Long-term biopersistence of tangled oxidized carbon nanotubes inside and outside macrophages in rat subcutaneous tissue.

Because of their mechanical strength, chemical stability, and low molecular weight, carbon nanotubes (CNTs) are attractive biological implant materials. Biomaterials are typically implanted into subcutaneous tissue or bone; however, the long-term biopersistence of CNTs in these tissues is unknown. Here, tangled oxidized multi-walled CNTs (t-ox-MWCNTs) were implanted into rat subcutaneous tissues and structural changes in the t-ox-MWCNTs located inside and outside of macrophages were studied for 2 years post-implantation. The majority of the large agglomerates were present in the intercellular space, maintained a layered structure, and did not undergo degradation. By contrast, small agglomerates were found inside macrophages, where they were gradually degraded in lysosomes. None of the rats displayed symptoms of cancer or severe inflammatory reactions such as necrosis. These results indicate that t-ox-MWCNTs have high biopersistence and do not evoke adverse events in rat subcutaneous tissue in vivo, demonstrating their potential utility as implantable biomaterials.

Activation of the FGF signaling pathway and subsequent induction of mesenchymal stem cell differentiation by inorganic polyphosphate.

Inorganic polyphosphate [poly(P)] is a biopolymer existing in almost all cells and tissues, although its biological functions in higher eukaryotes have not been completely elucidated. We previously demonstrated that poly(P) enhances the function of fibroblast growth factors (FGFs) by stabilizing them and strengthening the affinity between FGFs and their cell surface receptors. Since FGFs play crucial roles in bone regeneration, we further investigated the effect of poly(P) on the cell differentiation of human stem cells via FGF signaling systems. Human dental pulp cells (HDPCs) isolated from human dental pulp show the characteristics of multipotent mesenchymal stem cells (MSCs). HDPCs secreted FGFs and the proliferation of HDPCs was shown to be enhanced by treatment with poly(P). Cell surface receptor-bound FGF-2 was stably maintained for more than 40 hours in the presence of poly(P). The phosphorylation of ERK1/2 was also enhanced by poly(P). The effect of poly(P) on the osteogenic differentiation of HDPCs and human MSCs (hMSCs) were also investigated. After 5 days of treatment with poly(P), type-I collagen expression of both cell types was enhanced. The C-terminal peptide of type-I collagen was also released at higher levels in poly(P)-treated HDPCs. Microarray analysis showed that expression of matrix metalloproteinase-1 (MMP1), osteopontin (OPN), osteocalcin (OC) and osteoprotegerin was induced in both cell types by poly(P). Furthermore, induced expression of MMP1, OPN and OC genes in both cells was confirmed by real-time PCR. Calcification of both cell types was clearly observed by alizarin red staining following treatment with poly(P). The results suggest that the activation of the FGF signaling pathway by poly(P) induces both proliferation and mineralization of stem cells.

CaTiO(3) coating on titanium for biomaterial application--optimum thickness and tissue response.

The objectives of this study were to determine the optimum thickness of a CaTiO(3) film for biomaterial applications and to investigate the biocompatibility and bone formation of titanium with a CaTiO(3) film. First, CaTiO(3) films of 10, 20, 30, and 50 nm in thickness were deposited on titanium substrates using radiofrequency magnetron sputtering followed by annealing at 873 K in air for 7.2 ks. The optimum thickness of the CaTiO(3) film for bone formation was determined by comparison with its performance regarding calcium phosphate formation in Hanks' balanced saline solution (HBSS). Regarding calcium phosphate formation, the performance of the specimen with a 50-nm-thick CaTiO(3) film was superior to those of specimens with other thicknesses. A titanium prism with a CaTiO(3) film of 50-nm in thickness was surgically inserted in both soft and hard rat tissues. The biocompatibility of CaTiO(3)-deposited titanium and bone formation on it was investigated by histological observations. A slight inflammatory reaction was observed around the titanium with the 50-nm-thick CaTiO(3) film, while no severe response, such as degeneration and necrosis, was observed in either soft or hard rat tissue. New bone formation on the titanium plate with the CaTiO(3) film was more active than that without the film. The 50-nm-thick CaTiO(3) film has biocompatibility and can facilitate new bone formation in vivo, and, consequently, it is an excellent surface modification method for biomaterial applications.

Participation of the Fas and Fas ligand systems in apoptosis during atrophy of the rat submandibular glands.

Most acinar cells and some duct cells undergo apoptosis during atrophy of the submandibular gland. The present study was designed to elucidate whether Fas and its receptor ligand (FasL) are involved during apoptotic atrophy of the gland. The excretory duct of the right submandibular gland of rats was doubly ligated with metal clips from 1 to 14 days for induction of gland atrophy. Control rats were untreated. Fas and FasL expression in the atrophied submandibular gland was detected using immunohistochemistry (IHC) and Western immunoblot. Expression of activated caspase 8 and activated caspase 3 was also detected with IHC. Fas-positive acinar and duct cells and FasL-positive duct cells increased in the atrophic glands at 3 and 5 days after duct ligation when apoptotic cells were commonly observed. Thereafter, Fas- and FasL-positive cells declined in number. Patterns of expression of Fas and FasL using Western immunoblots concurred with the IHC results. Activated caspase 8-positive cells were present at every time interval but peaked at 3 and 5 days following duct ligation. The cells showing immunoreaction for activated caspase 3 first appeared on day 3, with the peak in apoptosis, after which they decreased. The results indicate that the Fas/FasL systems likely play an important role in apoptotic pathways during atrophy of the submandibular gland.

Biomimetic porous scaffolds with high elasticity made from mineralized collagen--an animal study.

Histological investigations of a new hydroxyapatite-collagen composite material were carried out to evaluate its possible suitability as a bone substitute. The three-dimensional scaffolds made from biomimetically mineralized collagen exhibit an interconnecting pore structure and elastic mechanical properties. They were implanted into the subcutaneous tissue and bone defects made in the femur of rats and harvested with the surrounding tissue at 1, 2, 4, 8, and 12 weeks after surgery. The materials implanted in the subcutaneous tissue were covered by fibrous connective tissue with a slight inflammatory response, and many foreign-body giant cells were observed on the surface of the scaffolds. Most of the material implanted in the subcutaneous tissue was resorbed at 8 weeks by phagocytosis. In the bone defects, new bone formation was observed on the surface of the material at 1 week. New bone increased with time, and osteoclasts were seen on the surface of the scaffolds at 2 weeks. Resorption and replacement by new bone of many parts of the materials implanted in the femur were observed by 12 weeks. These responses occurred faster than those of other hydroxyapatite-collagen composites. The results suggested that the new biomimetically mineralized collagen scaffolds were suitable as an implant material for bone-tissue reconstruction.

Adenovirus E4orf6 targets pp32/LANP to control the fate of ARE-containing mRNAs by perturbing the CRM1-dependent mechanism.

E4orf6 plays an important role in the transportation of cellular and viral mRNAs and is known as an oncogene product of adenovirus. Here, we show that E4orf6 interacts with pp32/leucine-rich acidic nuclear protein (LANP). E4orf6 exports pp32/LANP from the nucleus to the cytoplasm with its binding partner, HuR, which binds to an AU-rich element (ARE) present within many protooncogene and cytokine mRNAs. We found that ARE-mRNAs, such as c-fos, c-myc, and cyclooxygenase-2, were also exported to and stabilized in the cytoplasm of E4orf6-expressing cells. The oncodomain of E4orf6 was necessary for both binding to pp32/LANP and effect for ARE-mRNA. C-fos mRNA was exported together with E4orf6, E1B-55kD, pp32/LANP, and HuR proteins. Moreover, inhibition of the CRM1-dependent export pathway failed to block the export of ARE-mRNAs mediated by E4orf6. Thus, E4orf6 interacts with pp32/LANP to modulate the fate of ARE-mRNAs by altering the CRM1-dependent export pathway.

Biological behavior of hat-stacked carbon nanofibers in the subcutaneous tissue in rats.

The tissue response to hat-stacked carbon nanofibers (H-CNFs) was evaluated. H-CNFs were implanted in the subcutaneous tissue of rats. Histological and ultrastructural investigations were carried out by transmission electron microscopy. Although many macrophages and foreign body giant cells were seen around H-CNFs, no severe inflammatory response such as necrosis was observed. Some H-CNFs were observed in lysosomal vacuoles of phagocytes. These results showed that H-CNFs were not strong prophlogistic substances and were englobed in vivo.

E1AF expression is closely correlated with malignant phenotype of tongue squamous cell carcinoma through activation of MT1-MMP gene promoters.

E1AF is an ets-oncogene family transcription factor that has been shown to up-regulate multiple MMPs whereas MMP-2, a potent extracellular matrix degrading enzyme, is not up-regulated. We investigated the activation mechanism of MMP-2 in oral squamous cell carcinoma. Chloramphenicol acetyltransferase (CAT) assay was utilized to investigate whether E1AF is able to up-regulate membrane type-1 matrix metalloproteinase (MT1-MMP), which is known to activate MMP-2. Expression of the CAT reporter gene under the control of the MT1-MMP promoter was increased approximately 40-fold by co-transfection with the E1AF expression vector. Real-time RT-PCR study was carried out in 25 patients with tongue squamous cell carcinoma, and the mRNA expression level of E1AF and MT1-MMP was synergistically increased. These results indicate that E1AF positively regulates transcription from MT1-MMP genes, which plays an important role in invasion and metastasis of squamous cell carcinoma of the tongue by converting pro-MMP-2 into active-MMP-2.

E1AF degradation by a ubiquitin-proteasome pathway.

E1AF is a member of the ETS family of transcription factors. In mammary tumors, overexpression of E1AF is associated with tumorigenesis, but E1AF protein has hardly been detected and its degradation mechanism is not yet clear. Here we show that E1AF protein is stabilized by treatment with the 26S protease inhibitor MG132. We found that E1AF was modified by ubiquitin through the C-terminal region and ubiquitinated E1AF aggregated in nuclear dots, and that the inhibition of proteasome-activated transcription from E1AF target promoters. These results suggest that E1AF is degraded via the ubiquitin-proteasome pathway, which has some effect on E1AF function.

Influence of length on cytotoxicity of multi-walled carbon nanotubes against human acute monocytic leukemia cell line THP-1 in vitro and subcutaneous tissue of rats in vivo.

Carbon nanotubes (CNTs) are single- or multi-cylindrical graphene structures that possess diameters of a few nanometers, while the length can be up to a few micrometers. These could have unusual toxicological properties, in that they share intermediate morphological characteristics of both fibers and nanoparticles. To date, no detailed study has been carried out to determine the effect of length on CNT cytotoxicity. In this paper, we investigated the activation of the human acute monocytic leukemia cell line THP-1 in vitro and the response in subcutaneous tissue in vivo to CNTs of different lengths. We used 220 nm and 825 nm-long CNT samples for testing, referred to as "220-CNTs" and "825-CNTs", respectively. 220-CNTs and 825-CNTs induced human monocytes in vitro, although the activity was significantly lower than that of microbial lipopeptide and lipopolysaccharide, and no activity appeared following variation in the length of CNTs. On the other hand, the degree of inflammatory response in subcutaneous tissue in rats around the 220-CNTs was slight in comparison with that around the 825-CNTs. These results indicated that the degree of inflammation around 825-CNTs was stronger than that around 220-CNTs since macrophages could envelop 220-CNTs more readily than 825-CNTs. However, no severe inflammatory response such as necrosis, degeneration or neutrophil infiltration in vivo was observed around both CNTs examined throughout the experimental period.

E1AF, an ets-oncogene family transcription factor.

E1AF is an ets-oncogene family transcription factor. E1AF was shown to upregulate multiple matrix metalloproteinase (MMP) genes and contribute to the malignant phenotype of cancer cells by inducing invasive and metastatic activities. E1AF is upregulated by hepatocyte growth factor (HGF) stimulation, which indicates that E1AF would participate in cell motility by HGF/scatter factor. On the other hand, E1AF upregulates p21waf1/cip1 to induce cell cycle arrest when cells are exposed to stress. EWS/ETS fusions are frequently observed in Ewing's sarcoma, and we have revealed that EWS/ETS chimeric protein activates telomerase activity by upregulating hTERT. However, substitution ets binding site (EBS) mutants did not affect the responsiveness to EWS/E1AF. DNA-IP assay showed that the complexes contained EWS/E1AF bound to the hTERT promoter, which suggested that EWS/ETS functions as a co-activator for TERT transcription. Our findings that EWS/ETS acts as a transcriptional co-factor may imply that the transcription pathway is regulated by the interaction of transcription factors.

Bisphenol A from dental polycarbonate crown upregulates the expression of hTERT.

Bisphenol A (BPA) is one of the endocrine-disrupting chemicals (EDCs) that possess estrogen-like biologic activity. Many dental materials have been reported to release BPA. However, there are few reports available on the release of BPA from dental polycarbonates. The purpose of this study was to investigate the release of BPA from dental polycarbonate crowns and to evaluate the estrogenic activity of BPA. Polycarbonate crowns were immersed in five solvents (water, ethanol, n-hepthane, acetic acid, and acetonitrile) at 37 or 65 degrees C for 24 h. The elution from the material was analyzed by high-performance liquid-chromatography (HPLC) and mass-spectrometry (MS) analysis. BPA release was detected corresponding to the degradation of dental polycarbonates under the some storage conditions (ethanol, acetic acid, and acetonitrile). A previous report proved that estrogen increased human telomerase catalytic subunit (hTERT) mRNA, whereas the effect of EDCs on the hTERT promoter has never been reported. The estrogenic activity of BPA was analyzed by luciferase assay with the use of the hTERT promoter. This assay revealed that BPA was a positive regulator of hTERT transcription. In addition, quantitative real-time PCR analysis showed that BPA increased the expression level of hTERT mRNA in MCF7 cells. Herein, it is demonstrated that hTERT is a new target of BPA.

EWS/ETS fusions activate telomerase in Ewing's tumors.

EWS/ETS is a chimeric protein identified in most Ewing's sarcomas. Although EWS/ETS has been shown to activate transcription as a transcription factor, the detailed targets of EWS/ETS in transformed cells have not been clarified. Herein, we demonstrate that telomerase is a new target of EWS/ETS fusions. Both telomerase activity and the expression level of telomerase reverse transcriptase (TERT) mRNA were up-regulated in NIH3T3 cells transformed by EWS/E1AF and EWS/FLI1 as well as in two Ewing's sarcoma cell lines. Luciferase assay using the TERT promoter revealed that EWS/E1AF and EWS/FLI1 function as positive regulators of TERT transcription in an ETS binding site-independent manner. EWS/ETS appeared to be included in the initiation complex of TERT transcription and to cooperate with CREB-binding protein (CBP)/p300. When EWS/FLI1 was knocked down in Ewing's sarcomas cells by RNA interference, the expression level of TERT mRNA and the telomerase activity were significantly decreased. These findings indicate that EWS/ETS fusion proteins activate human telomerase activity in Ewing's tumors through up-regulation of TERT gene expression, probably as a transcriptional coactivator.

Ultrastructure of ceramic-bone interface using hydroxyapatite and beta-tricalcium phosphate ceramics and replacement mechanism of beta-tricalcium phosphate in bone.

Hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP) are useful for grafting and augmentation of bone tissue. Observation by transmission electron microscopy (TEM) was done to investigate the ultrastructures at the interfaces between the biomaterials and the adjacent tissue, and osteogenesis around the biomaterials in the present study. HA and beta-TCP ceramics were used in disk forms which had macropores and micropores, and were implanted between the parietal bone and the cranial periosteum of rats. Specimens were prepared for observation at 4 and 8 weeks postoperatively. The microscopic results indicated that an intervening layer was present on the surface of HA, whereas it was not present on the surface of beta-TCP. A characteristic fibrillar structure was observed in the intervening layer between HA and bone under decalcification by HCl. In beta-TCP, in reticular structures observed close to the bone tissue by optical microscopy, calcification and sparse collagen fibers were interspersed among the granules of beta-TCP. In addition, close to the interface between beta-TCP and bone, many osteocytes with numerous processes were present. Some processes were elongated towards the interface. These results revealed the difference in the ultrastructures of the interfaces between HA and beta-TCP, and the dissolution mechanism of beta-TCP in bone.

Immunohistochemical localization of macrophage migration inhibitory factor (MIF) in human gingival tissue and its pathophysiological functions.

Recent reports have indicated that macrophage migration inhibitory factor (MIF) plays a key role in systemic as well as local inflammatory and immune responses. In this study, the presence and localization of MIF in human gingival tissue were examined. The expression of MIF was confirmed by western blot analysis, which demonstrated the same band at 12.5 kDa in different gingival tissues. Immunohistochemical studies showed that MIF protein existed in the cytoplasm of keratinocytes, especially in free gingival epithelium and junctional epithelium. It was also found in basal cells, fibroblasts, and various cells. These cells were considered to be stimulated mechanically at all times. To determine the effect of mechanical stimuli, Gin-1 cells were cyclically stretched for a short time by using a Flexercell Strain Unit. RT-PCR analysis demonstrated upregulation of MIF mRNA in these Gin-1 cells. In this study, MIF existed not only in inflammatory parts but also in those areas with high cell proliferative activity subjected to external stimulus. Moreover, the finding that MIF protein levels of the control determined by immunohistochemical detection were quite similar to those for grown and stretched Gin-1 cells suggested that MIF protein was stored in the cytoplasm for some time and that MIF is an important autocrine mediator of homeostatic-dependent signaling events. These results suggest that MIF plays an important role in the homeostatic process of periodontal inflammation.

Radioresistance in oral squamous cell carcinoma with p53 DNA contact mutation.

Reliable variables to predict the radiosensitivity of each tumor have not been identified. Recent studies have demonstrated that specific regions of mutations within the core domain of p53 protein correlate with responses to chemotherapy and radiotherapy in some tumor types. In this study, we evaluated the relationship between specific p53 mutations and radiosensitivity in 49 patients with oral squamous cell carcinomas (SCCs) who underwent preoperative radiotherapy. Exons 5 through 8 of the p53 gene were examined by polymerase chain reaction-single-strand conformation polymorphism and direct sequencing. We detected p53 mutations in 27 (55.1%) cases. DNA contact mutations were detected in 11 (40.7%) of these 27 cases in L3 loop, loop-sheet-helix motif, and zinc-binding residues. Tumors containing p53 DNA contact mutations had significantly poorer responses to radiation than the other tumors, although no statistically significant difference between tumors with and without p53 mutations was found. These data indicate that DNA contact mutation of p53 could be a useful marker to predict the radioresistance of oral SCCs.

Nuclear export of adenovirus E4orf6 protein is necessary for its ability to antagonize apoptotic activity of BH3-only proteins.

The adenovirus E4orf6 is a viral oncoprotein known to cooperate with the E1A gene product in transforming primary murine cells. It has been shown to inhibit the apoptotic activities of p53 and p73 through direct binding to these proteins. Here, we demonstrate that the adenovirus E4orf6 protein inhibits apoptosis mediated by BNIP3 and Bik, which are BH3-only proteins of the Bcl-2 family. This activity was not mediated by p53 and p73 because E4orf6 had the same effect on the apoptosis in Saos-2 cells that do not express p53-related genes. It was also ascertained that E4orf6 could change the mitochondrial localization of BNIP3 and Bik. A mutant lacking the nuclear export signal of E4orf6 failed to inhibit apoptosis and to translocate BNIP3 protein from the mitochondria. Moreover, it was also established that E4orf6 was able to interact with BNIP3 and Bik. In BNIP3 protein, the region required for the interaction included the transmembrane domain, which is required for the localization of BNIP3 to the mitochondria. These results suggest that E4orf6 is exported from the nucleus to the cytoplasm, enabling it to interact with BH3-only proteins, eventually leading to the inhibition of apoptotic activity.

Bone augmentation osteogenesis using hydroxyapatite and beta-tricalcium phosphate blocks.

PURPOSE: In this study, we investigated the differences in osteogenesis and resorption between hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP) implanted on the parietal bone of rats. MATERIALS AND METHODS: HA and beta-TCP were used in blocks with macropores and micropores. They were implanted between the parietal bone and the cranial periosteum in rats. Osteogenesis around the implanted materials was investigated histopathologically and histomorphometrically at 1, 2, 4, 8, and 24 weeks after surgery. RESULTS: At 2 weeks, osteogenesis from the parietal bone was observed around both materials, and new bone had attached directly to the surfaces of both materials. New bone grew into the pores of the upper regions of both materials with time. The beta-TCP block had a characteristic basophilic reticular structure in which the dissolution of the materials was observed close to the new bone. The HA blocks were stable for 24 weeks, whereas parts of the beta-TCP blocks were fractured and resorbed at 24 weeks. Histomorphometrically, the volume of new bone around HA was larger than that around beta-TCP. There was no remarkable change in the amount of remaining HA, but that of beta-TCP was decreased. CONCLUSION: HA blocks in this model are suitable for onlay grafts because of its stability and osteogenesis, beta-TCP is not stable. Therefore, when beta-TCP blocks are used for onlay grafts, the mechanical stress on the recipient site should be taken into consideration because of resorption and fracture.

Tissue response to a newly developed calcium phosphate cement containing succinic acid and carboxymethyl-chitin.

We developed a new calcium phosphate cement containing succinic acid and carboxymethyl-chitin in the liquid component. In this study, the biocompatibility and osteoconductivity of this new cement were investigated. After mixing, cement in putty form was implanted immediately between the periosteum and parietal bone and in the subcutaneous tissues of rats. In control cement, distilled water was used instead of the liquid component. In addition to histological evaluations, analyses with X-ray diffraction and Fourier transform infrared were performed for the subcutaneously implanted cements. Histological examination showed slight inflammation around the new cement on the bone and in the subcutaneous tissue at 1 week after surgery. At 2 weeks, the cement was partially bound to the parietal bone. The extent of the surface of the new cement directly in contact with the bone increased with time, and most of the undersurface of the new cement bound to the host parietal bone by 8 weeks. Analysis by X-ray diffraction showed that the new cement in the subcutaneous tissue was transformed into hydroxyapatite by 8 weeks. These results indicate that this new calcium phosphate cement is useful as a bone substitute material.

Mucinous adenocarcinoma of probable minor salivary gland origin.

Mucinous adenocarcinoma is characterized by large pools of extracellular mucin. The tumor has been reported in the nasal cavity, paranasal sinus, breast, colon, stomach, prostate, skin, and lung. Mucinous adenocarcinoma also arises as a primary tumor of the major salivary glands. However, its occurrence is exceedingly rare and has only recently been recognized. Only 9 cases in the major salivary glands have been reported. We present an additional case of MAC in the maxilla that was considered to have developed from a palatal minor salivary gland.