The feasibility of an in situ tissue-engineering method employing cell-based therapy with autologous periodontal ligament-derived cells was investigated. Periodontal ligament cells were obtained from six beagle dogs. Periodontal fenestration defects (6 x 4 mm) were created bilaterally at a location 6 mm apical to the marginal alveolar crest in the maxillary canines. Alkaline phosphatase-positive periodontal ligament cells (3 x 10(5) cells) were seeded onto a collagen sponge scaffold just before implantation. One defect was filled with the cell-scaffold construct, and another was left empty as the control. All animals were killed 4 weeks after surgery, and specimens were evaluated histomorphometrically. All the histomorphometrical data were analyzed by three-way analysis of variance with the Bonferroni multiple comparisons test. Regeneration of apical tissue was faster than that of coronal and isolated tissues on the control side (apical > coronal > isolated; p < 0.0001). On the other hand, on the cell-seeded side, regeneration of the cementum was observed uniformly on the root surface. Our data suggest that the seeded cells induced cementum regeneration on the root surface, indicating the potential of in situ tissue engineering using autologous cells for the regeneration of periodontal tissues.

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SUMMARYExposure to foreign particles sometimes causes inflammatory reactions through production of cytokines and chemoattractants by phagocytic cells. In this study, we focused on macrophage migration inhibitory factor (MIF ) to evaluate its pathophysiological role in the phagocytic process. Immunohistochemical analysis of human pseudosynovial tissues retrieved at revision of total hip arthroplasty showed that infiltrating mononuclear and multinuclear cells were positively stained by both an anti-CD68 antibody and anti-human MIF antibody. For in vitro study, MIF was released from murine macrophage-like cells (RAW 264.7) in response to phagocytosis of fluorescent-latex beads in a particle dose-dependent manner. Northern blot analysis showed marked elevation of the MIF mRNA level in the phagocytic macrophage-like cells. Moreover, pretreatment of RAW 264.7 cells with rat recombinant MIF increased the extent of phagocytosis by 1·6-fold compared with the control. Taken together, these results suggest that MIF plays an important role by activating macrophages in autocrine and paracrine fashion to phagocytose foreign particles.

Green fluorescent protein (GFP), which was originally isolated from jellyfish, is a widely used tool in biological research, and homologs from other organisms are available. However, researchers must determine which GFP is the most suitable for a specific host. Here, we expressed GFPs from several sources in codon-optimized and non-codon-optimized forms in the yeast Saccharomyces cerevisiae, which represents an ideal eukaryotic model. Surprisingly, codon-optimized mWasabi and mNeonGreen, which are typically the brightest GFPs, emitted less green fluorescence than did the other five codon-optimized GFPs tested in S. cerevisiae. Further, commercially available GFPs that have been optimized for mammalian codon usage (e.g., EGFP, AcGFP1 and TagGFP2) unexpectedly exhibited extremely low expression levels in S. cerevisiae. In contrast, codon-optimization of the GFPs for S. cerevisiae markedly increased their expression levels, and the fluorescence intensity of the cells increased by a maximum of 101-fold. Among the tested GFPs, the codon-optimized monomeric mUkG1 from soft coral showed the highest levels of both expression and fluorescence. Finally, the expression of this protein as a fusion-tagged protein successfully improved the reporting system’s ability to sense signal transduction and protein–protein interactions in S. cerevisiae and increased the detection rates of target cells using flow cytometry.

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.Hepatitis C virus (HCV) infection represents a major public health problem in the world today. The infection primarily causes liver disease; however, HCV infection has also been associated with extrahepatic abnormalities, including mixed cryoglobulinemia, malignant lymphoma, Sjögren's syndrome, and oral lichen planus (2,12,18,19,34,39). Lymphotropism of HCV has been observed, and several laboratories have detected the virus in blood mononuclear cells (BMC) (16,22,26,28,35,38). Common risk factors for HCV infection include blood transfusion from unscreened donors as well as injection drug use. Although sexual and vertical transmissions have also been reported, there remain a large number of HCV carriers in whom no route of infection has been identified.Epidemiological surveys demonstrate that body fluids other than blood, including saliva, might be potential sources of HCV infection. Experimental inoculation of saliva obtained from chronic HCV carrier chimpanzees has been reported to transmit hepatitis to recipient animals (1). Several studies have demonstrated HCV RNA in the saliva of hepatitis C patients by reverse transcription (RT)-nested PCR. However, the detection rates of viral RNA within saliva have varied widely, and some groups have failed to demonstrate HCV RNA within saliva (6-11, 14, 17, 23, 25, 27, 29-33, 36-38). A potential source of HCV RNA within saliva includes gingival crevicular fluid (GCF), which might contain HCV-infected BMC in the setting of periodontal inflammation. To our knowledge, only one study has qualitatively identified HCV in GCF; HCV RNA was detected in 59% of GCF specimens from hepatitis C patients in the study (20). Since the efficiency of HCV transmission is likely related to its viral load, it is important to quantitate viral RNA levels within body fluids in order to properly evaluate possible nonparenteral routes of HCV infection.Thus, we examined the presence of HCV RNA in the saliva and GCF of anti-HCV antibody-positive patients using realtime quantitative RT-PCR. MATERIALS AND METHODSSample collection. Twenty-six dental patients attending the ...

Synthetic biomaterials have been developed and used for bone grafting. Here, we developed a biodegradable sponge composite for bone tissue engineering by combining β-tricalcium phosphate（β -TCP）and collagen. In addition, we sought to determine the optimal β-TCP granules/collagen ratio by evaluating and bone formation in vivo. Porous β-TCP granules were mixed with atelocollagen hydrochloride solution at various ratios -0.02, 0.05, 0.1, and 0.2 g/mL. The resultant mixtures were freeze-dried and subjected to dehydrothermal treatment in vacuo. The final composites obtained were designated β -TCP/collagen sponge composites（β-TCP/CS） . Through compression testing, it was found that the stress values for β-TCP/CS（0.2 g/mL）were higher than those of the other three composites over the whole strain range. Histological evaluation at four weeks after implantation revealed that the collagen sponge had degraded and newly formed bone was present on the surface of the β-TCP granules. At 12 weeks, the β-TCP granules were completely degraded and remodeling of the lamellar bone was observed.

A physiologically active substance has been isolated from Brazilian propolis and charac­terized as a new clerodane diterpenoid, as indicated by human hepatocellular carcinoma HuH 13 cell cytotoxicity assays. This compound inhibited growth of the hepatoma cells at a concentration around 10 μg/ml and arrested the tumor cells at S phase as revealed by flow cytometry. At higher concentrations it exerted lethal damage. The compound showed cyto­toxicity on human lung carcinoma HLC-2, HeLa, KB and rat W3Y cells, whereas human diploid foreskin and primary rabbit kidney cells were less affected.