electron microscope tomography to determine the characteristics of crack propagation in the young and aged teeth. The results show that, in young teeth, crack propagation tended to pass through the dentinal tubules, while in aged teeth, it tended to deflect to the outer side of peritubular dentine (PTD), especially in coronal dentine. The advantage of this method is that it allows visualization and evaluation of the ultrastructural propagation of microcracks in dentine. The differences in crack propagation between young and aged dentine could be explained by differences in PTD thickness.

Ultra-structural observation of human enamel and dentin by ultra-high-voltage electron tomography and the focus ion beam technique.

Human enamel and dentin are hard and brittle mineralized tissues. It is difficult to prepare thin specimens (under 200 nm) of these tissues for transmission electron microscope observation without demineralizing them. We present a novel method of creating three-dimensional ultra-structural images of human enamel and dentin, using the focus ion beam (FIB) method and ultra-high-voltage electron microscope tomography. Thin specimens (less than 2 µm) of enamel and dentin were prepared using the FIB method. This method is appropriate for nano-fabrication of thin specimens for brittle materials such as enamel and dentin. It allows penetration of an ultra-high-voltage electron beam of a 3000 kV maximal acceleration voltage into a specimen, enabling projections of the specimen to be taken. It facilitates tomography of the enamel rods, sheaths, dentinal tubules and collagen fibrils with a high resolution of 2 nm. The component percentages in ultra-structures of dentin can be expressed numerically by using three-dimensional reconstruction images of tomograms. The matrix of peritubular dentin differed from that of intertubular dentin by having relatively fewer collagen fibrils. The major advantage of this method is its ability to visualize ultra-structural tomograms of highly calcified specimens without demineralization.

Potent anti-obese principle from Rosa canina: structural requirements and mode of action of trans-tiliroside.

The 80% aqueous acetone extracts from the fruit (50 mg/kg/d) and seeds (12.5 and 25 mg/kg/d) of Rosa canina L., but not from the pericarps, were found to show substantial inhibitory effect on the gain of body weight and/or weight of visceral fat without affecting food intake in mice for 2 weeks after administration of the extracts. With regard to the active constituents, the principal constituent, trans-tiliroside (0.1-10 mg/kg/d), potently inhibited the gain of body weight, especially visceral fat weight, and significantly reduced blood glucose levels after glucose loading (1 g/kg, ip) in mice. On the other hand, kaempferol and p-coumaric acid lacked such effect and kaempferol 3-O-beta-D-glucopyranoside tended to reduce the gain of body weight and visceral fat weight, but not significantly, at a dose of 10 mg/kg/d. These results indicate the importance of both kaempferol 3-O-beta-D-glucopyranoside and p-coumaroyl moieties for anti-obese effects. Furthermore, a single oral administration of trans-tiliroside at a dose of 10 mg/kg increased the expression of PPAR-alpha mRNA of liver tissue in mice.

Inhibitory effects of coumarin and acetylene constituents from the roots of Angelica furcijuga on D-galactosamine/lipopolysaccharide-induced liver injury in mice and on nitric oxide production in lipopolysaccharide-activated mouse peritoneal macrophages.

The methanolic extract (200 mg/kg, p.o. and i.p.), principal coumarin constituents (isoepoxypteryxin, anomalin, and praeroside IV), and a polyacetylene constituent (falcarindiol) (25 mg/kg, i.p.) from the roots of Angelica furcijuga protected the liver injury induced by D-galactosamine (D-GalN)/lipopolysaccharide (LPS) in mice. In in vitro experiments, coumarin constituents (hyuganins A-D, anomalin, pteryxin, isopteryxin, and suksdorfin) and polyacetylene constituents [(-)-falcarinol and falcarindiol] substantially inhibited LPS-induced NO and/or TNF-alpha production in mouse peritoneal macrophages, and isoepoxypteryxin inhibited D-GalN-induced cytotoxicity in primary cultured rat hepatocytes. Furthermore, hyuganin A, anomalin, and isopteryxin inhibited the decrease in cell viability by TNF-alpha in L929 cells.