Relationship between hyposalivation and oxidative stress in aging mice.

The increase in oxidative stress that accompanies aging has been implicated in the abnormal advance of aging and in the onset of various systemic diseases. However, the details of what effects the increase in oxidative stress that accompanies aging has on saliva secretion are not known. In this study, naturally aging mice were used to examine the stimulated whole saliva flow rate, saliva and serum oxidative stress, antioxidant level, submandibular gland H-E staining, and immunofluorescence staining to investigate the effect of aging on the volume of saliva secretion and the relationship with oxidative stress, as well as the effect of aging on the structure of salivary gland tissue. The stimulated whole saliva flow rate decreased significantly with age. Also, oxidative stress increased significantly with age. Antioxidant levels, however, decreased significantly with age. Structural changes of the submandibular gland accompanying aging included atrophy of parenchyma cells and fatty degeneration and fibrosis of stroma, and the submandibular gland weight ratio decreased. These results suggest that oxidative stress increases with age, not just systemically but also locally in the submandibular gland, and that oxidative stress causes changes in the structure of the salivary gland and is involved in hyposalivation.

Enhanced bone regeneration by gelatin-ß-tricalcium phosphate composites enabling controlled release of bFGF.

The objective of this study was to investigate the feasibility of biodegradable gelatin-ß-tricalcium phosphate (ß-TCP) composites as a cell scaffold and controlled-release carrier of basic fibroblast growth factor (bFGF) suitable for inducing bone regeneration at a segmental bone defect. The composite of gelatin sponge and ß-TCP granules had an interconnected pore structure with an average size of 340 µm. The composite provided the controlled release of bFGF over 2 weeks. Segmental, critical-sized, bone defects of 20 mm length were created in the ulnas of New Zealand white rabbits and the gelatin-ß-TCP composites, with or without incorporated bFGF, were implanted into the defects. Bone regeneration and ß-TCP resorption profiles were evaluated by microcomputed tomography scanner analysis and haematoxylin and eosin staining. The composites incorporating bFGF promoted significantly higher bone regeneration at the defect site as compared to the bFGF-free composites. The controlled release of biologically active bFGF from the composites may possibly be achieved through the biodegradation of the composites, resulting in the promotion of bone regeneration. We conclude that the biodegradable gelatin-ß-TCP composite is a promising scaffold for bone regeneration that enables the controlled release of bFGF.

Effect of an injectable 3D scaffold for osteoblast differentiation depends on bead size.

The objective of this study was to evaluate the effect of beta-tricalcium phosphate (beta-TCP) bead size on the behavior of KUSA/A1 mouse osteoblasts when the beta-TCP beads are used as the solid phase of a scaffold in which alginate was used as the gel phase. KUSA/A1 cells were loaded onto a three-dimensional (3D) scaffold fabricated from beta-TCP beads with diameters ranging from 300 to 500 microm (small beads), 500-700 microm (medium beads) and 700-850 microm (large beads); cells were cultured for 3, 7 and 14 days. Scanning electron microscope observations showed that each bead was connected in a network consisting of the alginate gel and KUSA/A1 cellular matrix that was tightly bonded to form a 3D structure. After 3 days, cells in the 3D scaffold with medium beads had a significantly higher alkaline phosphatase activity (ALP) than cells in the other scaffolds. However, by 7 and 14 days in culture there was no significant difference in DNA levels, ALP activity or osteocalcin expression. At 8 weeks, only the composite containing small beads and KUSA/A1 cells had turned completely into bone in vivo. Thus, bead size may influence the success of bone formation in this context.

Identification and characterization of the human inhibitor of caspase-activated DNase gene promoter.

DNA fragmentation factor is a heterodimer complex of the nuclease CAD and its specific inhibitor ICAD, which can be activated during apoptosis to induce DNA fragmentation. Although ICAD expression levels have been quantified in a variety of human cancer cells, the mechanism of ICAD gene regulation remains unknown. In this study, we identified a 106-bp TATA-less region upstream of the transcription start site as a basal promoter of the human ICAD gene. An E-Box motif, which binds transcription factors of the basic helix-loop-helix/leucine zipper family, is responsible for transcriptional activity, as demonstrated using mutated promoter-reporters. A chromatin immunoprecipitation assay further demonstrated that Myc binds to an endogenous ICAD promoter. The functional importance of Myc in the regulation of ICAD transcription was also demonstrated by knock-down of c-Myc and N-Myc gene expression, as well as their ectopic expression. Structural analysis of the human ICAD promoter and identification of factors which regulate its activity might further our understanding of the biological role of ICAD with respect to regulation of apoptosis and cancer development.

Dynamic behavior of hepatitis C virus quasispecies in a long-term culture of the three-dimensional radial-flow bioreactor system.

Hepatitis C virus (HCV) exists in infected individuals as quasispecies, usually consisting of a dominant viral isolate and a variable mixture of related, yet genetically distinct, variants. A prior HCV infection system was developed using human hepatocellular carcinoma cells cultured in the three-dimensional radial-flow bioreactor (RFB), in which the cells retain morphological appearance and their differentiated hepatocyte functions for an extended period of time. This report studies the selection and alteration of the viral quasispecies in the RFB system inoculated with pooled serum derived from HCV carriers. Monitoring the viral RNA and core protein in the culture supernatants, together with nucleotide sequencing of hypervariable region 1 of the HCV genome, demonstrated that (1) the virus production intermittently fluctuated in the cultures, (2) the viral genetic diversity was markedly reduced 3 days post-infection (p.i.), and (3) dominant species changed on days 19-33p.i., suggesting that the virus populations can be selected according to susceptibility to the viral infection and replication. A therapeutic effect of interferon-alpha also demonstrated the inhibition of HCV expression. Thus, this HCV infection model in the RFB system should be useful for investigating the dynamic behavior of HCV quasispecies in cultured cells and evaluating anti-HCV compounds.

Preparation of injectable 3D-formed beta-tricalcium phosphate bead/alginate composite for bone tissue engineering.

A novel, injectable bone tissue engineering material was developed that consisted of beta-tricalcium phosphate (beta-TCP) beads as the solid phase and alginate as the gel phase. To prepare the instantaneously formed composite scaffold, an aqueous calcium chloride solution was dried on the surface of beta-TCP beads and crosslinked with an alginic acid sodium solution, thereby forming stable beta-TCP beads and alginate gel which were injectable via a syringe. This biodegradable composite was a three-dimensional (3D) material that could be used as an injectable scaffold for bone tissue engineering. In particular, the composite with 2.0 wt% alginate concentration exhibited a compressive strength of 69 kPa in dry conditions, which was significantly higher than that exhibited by 1.0 wt%. Furthermore, mesenchymal stem cells (MSC) were 3D-cultured within the composite and then investigated for osteogenic markers. MSC-loaded composite was subjected to scanning electron microscope (SEM) examination and implanted subcutaneously for in vivo experiment. Results showed that the scaffold provided support for osteogenic differentiation. In light of the encouraging results obtained, this novel injectable composite material may be useful for bone tissue engineering.

Quantitative detection of hepatitis C virus (HCV) RNA in saliva and gingival crevicular fluid of HCV-infected patients.

The search for hepatitis C virus (HCV) in body fluids other than blood is important when assessing possible nonparenteral routes of viral transmission. However, the role of oral fluids in HCV transmission remains controversial. Here we quantitatively determined HCV RNA in saliva and gingival crevicular fluid (GCF) of anti-HCV-positive patients. Most patients (14 of 18; 78%) whose saliva specimens were negative had HCV RNA in their GCF. Most patients (20 of 26; 77%) had higher HCV RNA levels in their GCF than in their saliva. Although there was not a statistically significant correlation between the serum viral load and HCV level in saliva or GCF, patients with low serum HCV loads were less likely to have detectable HCV in their saliva. These findings have important implications for medical personnel and suggest that epidemiological studies designed to understand the significance of the oral route of transmission of HCV are warranted.