pH changes of self-etching primers mixed with powdered dentine.

OBJECTIVES: The purpose of this study was to examine the pH changes of self-etching primers mixed with dentine powder. METHODS: Four self-etching primer adhesive systems were used: Clearfil SE Bond, Imperva Fluoro Bond, Mac Bond II, and Unifil Bond. Dentine discs obtained from extracted bovine incisors were milled and pulverized into a fine powder. The dentine powder was then mixed with solutions of self-etching primers diluted with distilled water. The pH changes of the primer-dentine powder mixtures were measured by a solid-state pH sensor connected to a pH meter at time points 10, 20, 30, 60, 120, 180, 300, and 600 s after the start of mixing. Data were analyzed by the Tukey HSD test and the Dunnett test at a significance level of 0.05. RESULTS: The baseline pH values of the self-etching primers ranged from 1.83 to 2.34, with Mac Bond II exhibiting a significantly lower value than the other three products. After mixing with the dentine powder, the pH values significantly increased, ranging from 6.95 to 7.37 at 600 s after mixing; there were no significant differences in these values among the self-etching primers used. An insoluble precipitate was formed in the case of Clearfil SE Bond, indicating a chemical reaction between the functional monomer and the dentine powder. CONCLUSIONS: The dentine has a strong buffering capacity against the acidity of self-etching primers.

BIOCOMPATIBLE TARGETING HYDROGELS FOR BREAST CANCER TREATMENT.

Hydrogels have received growing attention as materials for drug delivery systems (DDS) because of their biodegradable and biocompatible properties. DDS were developed to optimize the therapeutic properties of drug products and to render them more safe, effective, and reliable. In the past, drugs were frequently administered orally, as liquids or in powder forms. To avoid problems incurred through the utilization of the oral route of administration, new dosage forms, DDS, containing the drugs were introduced. They can deliver drugs directly to the intended site of action and can also improve treatment efficacy, while minimizing unwanted side effects elsewhere in the body, which often limit the long-term use of many drugs, and provide better efficacy of treatment. Biocompatible hydrogels are an example of such systems available for therapeutic use. In this review, results from recent publications concerning these systems are discussed. Hydrogels show superior effectiveness over conventional methods of treatment providing controlled release of active substances. They are of interest in medical applications such as breast cancer treatment.

Effect of lipidic factors on membrane cholesterol topology--mode of binding of theta-toxin to cholesterol in liposomes.

We have previously suggested the existence of two distinct states for cholesterol in cell membranes as revealed by high- and low-affinity binding sites for theta-toxin of Clostridium perfringens. In liposomes, phospholipid and cholesterol compositions, but not membrane protein composition, have been shown to be major determinants for the topology of membrane cholesterol. The effects of lipidic factors on cholesterol topology were investigated in detail by analyzing toxin binding to large unilamellar liposomes composed of cholesterol and phospholipids (neutral phospholipids/phosphatidylglycerol = 82:18, mol/mol). The numbers of high- and low-affinity toxin-binding sites depend strictly on the cholesterol mole percentage in liposomes. High-affinity toxin-binding sites appear only in liposomes with high cholesterol contents. Liposomes whose cholesterol/phospholipid ratio is 0.4 or less have no high-affinity sites regardless of their phospholipid compositions, while low-affinity sites appear in liposomes with lower cholesterol contents. The threshold values for the cholesterol mole percentage above which high-affinity toxin-binding sites appear were examined. The values decrease in accordance with the increase in the mole fraction of 18-carbon hydrocarbon chains among the total 14-18 carbon-hydrocarbon chains of the liposomal phospholipids. Furthermore, both the partial replacement of phosphatidylcholine with phosphatidylethanolamine and the digestion of phospholipids with phospholipase C also affect the threshold values. Thus the cholesterol mole percentage, in combination with phospholipid chain length and other factors, determines the topology of membrane cholesterol providing distinctively different affinity sites for theta-toxin.

Membrane disorganization induced by perfringolysin O (theta-toxin) of Clostridium perfringens--effect of toxin binding and self-assembly on liposomes.

theta-Toxin (perfringolysin O) of Clostridium perfringens binds to membrane cholesterol with high (Kd approximately 10(-9) M) and low (Kd approximately 10(-7) M) affinities and causes membrane lysis of intact cells and liposomes. In order to understand the lytic process at the molecular level, the lysis of liposomes was investigated in comparison with that of intact cells. The toxin dose required to cause 50% lysis (RD50) of phosphatidylcholine/phosphatidylglycerol (82:18, mol/mol) liposomes containing 36-40 mol% cholesterol was 300-1400-times higher than the RD50 value for sheep or human erythrocytes when samples with the same cholesterol concentration were compared. However, the average number of toxin molecules bound per liposome vesicle at 50% lysis was estimated as 10-18 from the RD50 values, close to the number on erythrocytes at 50% lysis, suggesting that the number of toxin molecules adsorbed per vesicle is important for lysis. As to the toxin dose required for membrane lysis, no significant difference was observed between liposomes containing both high- and low-affinity toxin-binding sites and those containing only low-affinity sites, suggesting that theta-toxin molecules bound to low-affinity sites can assemble and cause membrane lysis as well as those bound to high-affinity sites. theta-Toxin assembles on liposomal membranes, as on erythrocytes, in a high-molecular-weight polymeric form as judged from sedimentation patterns in sucrose density-gradient centrifugation. The high-molecular-weight polymers were detected only under conditions where cell or liposome lysis occurred. At low toxin doses, slower sedimenting toxin oligomers and monomers were predominant on liposomal membranes. These results indicate that toxin assembly on membranes is essential for liposome lysis as it is for cell lysis and that assembly occurs on membranes without membrane proteins.

Antimicrobial specificity and hemolytic activity of cyclized basic amphiphilic beta-structural model peptides and their interactions with phospholipid bilayers.

We synthesized a series of cyclic antiparallel beta-sheet model peptides with various ring sizes, which were designed on the basis of a cyclic beta-structural antibiotic, gramicidin S (GS); cyclo(Val-Orn-Leu-D-Phe-Pro)2, and investigated in terms of their antimicrobial activity and specificity against Gram-positive and Gram-negative bacteria and lytic activity for human erythrocytes. In our planning, in order to compare the peptides with GS, D-Phe-Pro sequence forming beta-turn in GS molecule remained unaltered and repeating sequences of alternately hydrophobic (Leu)-hydrophilic (Orn) residue were introduced into the beta-structural parts. CD study in acidic liposomes as well as leakage study of carboxyfluorescein encapsulated in phospholipid vesicles indicated that the peptides strongly interacted with lipid bilayers by taking an amphiphilic beta-structure. Antimicrobial study showed that although GS is active only against Gram-positive bacteria, the antimicrobial spectra of the model peptides transformed gradually to be active against Gram-negative ones and finally only against Gram-negative bacteria whose repeating sequences increased. It should be noted that the designed cyclic model peptides show antibacterial activity but accompany no hemolysis. This indicates that an appropriate hydrophobicity together with a proper orientation of hydrophilic (cationic) and hydrophobic groups in cyclic beta-structural molecules can hold antimicrobial activity against both types of bacteria without damaging eukaryotic cells.

Polymorphisms in PTCH1 affect the risk of ameloblastoma.

Ameloblastoma is the most common odontogenic tumor, but the genetic nature of the changes in the tumor cells has been unclear. Mutations of CTNNB1 or PTCH1 are observed in many human tumors. Both CTNNB1 and PTCH1 are important in tooth development and are expressed in ameloblastoma. The aim of this study was to investigate whether genetic alterations of CTNNB1 and PTCH1 are present in ameloblastoma. We investigated 14 cases of ameloblastoma. The polymorphisms found in the ameloblastoma patients were further examined in a subsequent case-control study. We found a CTNNB1 mutation in one case of plexiform-type ameloblastoma. CGG triplet repeat-number polymorphism (CGG7/CGG8) in the 5'-untranslated region of PTCH1 was observed. The proportion of CGG8 alleles was significantly higher in the ameloblastoma group. The results of this study indicate a possible relationship between the CGG8 allele in PTCH1 and the risk for ameloblastoma.

Age-development changes in susceptibility of erythrocytes to perfringolysin O.

Susceptibility to perfringolysin O of erythrocytes from mice of different ages was examined. Erythrocytes of mice younger than 5 weeks' old were more resistant to the toxin than those of young adult and adult mice. Erythrocytes of aged mice were about 3.5 times more susceptible to the toxin than erythrocytes from 4-week-old mice. The membrane cholesterol content of erythrocytes appeared to be maintained at a constant level throughout the ages of mice examined. About 5% of the total membrane cholesterol was supposed to provide receptor sites for the toxin from an experiment in which cholesterol was specifically extracted by liposomes. It was demonstrated in this experiment that susceptibility of erythrocytes to the toxin was lost in proportion to the reduction in the toxin binding. The susceptibility, however, of erythrocytes from young or aged mice was much lower or higher than expected from the changes in toxin binding. Therefore, two possibilities were raised to account for age-related alterations in the susceptibility of erythrocytes; not only expansion of a particular compartment of membrane cholesterol as a toxin receptor but also some activation of intracellular reactions leading to hemolysis might occur in senescence.

A cytolysin, theta-toxin, preferentially binds to membrane cholesterol surrounded by phospholipids with 18-carbon hydrocarbon chains in cholesterol-rich region.

We have previously suggested the existence of two distinctive states of cholesterol in erythrocyte and lymphoma cell membranes as revealed by high- and low-affinity binding sites for theta-toxin of Clostridium perfringens [Ohno-Iwashita, Y., Iwamoto, M., Mitsui, K., Ando, S., & Nagai, Y. (1988) Eur. J. Biochem. 176, 95-101; Ohno-Iwashita, Y., Iwamoto, M., Ando, S., Mitsui, K., & Iwashita, S. (1990) Biochim. Biophys. Acta 1023, 441-448]. To understand factor(s) which determine membrane cholesterol heterogeneity, we analyzed toxin binding to large unilamellar liposomes composed of cholesterol and phospholipids (phosphatidylcholine/phosphatidylglycerol = 82:18, mol/mol). Liposomes containing phospholipids with 18-carbon hydrocarbon chains at both positions 1 and 2 of the glycerol have both high- and low-affinity toxin-binding sites with Kd values similar to those of intact erythrocytes, whereas liposomes with hydrocarbon chains containing 16 or fewer carbons at either position 1 or 2 have only low-affinity toxin-binding sites. The cholesterol/phospholipid ratio, in addition to the length of phospholipid hydrocarbon chain, also determines the number of toxin-binding sites, indicating that at least these two factors determine the topology of membrane cholesterol by creating distinctively different affinity sites for the toxin. Since theta-toxin binding detects specific populations of membrane cholesterol that are not detectable by the measurements of susceptibility to cholesterol oxidase and cholesterol desorption from membranes, the toxin could provide a unique probe for studying the organization of cholesterol in membranes.

Characteristics and biodistribution of cationic liposomes and their DNA complexes.

We have developed some novel liposome formulations for gene transfection. The formulations consisting of O,O'-ditetradecanoyl-N-(alpha-trimethyl ammonio acetyl) diethanolamine chloride (DC-6-14) as a cationic lipid, phospholipid and cholesterol showed effective gene transfection activity in cultured cells with serum and in vivo, i.e., intraperitoneal injection in mice. In this report, the physicochemical characteristics and biodistribution of the liposomes containing DC-6-14 (DC-6-14 liposomes) as a drug (gene) carrier for gene therapy were investigated in vitro and in vivo. DC-6-14 liposome-DNA complexes were usually thought to have positive surface charge. However, depending on the ratio of DNA to liposomes, zeta-potential of the complexes became negative. The diameter of the complexes also depended on the DNA-liposome ratio, and showed a maximum when their surface potential was neutral. When biodistribution of the complexes was determined after intravenous injection, positively charged complexes showed an immediate lung accumulation. On the other hand, negatively charged complexes did not show lung accumulation. These results have suggested that biodistribution of the DNA-liposome complexes, prepared with DC-6-14 liposomes, depends on their surface charge. Therefore, some surface modification of DC-6-14 liposomes may improve the biodistribution and hence the targetability of their DNA complexes.

DNA-binding characteristics of aclarubicin as compared with daunorubicin and doxorubicin.

The effect of ionic strength on aclarubicin - DNA complexes was studied in comparison with its effect on daunorubicin - or doxorubicin - DNA complexes, using the spectrophotometric method. The hypochromic shift of aclarubicin, induced by its binding to native DNA, decreased to a lesser extent by the addition of Na+ than those of daunorubicin and doxorubicin, which suggests that aclarubicin-native DNA complexes are the most stable at high ionic strength. Similar examinations were made with heat-denatured DNA and polyvinyl sulfate (PVS). Aclarubicin-denatured DNA complexes showed a greater decrease in the hypochromic shift by the addition of Na+ than the corresponding complexes with native DNA. However, for daunorubicin and doxorubicin, there were no significant differences between the complexes of anthracyclines with native and denatured DNAs. In addition, the hypochromic shift of aclarubicin-PVS complexes decreased more prominently by the addition of Na+ than those of daunorubicin - and doxorubicin - PVS complexes. These results suggest that the electrostatic interaction of aclarubicin with DNA is more labile than that of daunorubicin and doxorubicin, since anthracyclines bind to single-stranded DNA and polyelectrolytes primarily by electrostatic interaction. Therefore, the other types of interaction, which may be stronger than that of daunorubicin and doxorubicin, seem to be associated with the higher stability of aclarubicin-native DNA complexes at high ionic strength. The structural differences between aclarubicin and daunorubicin or doxorubicin are considered to contribute to the differences in DNA-binding characteristics observed in this study.

PLGA microspheres containing plasmid DNA: preservation of supercoiled DNA via cryopreparation and carbohydrate stabilization.

Biodegradable microspheres containing plasmid DNA have potential uses as mediators of transfection in cells, particularly phagocytic cells such as macrophages. However, the hydrophilic nature and the structural instability of supercoiled DNA preclude its facile encapsulation in polymer matrixes such as poly(d, l-lactic-co-glycolic acid) (PLGA) by traditional methods. We initially studied the microencapsulation of plasmid DNA using the established water-in-oil-in-water double-emulsion solvent-evaporation method and found that (1) the encapsulation efficiency was low (about 20%), (2) the microencapsulation procedure nicked (degraded) the supercoiled DNA, and (3) lyophilization of the microsphere also nicked the DNA. We have therefore designed a new microsphere preparation method (called cryopreparation) to specifically address these concerns. Using the cryopreparation method, the aqueous phase of the primary emulsion containing the plasmid DNA is frozen and then subjected to homogenization. Because there is no shear stress inside a solid, we hypothesized that freezing the aqueous phase of the primary emulsion would help to preserve the supercoiled plasmid DNA during formation of the secondary emulsion. We also hypothesized that the formation of crystals from buffers within the primary emulsion was a causative factor for nicking during freezing or lyophilization, and that disruption of the crystal formation by the addition of saccharides into the primary emulsion would improve the supercoiled-DNA content of the spheres. Our results support the two hypotheses. Not only was the supercoiled-DNA content increased from 39% to over 85%, but the encapsulation efficiency was also elevated from 23% to over 85%.

A method for determining the distribution of fluoride, calcium and phosphorus in human dental plaque and the effect of a single in vivo fluoride rinse.

A new sampling method, capable of sampling plaque from its surface to its interior for quantitative studies, was modified to meet some of the requirements for the determination of the fluoride and mineral (Ca and P) profiles within dental plaque formed in vivo. Plaque samples were repeatedly collected from the same individual, using special devices, before a single fluoride rinse (900 parts/10(6) fluoride) and 10 min and 24 hr after rinse. The method allowed examination of fluoride, calcium and phosphorus distribution along the entire thickness of plaque. Fluoride content significantly increased throughout the sample 10 min after rinsing, indicating the fluoride had rapidly penetrated into the plaque. Although the elevated fluoride concentrations diminished almost to baseline with 24 hr, a high correlation was found between fluoride and minerals in each plaque fraction. It is concluded that this technique will be useful for evaluating the fluoride and mineral behaviour in the saliva/plaque and plaque/enamel interfaces, and the anti caries efficacy of fluoride applications.

Fluoride profiles in dental calculus from Japanese, Chinese and British residents.

Whether the fluoride concentrations and profiles differ in human dental calculus obtained from different countries was investigated. A total of 203 dental calculus deposits on 203 permanent teeth from residents (mean age, 52.1 years) of Nagoya (Japan), Shanghai (China), Leeds (Great Britain) and the Wuhan mountainous area (China, fluoridated area) were analysed. An abrasive microsampling procedure was used to examine fluoride distribution. There were five types of fluoride profiles in dental calculus in each area/country (designated types L, J, U, T, W). In supragingival calculus, type L (highest in the outermost layers) and type J (highest in the innermost layers) both had significantly higher values than type U (high in the surface and innermost layers) but were relatively identical. In subgingival calculus, type W (high in the outermost, mid and innermost layers) was characteristics. Calculus from the Wuhan mountainous area (fluoridated) had the highest fluoride concentration, followed by Leeds (non-fluoridated), and Nagoya and Shanghai (non-fluoridated) calculus had the lowest. Fluoride concentrations in supragingival calculus were higher in teeth extracted because of periodontal diseases than dental caries. It is concluded that fluoride concentrations and distribution in dental calculus differ from country to country, probably due to different fluoride environments.

Isolation of low density lipoprotein subfraction containing apolipoprotein B-like protein from Japanese eel Anguilla japonica plasma using dextran sulfate cellulose.

Japanese eel (Anguilla japonica) possessed a unique lipoprotein profile in their plasma, reflecting high utilization of lipids. Low density lipoprotein (LDL) fraction isolated at the densities from 1.006 to 1.085 g/ml comprised the heterogeneous components with molecular weight (Mr) 1200 K, 470 K, and 250 K. LDL subfraction with Mr 1200 K was completely adsorbed to dextran sulfate cellulose (DSC) column which had been developed for LDL apheresis treatment of the patients with familial hypercholesterolemia, while LDL subfractions with Mr 470 K and 250 K had no affinity for the DSC column. LDL subfractions with Mr 470 K and 250 K were floated and settled, respectively, by centrifuging the unbound fraction of DSC column at a density of 1.063 g/ml. LDL subfraction with Mr 1200 K possessed apolipoprotein (apo) B-like protein of Mr 230 K, while apo A-I- and A-II-like proteins of Mr 25 K and 14 K were the main components in LDL subfractions with Mr 470 K and 250 K. The presence of apo B-like protein seemed to be responsible for the adsorption of LDL subfraction with Mr 1200 K for the DSC column. LDL subfractions with Mr 470 K and 250 K seemed to belong to high density lipoprotein (HDL) with respect to molecular weights and apolipoprotein features. To our knowledge, this is the first report of the separation of LDL and HDL from the plasma of Japanese eels using the DSC column.

Influence of filler addition to bonding agents on shear bond strength to bovine dentin.

OBJECTIVES: The purpose of this study was to investigate the influence of adding filler particles to a bonding agent on dentin bond strength and of the temperature change during curing in order to determine the optimum filler level for an experimental bonding agent. METHODS: Experimental light-cured bonding agents with microfiller (average size: 0.05 micrometers) content of 0, 10, 20, 30, 40, 50, 60, and 70 wt% were used with the Imperva Bond / Lite-Fil II A (Shofu) restorative material. Bovine incisors were mounted in self-cured resin, and the facial surfaces were prepared with 600-grit SiC paper. After dentin surface pretreatment with dentin primer, experimental bonding agents were applied to the dentin surface and bonded with resin composite. Ten samples per test group were stored in 37 degrees C water for 24 h, then shear tested at 1.0 mm/min. The temperature change of the bonding agent was monitored during the exthothermic polymerization reaction according to the method of ISO standard #4049. The peak temperature and the time required to reach peak temperature were recorded. RESULTS: Bond strength to dentin and the temperature change were greatly affected by the filler level. Maximum dentin bond strength (14.3 +/- 2.3 MPa) was obtained with a filler level of 10 wt% and decreased with filler level higher than 30 wt% (10.4 +/- 1.7 MPa - 5.3 +/- 2.6 MPa). Peak temperature decreased and the time required to reach peak temperature increased with the higher filler levels. There were strong correlations between the bond strength and temperature change of experimental bonding agents. SIGNIFICANCE: The initial setting behavior of bonding agents containing filler particles may be one of the important factors influencing dentin bond strength. When bonding agents with filler particles are used, it is important to determine if optimum filler levels exist in order to optimize the dentin bond strength.

Influence of environmental conditions on bond strength of a resin-modified glass ionomer.

PURPOSE: To investigate the influence of temperature and relative humidity (RH) on the dentin bond strength of a resin-modified glass ionomer cement (Fuji II LC) and to evaluate the bonding efficacy of dentin primer. MATERIALS AND METHODS: Four environmental conditions: (A) 25 +/- 1 degrees C, 50 +/- 5% RH, (B) 25 +/- 1 degrees C, 95 +/- 5% RH, (C) 37 +/- 1 degrees C, 50 +/- 5% RH, (D) 37 +/- 1 degrees C, 95 +/- 5% RH, were used to make the specimens. Bovine mandibular incisors were mounted in self-curing resin and the facial surfaces were ground on wet #600 SiC paper to expose dentin. Dentin Conditioner and OptiBond Prime were employed as treatment agents. After treating the dentin surface, the cement was condensed into a vinyl mold (4 x 2 mm) placed on the dentin and light cured. Ten samples per test group were stored in 37 degrees C water for 24 hours, then shear tested at a crosshead speed of 1.0 mm/minute. One-way ANOVA followed by Duncan test (P < 0.05) were done. RESULTS: The dentin bond strengths of the no-conditioning group and Dentin Conditioner treatment group increased with increasing temperature but were not influenced by RH. With the use of the dentin primer, increased bond strengths and no influence of environmental conditions were observed. These data suggest the efficacy of employing dentin primer for application of the resin-modified glass ionomer cement regardless of temperature or RH.

[Studies on the flow properties of autopolymerizing hard-setting resins used as direct relining materials for denture bases].

Autopolymerizing hard-setting direct relining resin is currently being used in dentistry to attain denture bases that conform to the supporting tissues with a high degree of accuracy. The purpose of this study is to investigate the flow of six commercial hard-setting direct relining resins (Denture Liner, Kooliner, New Truliner, Rebaron, Swift, Tokuso Rebase) and several types of impression materials (zinc oxide-eugenol, polysulfide rubber, silicone rubber) using the parallel plate viscometer. The viscometer is used to measure the spread radius of samples (a volume of 0.5 cm3) between two parallel flat plates when a load of 750-gram weight is applied at 1, 10, 60, 100 seconds after loading has begun. In this experiment, the load was applied at 120, 150, 180, 210, 240, 300 seconds after mixing had begun. All the samples were mixed according to the manufacturer's recommendations. The findings were as follows; 1. The flow of the six commercial hard-setting direct relining resins could be compared with one another by the spread radius at 1 second after loading (r1) with no relation to time lapse after initiation of the mix. 2. Denture Liner, Kooliner, Rebaron and Swift had the same flow characteristic when r1 was the same. 3. Polysulfide rubber impression materials had similar flow characteristic to hard-setting direct relining resins.

Quality of the interface of primary tooth dentin bonded with antibacterial fluoride-releasing adhesive.

OBJECTIVE: Limited information is available on the physical properties and ultrastructure of resin-dentin interfaces created in primary teeth. The interfacial quality of sound and caries-affected primary tooth dentin bonded with an antibacterial fluoride-releasing self-etch adhesive was examined in the present study. METHODS: Primary molars were bonded with Clearfil Protect Bond (Kuraray Medical). A nano-indentation tester was employed for creating indentations vertically across resin-dentin interfaces of the bonded sound and caries-affected primary dentin for determination of hardness (H) and Young's modulus (Y). Statistical analysis were performed using one-way and two-way ANOVA and Fisher's PLSD test at p<0.05. Similar resin-dentin interfaces were examined with SEM/EDX, and with TEM using ammoniacal silver nitrate tracer for nanoleakage. RESULTS: In both sound and caries-affected dentin, compared to the underlying dentin, significantly lower values were seen in the H and Y values of the interfacial dentin except for the H in caries-affected dentin. No significant difference between the interfacial dentin and the underlying dentin was observed in the H of caries-affected dentin and the Ca and P contents in both sound and caries-affected dentin. TEM revealed extensive interfacial nanoleakage in bonded sound dentin, while no silver deposit in bonded caries-affected dentin. However, silver deposits were observed in the porous caries-affected dentin beneath the interface. CONCLUSION: Within the limitation of this study, Clearfil Protect Bond cannot demonstrate evidence of remineralization, does not increase the hardness and elasticity of the interfacial dentin, and does not prevent nanoleakage along the resin-dentin interface.