Yoshihiko Sugita

Human papillomavirus type 38 infection in oral squamous cell carcinomas.

In this study, 53 paraffin-embedded oral squamous cell carcinoma (OSCC) biopsy specimens were used. Human papillomavirus type 38 (HPV-38) infection was demonstrated in OSCCs using the PCR technique, DNA sequencing analysis, in situ hybridization, and immunohistochemical techniques. Additionally, the correlation between HPV-38 infection and expressions of proliferating cell nuclear antigens (PCNA) or p53 protein was analyzed immunohistochemically. Using consensus primers for the L1 region (L1-PCR), we identified 35 of 53 specimens (66%) as positive for HPV-38 DNA. Furthermore, specimens from patients over 60 years of age revealed a lower prevalence for the HPV-38 (56.7%) than did those below that age (78.3%). Immunohistochemically, positive stainings for PCNA and p53 protein were more frequently detected in HPV-38 positive OSCCs than HPV negative ones. These results indicate that HPV-38 positive OSCCs were higher in proliferative cellular activity than HPV negative ones. Moreover, the findings suggest that HPV-38 infection may cause malignant transformation of the oral mucosal epithelium.

Effects of pico-to-nanometer-thin TiO2 coating on the biological properties of microroughened titanium.

The independent, genuine role of surface chemistry in the biological properties of titanium is unknown. Although microtopography has been established as a standard surface feature in osseous titanium implants, unfavorable behavior and reactions of osteogenic cells are still observed on the surfaces. To further enhance the biological properties of microfeatured titanium surfaces, this study tested the hypotheses that (1) the surface chemistry of microroughened titanium surfaces can be controllably varied by coating with a very thin layer of TiO(2), without altering the existing topographical and roughness features; and (2) the change in the surface chemistry affects the biological properties of the titanium substrates. Using a slow-rate sputter deposition of molten TiO(2) nanoparticles, acid-etched microroughened titanium surfaces were coated with a TiO(2) layer of 300-pm to 6.3-nm thickness that increased the surface oxygen levels without altering the existing microtopography. The attachment, spreading behavior, and proliferation of osteoblasts, which are considered to be significantly impaired on microroughened surfaces compared with relatively smooth surfaces, were considerably increased on TiO(2)-coated microroughened surfaces. The rate of osteoblastic differentiation was represented by the increased levels of alkaline phosphatase activity and mineral deposition as well as by the upregulated expression of bone-related genes. These biological effects were exponentially correlated with the thickness of TiO(2) and surface oxygen percentage, implying that even a picometer-thin TiO(2) coating is effective in rapidly increasing the biological property of titanium followed by an additional mild increase or plateau induced by a nanometer-thick coating. These data suggest that a super-thin TiO(2) coating of pico-to-nanometer thickness enhances the biological properties of the proven microroughened titanium surfaces by controllably and exclusively modulating their surface chemistry while preserving the existing surface morphology. The improvements in proliferation and differentiation of osteoblasts attained by this chemical modification is of great significance, providing a new insight into how to develop new implant surfaces for better osseointegration, based on the established microtopographic surfaces.

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.

Bone integration capability of alkali- and heat-treated nanobimorphic Ti–15Mo–5Zr–3Al

The role of nanofeatured titanium surfaces in a number of aspects of in vivo bone-implant integration, and, in particular, their potential advantages over microfeatured titanium surfaces, as well as their specific contribution to osteoconductivity, is largely unknown. This study reports the creation of a unique nanobimorphic titanium surface comprised of nanotrabecular and nanotuft-like structures and determines how the addition of this nanofeature to a microroughened surface affects bone-implant integration. Machined surfaces without microroughness, sandblasted microroughened surfaces, and micro-nano hybrid surfaces created by sandblasting and alkali and heat treatment of Ti-15Mo-5Zr-3Al alloy were subjected to biomechanical, interfacial and histological analyses in a rat model. The presence of microroughness enabled accelerated establishment of biomechanical implant fixation in the early stages of healing compared to the non-microroughened surfaces; however, it did not increase the implant fixation at the late stages of healing. The addition of nanobimorphic features to the microroughened surfaces further increased the implant fixation by as much as 60-100% over the healing time. Bone area within 50 μm of the implant surface, but not beyond this distance, was significantly increased by the presence of nanobimorphic features. Although the percentage of bone-implant contact was also significantly increased by the addition of nanobimorphic features, the greatest improvement was found in the soft tissue intervention between the bone and the implant, which was reduced from >30% to <5%. Mineralized tissue densely deposited with calcium-binding globular proteins was observed in an extensive area of nanobimorphic surfaces after biomechanical testing. This study clearly demonstrates the nanofeature-enhanced osteoconductivity of titanium by an alkali- and heat-treated nanobimorphic surface compared to that by microfeatured surfaces, which results not only in an acceleration but also an improvement of bone-implant integration. The identified biological parameters that successfully detect the advantages of nanofeatures over microfeatures will be useful in evaluating new implant surfaces in future studies.

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.

Effects of UV photofunctionalization on the nanotopography enhanced initial bioactivity of titanium

This study addresses the control of the biological capabilities of titanium through specific nanosurface features and its potential modulation by UV photofunctionalization. Rat bone marrow derived osteoblasts were cultured on titanium disks with micropits alone, micropits with 100 nm nodules, micropits with 300 nm nodules, or micropits with 500 nm nodules, with or without UV treatment. After a 24 h incubation protein adsorption, as well as the attachment, retention, and spread of osteoblasts were examined in correlation with the topographical parameters of the titanium substrates. Each of the biological events was governed by a different set of multiple surface topographical factors with a distinctive pattern of regulation. For instance, without UV treatment the protein adsorption and cell attachment capability of titanium substrates increased linearly with increasing average roughness (Ra) and surface area of titanium disks, but increased polynomially with increasing nanonodule diameter. The cell retention capability increased polynomially with increasing nanonodular diameter and Ra, but increased linearly with increasing surface area. Consequently, the micropits with 300 nm nodules created the most favorable environment for this initial osteoblast behavior and response. UV treatment of the nanonodular titanium surfaces resulted in considerable enhancement of all biological events. However, the pattern of UV-mediated enhancement was disproportionate; exponential and overriding effects were observed depending upon the biological event and topographical parameter. As an example of overriding enhancement, the cell retention capability, which fluctuated with changes in various topographical parameters, became invariably high after UV treatment. The present data provide a basis for understanding how to optimize nanostructures to create titanium surfaces with increased biological capabilities and uncover a novel advantage of UV photofunctionalization of titanium substrates that synergistically increases its nanotopography enhanced biological capabilities whereby most of the initial biological events of osteoblasts were overwhelmingly enhanced beyond a simple proportional increase.

N-Acetyl Cysteine Protects TMJ Chondrocytes from Oxidative Stress

Temporomandibular joint (TMJ) inflammation is closely associated with oxidative stress. This study tested the potential of N-acetyl cysteine (NAC), an anti-oxidant amino-acid derivative, in alleviating oxidative stress-related damage in TMJ chondrocytes. The inflammatory condition was simulated by the addition of hydrogen peroxide (H2O2) to TMJ-derived chondrocyte cultures. Exposure to H2O2 decreased the cell population by half within 2 days as a result of induced apoptosis and reduced proliferation. Gene expression of aggrecan and collagen II, as well as glycosaminoglycan production, were reduced by more than 70%. These compromised chondrocyte viability and function were fully restored by the addition of NAC to the cultures. NAC reduced the H2O2-elevated intracellular reactive oxygen species to the normal level and increased cellular glutathione reserves. These results indicate that NAC restores oxidative stress-induced cell death and severe functional impairment in TMJ chondrocytes, and warrant in vivo testing to explore its therapeutic potential as an anti-inflammatory agent.

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.

Novel antioxidant capability of titanium induced by UV light treatment.

The intracellular production of reactive oxygen species (ROS) is a representative form of cellular oxidative stress and plays an important role in triggering adverse cellular events, such as the inflammatory reaction and delayed or compromised differentiation. Osteoblastic reaction to titanium with particular focus on ROS production remains unknown. Ultraviolet (UV) light treatment improves the physicochemical properties of titanium, specifically the induction of super hydrophilicity and removal of hydrocarbon, and eventually enhances its osteoconductivity. We hypothesized that there is a favorable regulatory change of ROS production within osteoblasts in contact with UV-treated titanium. Osteoblasts were cultured on titanium disks with or without UV-pretreatment. The intracellular production of ROS was higher on acid-etch-created rough titanium surfaces than on machine-prepared smooth ones. The ROS production was reduced by 40-50% by UV pretreatment of titanium regardless of the surface roughness. Oxidative DNA damage, as detected by 8-OHdG expression, was alleviated by 50% on UV-treated titanium surfaces. The expression of inflammatory cytokines was consistently lower in osteoblasts cultured on UV-treated titanium. ROS scavenger, glutathione, remained more without being depleted in osteoblasts on UV-treated titanium. Bio-burden test further showed that culturing osteoblasts on UV-treated titanium can significantly reduce the ROS production even with the presence of hydrogen peroxide, an oxidative stress inducer. These data suggest that the intracellular production of ROS and relevant inflammatory reaction, which unavoidably occurs in osteoblasts in contact with titanium, can be significantly reduced by UV pretreatment of titanium, implying a novel antioxidant capability of the particular titanium.

Bone regeneration by polyhedral microcrystals from silkworm virus.

Bombyx mori cypovirus is a major pathogen which causes significant losses in silkworm cocoon harvests because the virus particles are embedded in micrometer-sized protein crystals called polyhedra and can remain infectious in harsh environmental conditions for years. But the remarkable stability of polyhedra can be applied on slow-release carriers of cytokines for tissue engineering. Here we show the complete healing in critical-sized bone defects by bone morphogenetic protein-2 (BMP-2) encapsulated polyhedra. Although absorbable collagen sponge (ACS) safely and effectively delivers recombinant human BMP-2 (rhBMP-2) into healing tissue, the current therapeutic regimens release rhBMP-2 at an initially high rate after which the rate declines rapidly. ACS impregnated with BMP-2 polyhedra had enough osteogenic activity to promote complete healing in critical-sized bone defects, but ACS with a high dose of rhBMP-2 showed incomplete bone healing, indicating that polyhedral microcrystals containing BMP-2 promise to advance the state of the art of bone healing.