Fatigue and tensile properties of radicular dentin substrate.

The purpose of this study was to compare the fatigue and tensile strengths of radicular dentin. Forty bovine lower central incisors were used, twenty teeth for the fatigue test and twenty teeth for the tensile test. Bovine teeth were each sectioned into coronal and radicular portions. Dentin slabs of 1mm thickness were prepared along the radicular tooth using a low-speed cutting machine and trimmed into dumbbell-shaped specimens. A dentin slab was harvested from each tooth. Subsequently, fatigue and tensile tests were performed in Hank's balanced saline solution at 37 °C. The staircase method was employed to determine fatigue strength and its standard deviation. Fracture surfaces were observed by scanning electron microscopy. Mean fatigue strength and tensile strength were 44.3±5.0 and 84.4±8.3 MPa, respectively. The fatigue strength of radicular dentin was significantly lower than the tensile strength. The fatigue strength of radicular dentin was only approximately one half of the tensile strength.

Physicochemical and pharmacological characterization of alpha-tocopherol-loaded nano-emulsion system.

The main purpose of the present study is to develop a novel nano-emulsion (NE) formulation of alpha-tocopherol (alpha-TC) with enhanced oral bioavailability and pharmacological effects. Three NE formulations of alpha-TC at different loading amounts (10%, 30% and 50%) were prepared by a mechanochemical method. Physicochemical properties of NE formulations were characterized with a focus on the morphology by transmission electron microscopy (TEM), droplet size distribution and zeta-potential by dynamic light scattering (DLS), and long-term stability. According to the TEM images and DLS data, mean diameters of NE droplets ranged from 80 to 400nm, in proportion to the amount of loaded alpha-TC. Although all NE formulations of alpha-TC were found to be negatively charged with the zeta-potential of ca -40mV, NE formulations at alpha-TC content of 30% or higher exhibited severe aggregation of droplets in NE formulations during long-term storage. After oral administration of 10% alpha-TC-loaded NE formulation (30mg alpha-TC/kg) in rats, higher alpha-TC exposure was observed with a 2.6-fold increase of bioavailability as compared to the control mixture of oil and alpha-TC. In streptozotocin-induced diabetic rats, oral administration of the alpha-TC-loaded NE formulation (30mg alpha-TC/kg) exhibited a significant reduction of lipoperoxidant in several organs, especially the liver; however, the control mixture was less effective. With these findings, the NE approach might be efficacious to improve the oral bioavailability and anti-oxidative activities of alpha-TC.

Novel crystalline solid dispersion of tranilast with high photostability and improved oral bioavailability.

Tranilast (TL) is an anti-allergic agent and widely used in the clinical treatment of bronchial asthma, atopic rhinitis, atopic dermatitis and keloids. However, therapeutic potential of TL could be partly limited because of its poor solubility, bioavailability, and photostability. To overcome these drawbacks, crystalline solid dispersion of TL (CSD/TL) was prepared by wet-milling technique with aim of improving physicochemical and pharmacokinetic properties. Physicochemical properties of the formulations prepared were characterized by laser diffraction and dynamic light scattering for particle size analysis, scanning electron microscope for morphological analysis, and powder X-ray diffraction and differential scanning calorimetry for crystallinity assessment. TL particles in CSD/TL appeared to be crystalline with diameter of 122 nm, and CSD/TL exhibited marked improvement in the dissolution behavior as compared to crystalline TL. Under irradiation of UVA/B (250 W/m(2)), solution and amorphous solid dispersion of TL were found to be highly photodegradable, whereas high photochemical stability was seen in CSD/TL. After oral administration of CSD/TL, enhanced TL exposure was observed with increase of C(max) and AUC by 60- and 32-fold, respectively, as compared to crystalline TL. According to these observations, taken together with dissolution and pharmacokinetic behaviors, crystalline solid dispersion strategy would be efficacious to enhance bioavailability of TL with high photochemical stability.

Liposomal formulations of glucagon-like peptide-1: improved bioavailability and anti-diabetic effect.

Glucagon-like peptide-1 (GLP-1), an incretin hormone, is recognized to be potent drug candidate for treatment of diabetes, however its clinical application has been highly limited, because of rapid enzymatic degradation by dipeptidyl-peptidase IV. To protect GLP-1 from enzymatic degradation and improve pharmacological effects, liposomal formulations of GLP-1 were prepared using three types of lyophilized empty liposomes such as anionic, neutral and cationic liposomes. Electron microscopic and dynamic light scattering experiments indicated the uniform size distribution of GLP-1-loaded liposomes with mean diameter of 130-210 nm, and inclusion of GLP-1 did not affect the dispersibility and morphology of each liposome. Of all liposomal formulations tested, anionic liposomal formulation exhibited the highest encapsulation efficiency of GLP-1 (ca. 80%). In intraperitoneal glucose tolerance testing in rats, marked improvement of hypoglycemic effects were observed in anionic liposomal formulation of GLP-1 (100 nmol/kg) with 1.7-fold higher increase of insulin secretion, as compared to GLP-1 solution. In pharmacokinetic studies, intravenous administration of anionic liposomal formulation of GLP-1 (100 nmol/kg) resulted in 3.6-fold higher elevation of serum GLP-1 level as compared to GLP-1 injection. Upon these findings, anionic liposomal formulation of GLP-1 would provide the improved pharmacokinetics and insulinotropic action, possibly leading to efficacious anti-diabetic medication.

Comparison of nanohardness between coronal and radicular intertubular dentin.

This study investigated the hardness and Young's modulus of coronal and radicular intertubular dentin. Ten bovine teeth were each divided into coronal and radicular groups, and the flat surfaces of the coronal and radicular dentin were subsequently processed along the tooth axis. The hardness and Young's modulus of the coronal and radicular intertubular dentin were evaluated using nanoindentation tests, at two locations per tooth. Mean hardness and Young's modulus values were statistically compared by one-way ANOVA and Fisher's PLSD test. The hardness of coronal intertubular dentin was 0.81 +/- 0.05 GPa and that of radicular dentin was 0.55 +/- 0.02 GPa. Additionally, the Young's modulus of coronal intertubular dentin was 26.60 +/- 2.19 GPa and that of radicular dentin was 20.89 +/- 1.10 GPa. Findings of this study revealed that the hardness and Young's modulus of coronal intertubular dentin were greater than those of radicular intertubular dentin.

Is increased fat content of hindmilk due to the size or the number of milk fat globules?

BACKGROUND: It is known that the fat content of breast milk is higher in hindmilk than in foremilk. However, it has not been determined if this increased fat content results from an increase in the number of milk fat globules (MFGs), an increase in the size of MFGs, or both. This study aims to determine which factor plays the most important role. METHODS: Thirteen breastfeeding mothers were enrolled in the study and we obtained 52 samples from 26 breasts before (foremilk) and after (hindmilk) a breastfeeding session. The fat content was evaluated by creamatocrit (CrCt) values. MFG size was measured with the laser light scattering method. We compared CrCt values and MFG size between foremilk and hindmilk. RESULTS: Although the CrCt values were higher in the hindmilk (8.6 +/- 3.6%) than in the foremilk (3.7 +/- 1.7%), the MFG size did not change (4.2 +/- 1.0 mum and 4.6 +/- 2.1 mum, foremilk and hindmilk, respectively). There was no relationship between the changes in CrCt versus MFG size from foremilk to hindmilk. CONCLUSION: The results indicate that the increase in fat content results mainly from the increased number of MFGs, which may be released into the milk flow as the mammary lobe becomes progressively emptied.

Fine structure and mineral components of primary calculi in some human prostates.

The fine structure of prostatic calculi has not been elucidated yet, although the chemical components were reported in detail. We studied the primary or endogenous calculi removed from eight human prostates by secondary scanning electron microscopy, backscattered electron imaging, energy-dispersive X-ray microanalysis and X-ray diffraction. The primary calculi containing Mg, Zn and S, besides Ca and P were basically classified into four stone groups (I-IV) by fine structure and mineral components. Stone I had the core deposits of calcospherites showing concentric rings and the laminated deposits concentrically around the core. Their deposits were identified as apatite. Stone II was occupied with the calcospherite deposits of apatite although the stone growth showed a rough concentric formation. Stone III contained the core of calcospherites and concentric laminated structures, similar to a smaller type of group I, whereas the wider peripheral region was deposited with needle-like structures, identified as calcium oxalates. Stone IV had the core deposits containing small hexahedral structures, identified as whitlockite, which were surrounded with several incompletely concentric laminated bands of apatite. Whitlockite crystals were also found between the fused large calculi. The initial and formative calculi were basically observed as the deposition of mineralizing spherical structures suggesting variously sized corpora amylaceous bodies. Thus, the primary prostatic calculi of stones I-III will begin from the mineralization of amylaceous bodies as a core, while the organic substances, which form stone IV, might be derived from the simple precipitation of prostatic secretion.

Structural transition of glucagon in the concentrated solution observed by electrophoretic and spectroscopic techniques.

Glucagon, a polypeptide hormone consisting of 29 amino acid residues, tends to form gel-like fibrillar aggregates, and the glucagon fibril, as well as other pathologically related fibrils including prion, amylin, and beta-amyloid, have been found to be cytotoxic through the activation of apoptotic signaling pathways. To understand the aggregation properties of glucagon fibril, we have characterized and compared the physicochemical properties of glucagon, secretin, a member of the glucagon superfamily, and amylin using analytical techniques including capillary electrophoresis (CE), circular dichroism (CD), FT-IR, FT-Raman, transmission electron microscopy (TEM), and beta-sheet-imaging probe. Aging treatment of glucagon resulted in the formation of fibrillar aggregates in time- and concentration-dependent manner, and FT-IR and FT-Raman analyses showed the spectral shift of amide I band, suggesting the conformational changes from alpha-helix to beta-sheet structure. Interestingly, secretin, having high sequential and secondary structural homology with glucagon, did not generate the fibrillar aggregates at the conditions tested. In addition, we evaluated the association state of glucagon at various pHs raging from pH 2.0 to 3.5 using CE. Based on the CE data, the rate constants of glucagon aggregation were calculated to be 0.002 +/- 0.004/h and 0.080 +/- 0.011/h for aging at pH 2.0 and 3.5, respectively, suggesting the pH dependence of self-association. CE showed the potential to separate and detect the glucagon aggregates and intermediates during aging process.

Mishandling of the therapeutic peptide glucagon generates cytotoxic amyloidogenic fibrils.

PURPOSE: Some therapeutic peptides exhibit amyloidogenic properties that cause insolubility and cytotoxicity against neuronal cells in vitro. Here, we characterize the conformational change in monomeric therapeutic peptide to its fibrillar aggregate in order to prevent amyloidogenic formation during clinical application. METHODS: Therapeutic peptides including glucagon, porcine secretin, and salmon calcitonin were dissolved in acidic solution at concentrations ranging from 1 mg/ml to 80 mg/ml and then aged at 37 degrees C. Amyloidogenic properties were assessed by circular dichroism (CD), electron microscopy (EM), staining with beta-sheet-specific dyes, and size-exclusion chromatography (SEC). Cytotoxic characteristics were determined concomitantly. RESULTS: By aging at 2.5 mg/ml or higher for 24 h, monomeric glucagon was converted to fibrillar aggregates consisting of a beta-sheet-rich structure with multimeric states of glucagon. Although no aggregation was observed by aging at the clinical concentration of 1 mg/ml for 1 day, 30-day aging resulted in the generation of fibrillar aggregates. The addition of anti-glucagon serum significantly inhibited fibrillar conversion of monomeric glucagon. Glucagon fibrils induced significant cell death and activated an apoptotic enzyme, caspase-3, in PC12 cells and NIH-3T3 cells. Caspase inhibitors attenuated this toxicity in a dose-dependent manner, indicating the involvement of apoptotic signaling pathways in the fibrillar formation of glucagon. On the contrary to glucagon, salmon calcitonin exhibited aggregation at a much higher concentration of 40 mg/ml and secretin showed no aggregation at the concentration as high as 75 mg/ml. CONCLUSIONS: These results indicated that glucagon was self-associated by its beta-sheet-rich intermolecular structure during the aging process under concentrated conditions to induce fibrillar aggregates. Glucagon has the same amyloidogenic propensities as pathologically related peptides such as beta-amyloid (Abeta)1-42 and prion protein fragment (PrP)106-126 including conformational change to a beta-sheet-rich structure and cytotoxic effects by activating caspases. These findings suggest that inappropriate preparation and application of therapeutic glucagon may cause undesirable insoluble products and side effects such as amyloidosis in clinical application.

Specific biological functions of vacuolar-type H(+)-ATPase and lysosomal cysteine proteinase, cathepsin K, in osteoclasts.

We report the effects of specific and potent inhibitors of vacular-type H(+)-ATPase and lysosomal cysteine proteinases, cathepsins, on the ultrastructure, expression of these enzymes, and resorptive functions of cultured osteoclasts. Osteoclasts were formed by co-culture of marrow cells and calvarial primary osteoblasts of ddY mice. Formed osteoclasts were cultured on dentine slices for 6-48 hr with either an H(+)-ATPase inhibitor, bafilomycin A1, or a cysteine proteinase inhibitor, E-64. In control cultures with no additive, osteoclasts were structurally characterized by the development of ruffled borders and clear zones, and formed many resorption lacunae on dentine slices. Both H(+)-ATPase and cathepsin K were strongly expressed in the ruffled borders of these osteoclasts. In bafilomycin A1-treated cultures, osteoclasts lacked ruffled borders, and resorption lacuna formation was markedly diminished. This effect of bafilomycin A1 on osteoclast structure was reversible by removal of the compound. Bafilomycin A1 treatment altered the subcellular localization and decreased the expression of H(+)-ATPase molecules. H(+)-ATPase expression was observed throughout the cytoplasm, but not along the plasma membranes facing dentine slices. On the other hand, E-64 treatment did not affect the ultrastructure of osteoclasts and the expression of enzyme molecules. Although E-64 showed no effect on demineralization of dentine slices, it dose-dependently reduced resorption lacuna formation. Our results suggest that 1) bafilomycin A1 dose-dependently inhibits resorption lacuna formation via inhibition of ruffled border formation, 2) H(+)-ATPase expression is closely associated with the cytoskeleton of osteoclasts, and 3) E-64 treatment decreases the depth of resorption lacunae, by inhibition of secreted cathepsin K activity, but does not impair ruffled border formation and the associated expression of H(+)-ATPase and cathepsin K in osteoclasts.

Scanning electron microscopy and energy-dispersive X-ray microanalysis studies of afibrillar cementum and cementicle-like structures in human teeth.

We examined afibrillar cementum (AFC) and cementicle-like structures (CLS) in human teeth by scanning electron microscopy and energy-dispersive X-ray microanalysis. The AFC showed a spur- or island-, plate- and mass-like structure with appositional laminations, while large masses in the enamel fissures enclosed CLS showing concentric appositional rings. Such AFC was observed in enamel fissures, an abnormal enamel pit, dens invaginatus and root furcations with enamel droplets, as well as on the cervical enamel surfaces, where ameloblasts are differentiated at the later or last stage of enamel formation. Cementicle-like structures were occasionally found independent from AFC and some CLS contained epithelial cell-like or ameloblast-like remnants in the core, surrounded by a few or many concentric rings. In addition, cementicles (CEC) in the root furcations also contained the remnants of Malassez's epithelial-rest cells surrounded by a few concentric rings. In some areas, AFC was mixed with enamel structures. These results show that the organic material in some parts of AFC and CLS may be derived from epithelial cells similar to that of CEC. Calcification values of AFC and CLS were significantly higher than that of fibrillar cementum, and the minute crystals are probably apatite.

Effects of enamel matrix derivative to titanium implantation in rat femurs.

The effects of enamel matrix derivative (EMD; Emdogain) on new trabecular bone induction after pure bioinert titanium (Ti) implantation in the rat femur were examined by means of routine light and transmission electron microscopy, immunohistochemistry, and backscattered electron image analysis. Newly designed mini-Ti implants (3.5 mm in length and 1.6 mm in diameter) were placed in the corticotrabecular area of the femur with either EMD or its carrier, propylene glycol alginate, as control. On post-implantation days 4, 7, 14, and 30, the dissected femur was examined in the transverse direction through Ti implants. In both control and EMD-applied femurs, trabecular bone formation was recognized over the implant surfaces and within medullary cavities even at 4 days post-implantation. These newly formed bone trabeculae around the Ti implants were immunoreactive for bone sialoproteins as a bone matrix marker, and osteoclastic bone resorption became evident in these bone trabeculae after 7 days post-implantation. Although trabecular bone area around the implants was markedly decreased at 30 days post-implantation compared with those at 14 days, the trabecular bone areas in EMD-applied femurs were significantly greater than those in propylene glycol alginate-applied femurs at both 14 and 30 days post-implantation. Our results suggest that EMD is an effective biological matrix for enhancing new trabecular bone induction and resulting attachment of orthopedic prostheses to the recipient bone.