Flavinated SDHA underlies the change in intrinsic optical properties of oral cancers.

The molecular basis of reduced autofluorescence in oral squamous cell carcinoma (OSCC) cells relative to normal cells has been speculated to be due to lower levels of free flavin adenine dinucleotide (FAD). This speculation, along with differences in the intrinsic optical properties of extracellular collagen, lies at the foundation of the design of currently-used clinical optical detection devices. Here, we report that free FAD levels may not account for differences in autofluorescence of OSCC cells, but that the differences relate to FAD as a co-factor for flavination. Autofluorescence from a 70 kDa flavoprotein, succinate dehydrogenase A (SDHA), was found to be responsible for changes in optical properties within the FAD spectral region, with lower levels of flavinated SDHA in OSCC cells. Since flavinated SDHA is required for functional complexation with succinate dehydrogenase B (SDHB), decreased SDHB levels were observed in human OSCC tissue relative to normal tissues. Accordingly, the metabolism of OSCC cells was found to be significantly altered relative to normal cells, revealing vulnerabilities for both diagnosis and targeted therapy. Optimizing non-invasive tools based on optical and metabolic signatures of cancers will enable more precise and early diagnosis leading to improved outcomes in patients.

Effectively improved 3-dimensional structural stability of atelocollagen-gelatin sponge biomaterial by heat treatment.

Atelocollagen-gelatin (ACG) sponge was fabricated from atelocollagen and gelatin by lyophilization without introducing toxic substances. This study aimed to investigate the effects of heat treatment on the 3-dimensional structural stability of ACG sponge biomaterial. ACG sponge samples were fabricated and heat treated at 125oC for 12 h in the vacuum. The results revealed that heat treatment did not affect porosity, pore size and mechanical compressive strength. Heat-treated ACG sponge showed decreased absorbance and peak shift of amid I (C=O) stretches, slightly higher water uptake degree and significantly decreased in vitro degradation rate. Moreover, heat-treated ACG sponge maintained good 3-dimensional surface morphology and porous microstructure throughout 7 days, while non-heat-treated ACG sponge collapsed in less than 24 h. The human mesenchymal stromal cells (hMSCs) were shown to adhere and grow well on heat-treated ACG sponges. These results indicate that heat treatment is effective and safe to stabilize 3-dimensional ACG sponge biomaterial for tissue engineering.

Effects of 2% sodium fluoride solution on the prevention of streptococcal adhesion to titanium and zirconia surfaces.

Streptococci are associated with dental plaque formation as the early-colonizing bacteria that adhere to titanium (CpTi) and zirconia (TZP) implant abutment surfaces. Effective prevention of peri-implantitis may be possible by removing streptococci as target. This study aimed to evaluate the effects of 2% NaF on the prevention of streptococcal adhesion to CpTi and TZP. After immersion in 2% NaF for 90 min, surface characterization of mirror-polished CpTi and TZP disks were assesed using XPS, EPMA, and SEM. S. sanguinis, S. gordonii, and S. oralis were used as the streptococcal bacterial strains. After 24 h culture, bacterial adhesion was evaluated using an ATP-bioluminescent assay and SEM. In XPS, EPMA, and SEM analyses, fluoride was detected on the CpTi and TZP surfaces after 2% NaF immersion with no signs of localization, and no corrosion on the CpTi disks. Based on the adhesion assay, the adherences of S. sanguinis, S. gordonii, and S. oralis were significantly lower with NaF than without NaF in CpTi (p = 0.005, 0.001, and 0.001, respectively) and TZP (p = 0.003, 0.002, and 0.001). This was also confirmed by SEM. In conclusion, 2% NaF reduced the adhesion of streptococci to the CpTi and TZP surfaces.

Adhesion of streptococci to titanium and zirconia.

The purpose of this study was to evaluate the adherence of streptococci to disks of titanium (commercially pure titanium: CpTi) and zirconia (tetragonal zirconia polycrystals: TZP). CpTi and yttria-stabilized TZP disks with a mirror-polished surface were used as specimens. The arithmetic mean surface roughness (Ra and Sa) and the surface wettability of the experimental specimens were measured. For analyzing the outermost layer of the experimental specimens, X-ray photoelectron spectroscopy (XPS) analysis was performed. Streptococcus sanguinis, S. gordonii, S. oralis, and S. mutans were used as streptococcal bacterial strains. These bacterial cultures were grown for 24 h on CpTi and TZP. The number of bacterial adhesions was estimated using an ATP-bioluminescent assay, and scanning electron microscope (SEM) observation of the adhered bacterial specimens was performed. No significant differences in surface roughness or wettability were found between CpTi and TZP. In XPS analyses, outermost layer of CpTi included Ti0 and Ti4+, and outermost layer of TZP included Zr4+. In the cell adhesion assay, the adherences of S. sanguinis, S. gordonii, and S. oralis to TZP were significantly lower than those to CpTi (p < 0.05); however, significant difference was not observed for S. mutans among the specimens. The adherence to CpTi and TZP of S. mutans was significantly lower than that of S. sanguinis, S. gordonii, and S. oralis. These results were confirmed by SEM. S. sanguinis, S. gordonii, and S. oralis adhered less to TZP than to CpTi, but the adherence of S. mutans was similar to both surfaces. S. mutans was less adherent compare with the other streptococci tested in those specimens.

Bone marrow stromal cells from low-turnover osteoporotic mouse model are less sensitive to the osteogenic effects of fluvastatin.

This study aimed to investigate the effects of fluvastatin on the differentiation of bone marrow stromal cells (BMSCs) into osteoblasts in senescence-accelerated mouse prone 6 (SAMP6) compared with that in the normal senescence-accelerated-resistant mouse (SAMR1) model. SAMP strains arose spontaneously from the AKR/J background and display shortened life span and an array of signs of accelerated aging, compared with control SAMR strains. The dose effects of fluvastatin were also evaluated. BMSCs were cultured with/without fluvastatin (0 µM, 0.1 µM, 0.5 µM, and 1.0 µM). WST-1-based colorimetry was performed to evaluate cell proliferation. To evaluate cell differentiation, gene expression levels of bmp2 and runx2 were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR), and protein expression levels were determined using enzyme-linked immunosorbent assay (BMP2) and immunofluorescence staining (BMP2 and Runx2). Alkaline phosphatase (ALP) activity assay and histochemical detection were determined; the effect of noggin, a BMP-specific antagonist, was examined using ALP histochemical detection. To assess for mature osteogenic marker, gene expression levels of bglap2 were determined by qRT-PCR and mineralization was determined by alizarin red staining. RhoA activity was also examined by Western blotting. In SAMP6, BMP2, Runx2 and Bglap2 mRNA and protein expressions were significantly increased by fluvastatin, and ALP activity was increased by BMP2 action. RhoA activity was also inhibited by fluvastatin. The concentration of fluvastatin sufficient to increase BMP2 and Runx2 expression and ALP activity was 0.5 µM in SAMP6 and 0.1 µM in SAMR1. In conclusion, the present study revealed that fluvastatin promoted BMSC differentiation into osteoblasts by RhoA-BMP2 pathway in SAMP6. BMSCs of SAMP6 are less sensitive to the osteogenic effects of fluvastatin than SAMR1.

Release properties of atelocollagen-gelatin complexes as carriers for local administration of fluvastatin.

The aim of this study was to investigate properties of atelocollagen/gelatin complexes (AC/Gel) and their characteristics of sustained statin release, to assess the utility of AC/Gel. AC/Gel were prepared by changing the mixing ratio of AC (0 to 40% of AC). Analysis of spectra of fluvastatin (Flu), gelatin (Gel), and Flu with Gel complex using a Fourier transform-infrared spectrometer indicates that Flu was bound to Gel through a bond involving the carboxyl and amino groups. Evaluation of characteristics of sustained release of Flu from the AC/Gel using an ultraviolet-visible spectrophotometer showed that the release rate of Flu decreased with increasing the AC content. The histological evaluation using of Sprague-Dawley rats suggest that, unlike the pure Gel sponge, the AC/Gel was not absorbed in an early stage. Therefore, the present study showed that sustained Flu release can be controlled by using an AC/Gel, suggesting the utility of this composite material.

Influence of lyophilization factors and gelatin concentration on pore structures of atelocollagen/gelatin sponge biomaterial.

This study aimed to investigate influences of lyophilization factors and gelatin concentration on pore structures of ACG sponge. ACG sponges of different freezing temperatures (-30, -80 and -196oC), freezing times (1, 2 and 24 h), gelatin concentrations (0.6%AC+0.15%G, 0.6%AC+0.6%G and 0.6%AC+2.4%G), and with 500 µM fluvastatin were fabricated. Pore structures including porosity and pore size were analyzed by scanning electron microscopy and ImageJ. The cytotoxic effects of ACG sponges were evaluated in vitro. Freezing temperature did not affect porosity while high freezing temperature (-30oC) increased pore size. The high gelatin concentration group (0.6%AC+2.4%G) had decreased porosity and pore size. Freezing time and 500 µM fluvastatin did not affect pore structures. The cytotoxicity and cell proliferation assays revealed that ACG sponges had no cytotoxic effects on human mesenchymal stromal cell growth and proliferation. These results indicate that ACG sponge may be a good biomaterial scaffold for bone regeneration.

Effect of Physicochemical Surface Modifications on Bovine Serum Albumin Adsorption to Tetragonal Zirconia Polycrystal in vitro Through the change of the Zeta Potential.

The technology of physicochemical surface modification is available for enhancing the bioactivity and osseointegration capability of tetragonal zirconia polycrystal (TZP). Hydrophobicity index and electrical charge play important roles in protein adsorption. We previously studied the mechanism underlying the adsorption of bovine serum albumin (BSA) on the surfaces of dental materials and hydroxyapatite in vitro. The aim of the present study was to clarify the correlation among the adsorption of BSA to TZP and physicochemically modified TZP surfaces and the zeta potential of BSA and TZP. We used TZP that was sintered at 1350°C for 2 h in air because this kind of TZP is widely applied in the field of dentistry. Surface physicochemistry was modified with ultraviolet light (UV) and atmospheric-pressure plasma treatment. The zeta potentials were measured with ELSZ-1000 and ELSZ-2000 analyzers (Otsuka Electronics, Hirakata, Japan). All experiments were conducted in 10 mM NaCl (pH 7.0). The zeta potentials of as-received TZP and BSA were negative, but those of UV- and plasma-treated TZP were positive. The reason the zeta potentials of TZP changed positive by physicochemical modification is due to an increase in the amount of basic hydroxyl groups. The zeta potentials of UV- and plasma-treated TZP after BSA adsorption were negative. These results suggested that electrostatic interactions play an important role in BSA adsorption to TZP and modified TZP surfaces, so that this modified surface may control the adsorption of protein.

Antimicrobial effect of titanium dioxide after ultraviolet irradiation against periodontal pathogen.

We focused on the antimicrobial effects of titanium dioxide (TiO2) after stopping ultraviolet (UV) irradiation as an adjunctive treatment for peri-implantitis in this study. The aim was to determine the continuous photocatalytic effects of TiO2 after UV irradiation and its antimicrobial activity against periodontal pathogen. The continuous photocatalytic effects of TiO2 after UV irradiation were determined by electron spin resonance (ESR) spectroscopy using TiO2 particles of various sizes with various UV irradiation times. In addition, antimicrobial activity against Porphyromonas gingivalis was investigated by quantitation of colony-forming units (CFUs). The results showed that the ESR signal ratio for the UV-irradiated TiO2 was significantly higher than that of the non-irradiated TiO2. UV-irradiated TiO2 significantly reduced the number of P. gingivalis when compared with non-irradiated controls. These results suggest that TiO2 has a continuous photocatalytic effect even after stopping UV irradiation and that it showed antimicrobial activity against periodontal pathogen.

Proliferation and osteogenic differentiation of human mesenchymal stem cells on zirconia and titanium with different surface topography.

The purpose of this study was to elucidate behavior of human mesenchymal stem cells (hMSCs) on yttria stabilized tetragonal zirconia polycrystals (TZP) and commercial pure titanium (CpTi) with different surface topography. Mirror-polished (MS), sandblasted with 150-µm alumina (SB150) and SB150 acid-etched (SB150E) were prepared on TZP and CpTi. Proliferation, osteogenic differentiation of hMSCs was evaluated. The scanning electron microscopy showed that micro- and nano-topographies were created on both TZP and CpTi SB150E surfaces. The proliferation ability, ALP activity, expression of Runx2 on the both SB150E specimens was significantly higher than those on the other specimens. These results suggested that creation of micro- and nano-topographies on TZP and CpTi by blast and acid-etching may offer a promising method for enhancing the proliferation and differentiation of hMSCs in clinical application.

Influence of various superhydrophilic treatments of titanium on the initial attachment, proliferation, and differentiation of osteoblast-like cells.

The purpose of this study was to investigate the influence of superhydrophilic treatments of titanium on the behavior of osteoblastlike cells. Superhydrophilic specimens were prepared with sandblast and acid-etching (DW), oxygen plasma (Plasma) and ultraviolet light (UV), and were stored in distilled water for 3 days immediately after these treatments. Specimens stored in air for 3 weeks were used as a control Air group. Initial cell attachment, proliferation, alkaline phosphatase activity, and osteocalcin secretion of mouse osteoblast-like cells MC3T3-E1 were enhanced more on superhydrophilic groups than were Air specimens. On confocal laser scanning microscope images of cell morphology, the expression of actin filaments was observed on the superhydrophilic groups, whereas relatively little actin filament expression was seen on the Air surfaces on all culture periods. These results indicate that DW, Plasma, or UV treatment has potential for the creation and maintenance of superhydrophilic surfaces and the enhancement of the initial attachment, proliferation, and differentiation of osteoblast-like cells.

Influence of plasma and ultraviolet treatment of zirconia on initial attachment of human oral keratinocytes: expressions of laminin γ2 and integrin ß4.

Initial attachment of human oral keratinocytes cultured on yttria-stabilized tetragonal zirconia polycrystal (TZP) surfaces that were subjected to UV or oxygen plasma (O2-plasma) treatment was investigated. The viability of the attached cells, mRNA expression of laminin γ2 and integrin ß4, distribution of laminin γ2 and integrin ß4, cell area, and cell morphology were assessed. The results showed that no differences in the viability of attached cells were recognized among the conditions. However, expression of laminin γ2 and integrin ß4 as well as cell morphology were promoted only in O2-plasma specimens even though superhydrophilicity was obtained in both the UV and O2-plasma specimens compared with the untreated control specimen. The photocatalytic activity was believed to be closely involved in the above-mentioned differences. The results of this study suggest that TZP surface treated with oxygen plasma promotes the initial attachment capability of human oral keratinocytes with enhancing the extracellular matrix such as laminin γ2.

In vitro adherence of periodontopathic bacteria to zirconia and titanium surfaces.

Tetragonal zirconia polycrystals (TZP) has drawn attention as a potential alternative to titanium (Ti) in dental implant treatment, as it minimizes both allergic reactions and esthetic problems. It is also important for dental implants to maintain plaque-free surfaces to prevent peri-implantitis. The purpose of this study was to investigate in vitro adherence of periodontopathic bacteria to TZP comparing with Ti.Periodontopathic bacteria were cultured on polished discs of two kinds of TZP, and Ti as a control. After incubation, the numbers of adherent bacteria were estimated. No significant differences among specimens were observed in the initial attachment, although a decrease was observed in initial attachment to saliva-coated specimens. In the bacterial colonization, no significant differences were recognized among specimens. The adherence of the periodontopathic bacteria on TZP was similar to that on Ti. These results suggest that a strategy is required for inhibition of the bacterial adherence to TZP.

Osteogenic effect of fluvastatin combined with biodegradable gelatin-hydrogel.

The aim was to investigate the local osteogenic effect of fluvastatin incorporated into a biodegradable gelatin-hydrogel (GH) scaffold. The GH scaffolds were prepared through crosslinking by ultraviolet irradiation followed by freeze-drying. Two circular defects were surgically created on fifteen-week-old male rats calvaria. All defects of each rat were randomly filled with two of three treatments, specifically: fluvastatin incorporated into a GH disk (Flu-GH), distilled water incorporated into a GH disk, and no treatment. New bone formation was quantitatively analyzed after 7, 14, and 28 days using a micro-computed tomography (micro-CT) system, and histologically observed. Evaluation by micro-CT revealed a significant difference in new bone formation among the three kinds of defect. A highly osteogenic effect was observed in the Flu-GH group. The results showed that the fluvastatin incorporated into a biodegradable GH scaffold promoted osteogenesis in rat calvarial bone, indicating its potential for bone regeneration.

Siglec-15 protein regulates formation of functional osteoclasts in concert with DNAX-activating protein of 12 kDa (DAP12).

Osteoclasts are multinucleated giant cells that reside in osseous tissues and resorb bone. Signaling mediated by receptor activator of nuclear factor (NF)-κB (RANK) and its ligand leads to the nuclear factor of activated T cells 2/c1 (NFAT2 or NFATc1) expression, a critical step in the formation of functional osteoclasts. In addition, adaptor proteins harboring immunoreceptor tyrosine-based activation motifs, such as DNAX-activating protein of 12 kDa (DAP12), play essential roles. In this study, we identified the gene encoding the lectin Siglec-15 as NFAT2-inducible, and we found that the protein product links RANK ligand-RANK-NFAT2 and DAP12 signaling in mouse osteoclasts. Both the recognition of sialylated glycans by the Siglec-15 V-set domain and the association with DAP12 through its Lys-272 are essential for its function. When Siglec-15 expression was knocked down, fewer multinucleated cells developed, and those that did were morphologically contracted with disordered actin-ring structures. These changes were accompanied by significantly reduced bone resorption. Siglec-15 formed complexes with Syk through DAP12 in response to vitronectin. Furthermore, chimeric molecules consisting of the extracellular and transmembrane regions of Siglec-15 with a K272A mutation and the cytoplasmic region of DAP12 significantly restored bone resorption in cells with knocked down Siglec-15 expression. Together, these results suggested that the Siglec-15-DAP12-Syk-signaling cascade plays a critical role in functional osteoclast formation.

Detection of Campylobacter rectus in periodontitis sites by monoclonal antibodies.

Campylobacter rectus, a gram-negative, microaerophilic, and motile bacterium, has been proposed to play a pathogenic role in human periodontitis. Surface components, such as the flagellum, surface layer (S-layer), and cytotoxin, have been reported as possible virulence factors of the microorganism. In the present study, monoclonal antibodies against surface components of this bacterium were produced to detect and investigate the pathogenic potential of C. rectus in periodontitis. Two monoclonal antibodies, designated CRT-1 and CRT-2, recognized a peculiar 150 kDa S-layer protein by immunoblot analysis. The CRT-2 antibody reacted to all C. rectus strains tested, except for the S-layer negative strain of the species [C. rectus ATCC 33238 S-layer (-) strain]. The CRT-3 antibody reacted to a 60-kDa protein in C. rectus and also cross-reacted with Campylobacter showae ATCC 51164 and CCUG 11641 strains. Using the dot-blot method, we were able to detect C. rectus using the CRT-2 antibody when as few as 103 organisms were present in a subgingival dental plaque sample. Detection of C. rectus in plaque samples correlated significantly with clinical findings such as probing depth (P < 0.001), bleeding on probing (P < 0.001), and gingival index (P < 0.001). These findings indicate that infection by C. rectus may be an important indicator of periodontal disease status.

Heat shock protein of Mycoplasma salivarium and Mycoplasma orale strains isolated from HIV-seropositive patients.

It has been suggested that infection by some mycoplasma species can act as possible cofactors in the acceleration of immunodeficiency in HIV-infected patients. The present study was designed to examine infections by oral mycoplasma species in HIV-seropositive (HIV(+)) patients. Mycoplasma salivarium and Mycoplasma orale were isolated from 59.5% and 16.7% of 42 HIV(+) patients, respectively. Non-M. salivarium and non-M. orale species were isolated from 40.5% of saliva samples from the HIV(+) group and 20.8% of those from 24 HIV-seronegative (HIV(-)) subjects, respectively. Although the production of superantigen by human peripheral lymphocytes in the isolated mycoplasma species from HIV(+) and HIV(-) subjects was evaluated, none of the examined mycoplasma strains, including ATCC strains of M. salivarium, M. orale, Mycoplasma buccae and Mycoplasma penetrans, were found to produce superantigen. Production of heat shock proteins (HSPs) by isolated mycoplasma strains was examined by immunoblotting using monoclonal antibodies against Helicobacter pylori HSP60. It was found that all the strains of M. salivarium, M. orale, and unidentified mycoplasma species isolated from HIV(+) and HIV(-) groups produced heat shock proteins. HSP production by oral mycoplasma may play a role in the immunomodulation of HIV(+) patients.