Katsutoshi Kubo

Cellular behavior on TiO2 nanonodular structures in a micro-to-nanoscale hierarchy model

Biological tissues involve hierarchical organizations of structures and components. We created a micropit-and-nanonodule hybrid topography of TiO(2) by applying a recently reported nanonodular self-assembly technique on acid-etch-created micropit titanium surfaces. The size of the nanonodules was controllable by changing the assembly time. The created micro-nano-hybrid surface rendered a greater surface area and roughness, and extensive geographical undercut on the existing micropit surface and resembled the surface morphology of biomineralized matrices. Rat bone marrow-derived osteoblasts were cultured on titanium disks with either micropits alone, micropits with 100-nm nodules, micropits with 300-nm nodules, or micropits with 500-nm nodules. The addition of nanonodules to micropits selectively promoted osteoblast but not fibroblast function. Unlike the reported advantages of microfeatures that promote osteoblast differentiation but inhibit its proliferation, micro-nano-hybrid topography substantially enhanced both. We also demonstrated that these biological effects were most pronounced when the nanonodules were tailored to a diameter of 300nm within the micropits. An implant biomechanical test in a rat femur model revealed that the strength of bone-titanium integration was more than three times greater for the implants with micropits and 300-nm nanonodules than the implants with micropits alone. These results suggest the establishment of functionalized nano-in-microtitanium surfaces for improved osteoconductivity, and may provide a biomimetic micro-to-nanoscale hierarchical model to study the nanofeatures of biomaterials.

Enhanced osteoblast function on ultraviolet light-treated zirconia

Unlike titanium, surface roughening of zirconia for enhanced bone integration has been technically challenging. The photochemical reaction of semiconductor oxides, e.g., titanium dioxide, has earned considerable and broad interest in environmental and clean energy sciences. This study determined whether ultraviolet (UV) light treatment of zirconia enhances its bioactivity on osteoblasts. Machined zirconia disks were treated with UV light for various time periods up to 48 h. UV light treatment for 48 h increased the rates of attachment, spread, and proliferation of rat bone marrow-derived osteoblasts. Alkaline phosphatase-positive and mineralized nodule areas doubled on UV light-treated zirconia. The expression of osteoblastic genes, such as osteopontin and osteocalcin, was not modulated by UV light treatment. X-ray diffraction and X-ray photoelectron spectroscopy analyses showed that zirconia disks consisted of monoclinic and tetragonal phases of ZrO(2) and Y(2)O(3) having a wide light absorption band of 200-400 nm with its peak at <250 nm. UV light treatment transformed the zirconia surface from hydrophobic to hydrophilic status and reduced the atomic percentage of surface carbon in a UV light dose-dependent manner. These results suggest that UV treatment of yttrium-containing partially stabilized zirconia enhances its bioactivity on osteoblasts, in terms of their attachment, proliferation, and eventually mineralization. This biofunctionalization was associated with UV light-catalytic hydrophilic conversion of zirconia surfaces and progressive removal of hydrocarbons.

Human papillomaviruses in the normal oral cavity of children in Japan

The purpose of this study was to determine the frequency of human papillomavirus (HPV) infections in the normal oral cavity of children in Japan. Oral squamous cell specimens were collected from 77 children (44 boys and 33 girls), aged 3 and 5 years. Extracted DNA was evaluated for HPV infections by polymerase chain reaction (PCR) methods, using consensus primers for the L1 region, specific primers, and direct DNA sequencing analysis. Thirty-seven of 77 specimens (48.1%) were positive for HPV DNA. Positive rates of boys and girls in all specimens were 28.3 (22/77) and 19.5 (15/77)%, respectively. The positive rate in 3-year-old children was 45.2 (14/31)%, and positive rates in boys and girls were 52.6 (10/19) and 33.3 (4/12)%, respectively. The positive rate in 5-year-old children was 50.0 (23/46)%, and positive rates in boys and girls were 48.0 (12/25) and 52.4 (11/21)%, respectively. HPV types were determined by specific PCR and direct DNA sequencing analysis. Frequent HPV types in the specimens of all children were HPV-16 (11/37; 29.7%),-1 (6/37; 16.2%),-2 (6/37; 16.2%),-75 (6/37; 16.2%). The results of the present investigation indicate that many HPVs, including HPV-16 (a high-risk type for cancer), are present in the oral cavity of 3- and 5-year-old children. It is suggested, therefore, that the oral cavity is already a reservoir of HPVs in childhood where later HPV-associated diseases, such as oral cancer and other oral lesions, may develop.

Restored viability and function of dental pulp cells on poly-methylmethacrylate (PMMA)-based dental resin supplemented with N-acetyl cysteine (NAC)

This study examines cytotoxicity of poly-methylmethacrylate (PMMA)-based dental temporary filling resin to dental pulp cells, and the potential amelioration of the toxicity with an anti-oxidant amino-acid, N-acetyl cysteine (NAC). Dental pulp cells extracted from rat maxillary incisors were cultured on the resin material with or without NAC incorporation, or on the polystyrene. The cultures were supplied with osteoblastic media, containing dexamethasone. Forty five percent of cells on the PMMA dental resin were necrotic at 24h after seeding. However, this percentage was reduced to 27% by incorporating NAC in the resin, which was the level equivalent to that in the culture on polystyrene. The culture on the untreated resin was found to be negative for alkaline phosphate (ALP) activity at days 5 and 10 or von Kossa mineralized nodule formation at day 20. In contrast, some areas of the cultures on NAC-incorporated resin substrates were ALP and von Kossa positive. Collagen I and dentin sialoprotein genes were barely expressed in day 7 culture on the untreated resin. However, those genes were expressed in the culture on the resin with NAC. These results suggest that the decreased cell viability and the nearly completely suppressed odontoblast-like cell phenotype of dental pulp cells cultured on PMMA dental resin can be salvaged to a biologically significant degree by the incorporation of NAC in the resin.

TiO2 micro-nano-hybrid surface to alleviate biological aging of UV-photofunctionalized titanium.

Bioactivity and osteoconductivity of titanium degrade over time after surface processing. This time-dependent degradation is substantial and defined as the biological aging of titanium. UV treatment has shown to reactivate the aged surfaces, a process known as photofunctionalization. This study determined whether there is a difference in the behavior of biological aging for titanium with micro-nano-hybrid topography and titanium with microtopography alone, following functionalization. Titanium disks were acid etched to create micropits on the surface. Micro-nano-hybrid surfaces were created by depositioning 300-nm diameter TiO2 nodules onto the micropits using a previously established self-assembly protocol. These disks were stored for 8 weeks in the dark to allow sufficient aging, then treated with UV light for 48 hours. Rat bone marrow–derived osteoblasts were cultured on fresh disks (immediately after UV treatment), 3-day-old disks (disks stored for 3 days after UV treatment), and 7-day- old disks. The rates of cell attachment, spread, proliferation, and levels of alkaline phosphatase activity, and calcium deposition were reduced by 30%–50% on micropit surfaces, depending on the age of the titanium. In contrast, 7-day-old hybrid surfaces maintained equivalent levels of bioactivity compared with the fresh surfaces. Both micropit and micro-nano-hybrid surfaces were superhydrophilic immediately after UV treatment. However, after 7 days, the micro-nano- hybrid surfaces became hydrorepellent, while the micropit surfaces remained hydrophilic. The sustained bioactivity levels of the micro-nano-hybrid surfaces were nullified by treating these surfaces with Cl−anions. A thin TiO2 coating on the micropit surface without the formation of nanonodules did not result in the prevention or alleviation of the time-dependent decrease in biological activity. In conclusion, the micro-nano-hybrid titanium surfaces may slow the rate of time-dependent degradation of titanium bioactivity after UV photofunctionalization compared with titanium surfaces with microtopography alone. This antibiological aging effect was largely regulated by its sustained electropositivity uniquely conferred in TiO2 nanonodules, and was independent of the degree of hydrophilicity. These results demonstrate the potential usefulness of these hybrid surfaces to effectively utilize the benefits of UV photofunctionalization and provide a model to explore the mechanisms underlying antibiological aging properties.

DNA Vaccine against Hamster Oral Papillomavirus-associated Oral Cancer

Previously we developed a carcinogenesis model involving the combination of 9, 10-dimethyl-1, 2-benzanthracene (DMBA) application with physical wounding of hamster lingual mucosa. The presence of a novel hamster oral papillomavirus (HOPV) was demonstrated and its genome sequenced. In the present study, this HOPV hamster model was used to test whether vaccination with the L1 gene could prevent the development of oral carcinoma. DNA plasmids encoding the L1 gene or the vector alone were injected intramuscularly into 20 vaccinated and 20 control hamsters, respectively. The lingual tips of the hamsters were painted with DMBA for 8 weeks. A portion of the lingual tips was excised, and the tips were then painted daily with DMBA until the animals were killed 13 days later. All control hamsters developed lingual carcinoma, whereas 12 of the L1-vaccinated hamsters showed no lesions. These results suggest that immunization with L1 DNA vaccines may prevent the development of papillomavirus-associated oral cancer.

Osteoblast Mechanoresponses on Ti with Different Surface Topographies

During implant healing, mechanical force is transmitted to osteogenic cells via implant surfaces with various topographies. This study tested a hypothesis that osteoblasts respond to mechanical stimulation differently on titanium with different surface topographies. Rat bone-marrow-derived osteoblastic cells were cultured on titanium disks with machined or acid-etched surfaces. A loading session consisted of a 3-minute application of a 10- or 20-μm-amplitude vibration. Alkaline phosphatase activity and gene expression increased only when the cells were loaded in 3 sessions/day on machined surfaces, regardless of the vibration amplitude, whereas they were increased with 1 loading session/day on the acid-etched surface. The loading did not affect the osteoblast proliferation on either surface, but selectively enhanced the cell spreading on the machined surface. Analysis of the data suggests that osteoblastic differentiation is promoted by mechanical stimulation on titanium, and that the promotion is disproportionate, depending on the titanium surface topography. The frequency of mechanical stimulation, rather than its amplitude, seemed to have a key role.

N-Acetyl cysteine (NAC) inhibits proliferation, collagen gene transcription, and redox stress in rat palatal mucosal cells

OBJECTIVES Control of hyperplastic and invasively growing gingival tissue is crucial for maintaining normal oral function and for successful bone regenerative therapy. We tested the hypothesis that materials containing N-acetyl cysteine (NAC), an antioxidant cysteine derivative, can control proliferation and function of oral mucosal cells. METHODS Oral mucosal cells derived from the rat palatal tissue were cultured with or without NAC at different concentrations (2.5-10.0mM). To simulate inflammatory conditions, cultures were treated with hydrogen peroxide. NAC was also applied via collagen materials in membrane and sponge forms to explore the clinical applicability. The redox balance inside the cells was evaluated by measuring the concentration of intracellular glutathione (GSH). RESULTS Adding NAC into cultures of oral mucosal cells reduced their proliferation, transcriptional expression, and collagen production in an NAC-concentration-dependent manner without cytotoxic effects. Furthermore, NAC substantially reduced the hydrogen peroxide-induced elevation of cellular proliferation and collagen production. The controlling effects of NAC were also demonstrated in cells cultured on NAC-containing collagen materials and were associated with an increase in intracellular glutathione (GSH) reserves and a decrease in the oxidized form of glutathione (GSSG). SIGNIFICANCE These results indicate that NAC may abrogate inflammation- or oxidative-stress-induced hyperfunction of oral mucosal cells and that it can be delivered effectively via biodegradable materials. This study provides a basis to explore NAC-containing biomaterials that are functionalized to control oral soft tissue growth and function without cytotoxicity.

N-acetyl cysteine as an osteogenesis-enhancing molecule for bone regeneration

Bone regeneration often requires cues from osteogenesis-inducing factors for successful outcome. N-acetyl cysteine (NAC), an anti-oxidant small molecule, possibly modulates osteoblastic differentiation. This study investigated the potential of NAC as an osteogenesis-enhancing molecule in vitro and in vivo. Various concentrations of NAC (0, 2.5, 5.0, and 10 mM) were added to rat bone marrow stromal cell or osteoblastic cell culture in media with or without dexamethasone. The results showed marked enhancement of alkaline phosphatase activity and mineralized matrix formation together with consistent upregulation of bone-related gene markers such as collagen I, osteopontin, and osteocalcin in the osteoblastic culture with addition of 2.5 or 5.0 mM NAC regardless of the presence of dexamethasone. Micro-CT-based analysis and histological observation revealed that addition of NAC to a collagenous sponge implanted in a critical size cortical bone defect (3.0 mm × 5.0 mm) in rat femur yielded acceleration and completion of defect closure, with thick, compact, and contiguous bone after 6 weeks of healing. In contrast, with sponge alone, only sparse and incomplete bone regeneration was observed during the matching healing period. These results indicate that NAC can function as an osteogenesis-enhancing molecule to accelerate bone regeneration by activating differentiation of osteogenic lineages.

N-acetyl cysteine prevents polymethyl methacrylate bone cement extract-induced cell death and functional suppression of rat primary osteoblasts.

This study examines the cytotoxicity of bone cement extract to osteoblasts and the potential detoxification and restoration of osteoblastic function by an antioxidant amino acid, N-acetyl cysteine (NAC). The osteoblastic cells derived from rat femurs were cultured with extract from polymethyl methacrylate (PMMA)-based bone cement. The calcein and ethidium homodimer staining of the cells after 24-h incubation showed that 23.0% of the cells were dead in the culture with bone cement extract, while the addition of 5 mM NAC into the culture reduced the percentage to 4.3%. Annexin V and propidium iodide-based flow cytometric analysis also revealed that the apoptotic cells present at 15.8% in the culture with bone cement extract was reduced to 2.4% in the culture cotreated with bone cement extract and NAC. Severely suppressed alkaline phosphatase activity and matrix mineralization in the culture with bone cement extract (reduced to 10% and 5%, respectively, compared with the control culture) were restored to a normal level when treated with 5 mM NAC. The bone cement extract-induced, downregulated expression of osteoblastic genes, such as alkaline phosphatase, collagen I, and osteocalcin, was also restored to the baseline level by cotreatment with NAC. The data indicated that the addition of NAC into acrylic bone cement extract remarkably ameliorated the cytotoxicity to osteoblasts and restored their phenotype and function to a biologically significant degree, suggesting the potential usefulness of NAC in developing more biocompatible acrylic bone cement.