Ken Ishizaki

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.

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.

Morphological and molecular changes in denture-supporting tissues under persistent mechanical stress in rats

The purpose of this study was to determine the effects of mechanical compression on the palatal mucosa using an experimental palatal base. The palatal base was either pressed onto (stress group) or not pressed onto (fit group) rat palatal mucosa. Blood flow was measured and the animals were sacrificed 6-72 h later for analysis. The expression of heat shock protein 70 (HSP70), vascular endothelial growth factor (VEGF) and proliferation cell nuclear antigen (PCNA) was characterized by immunohistochemical staining. For morphometric analysis, connective tissues were divided into bone side and epithelial side tissues. The ratio of PCNA-positive cells (PCNA score) was calculated, and the expressions of mRNA encoding HSP70 and VEGF was evaluated. Whereas blood flow in the stress group showed ischaemia, none was found in the fit group. Proliferation cell nuclear antigen scores on the bone side were higher than on the epithelial side in the stress group (P < 0.05). Heat shock protein 70- and VEGF-positive cells were observed under compression conditions, particularly in the periosteum. In the stress group, the expressions of mRNA encoding HSP70 and VEGF were highest at 12 h (P < 0.05). These results suggest that mechanical compression of the palatal plate induces ischaemia, and that cells in the underlying denture-supporting tissue, which includes the periosteum, synthesize HSP70 and VEGF to maintain homeostasis under these conditions.

Nanometer-thin TiO2 enhances skeletal muscle cell phenotype and behavior

Background The independent role of the surface chemistry of titanium in determining its biological properties is yet to be determined. Although titanium implants are often in contact with muscle tissue, the interaction of muscle cells with titanium is largely unknown. This study tested the hypotheses that the surface chemistry of clinically established microroughened titanium surfaces could be controllably varied by coating with a minimally thin layer of TiO2 (ideally pico-to-nanometer in thickness) without altering the existing topographical and roughness features, and that the change in superficial chemistry of titanium is effective in improving the biological properties of titanium. Methods and results Acid-etched microroughened titanium surfaces were coated with TiO2 using slow-rate sputter deposition of molten TiO2 nanoparticles. A TiO2 coating of 300 pm to 6.3 nm increased the surface oxygen on the titanium substrates in a controllable manner, but did not alter the existing microscale architecture and roughness of the substrates. Cells derived from rat skeletal muscles showed increased attachment, spread, adhesion strength, proliferation, gene expression, and collagen production at the initial and early stage of culture on 6.3 nm thick TiO2-coated microroughened titanium surfaces compared with uncoated titanium surfaces. Conclusion Using an exemplary slow-rate sputter deposition technique of molten TiO2 nanoparticles, this study demonstrated that titanium substrates, even with microscale roughness, can be sufficiently chemically modified to enhance their biological properties without altering the existing microscale morphology. The controllable and exclusive chemical modification technique presented in this study may open a new avenue for surface modifications of titanium-based biomaterials for better cell and tissue affinity and reaction.

Response of Merkel cells in the palatal rugae to the continuous mechanical stimulation by palatal plate

The aim of the present study was to investigate the responses of Merkel cells that are numerous in the palatine rugae, due to the continuous mechanical stimulation exerted by the palatal plate. Forty golden hamsters were used in this experiment. The palatal plate was made of adhesive resin and it was set on the palate of the animal. To exert a continuous pressure, a 0.8 mm elevation on the internal surface of the palatal plate was created at the middle portion of the fourth palatine ruga. Thereafter, the number of Merkel cells in the mucosa was calculated by immunohistochemical observation. Morphological changes of Merkel cells were examined by electron microscopy. There was significant difference among the control and any of the treated groups on the number of CK20 positive Merkel cells (p < 0.05) and that numbers were decreased at the sites where continuous mechanical stimulation was exerted. Degeneration of the cytoplasm mitochondria and nerve endings, and a decrease in both the number of neurosecretory granules and cytoplasmic processes were observed. Furthermore, the presence of nuclear chromatin aggregation and fragmentation was recognized. The continuous mechanical stimulation by the palatal plate affected the responses of Merkel cells and nerve endings, thus inducing a decrease in the number of Merkel cells. A portion of these changes was also associated with the expression of apoptosis.

Influence of anterior palatal coverage on perception and retention in complete dentures

STATEMENT OF PROBLEM Oral perception is adversely affected by palatal coverage in complete denture wearers. PURPOSE The purpose of this study was to determine whether an opening in an anterior palatal base enhanced oral perception and the effect of the size of the opening on retention. MATERIAL AND METHODS Ten healthy dentate individuals (5 men, 5 women; age range: 22-29 years) participated in the perception test. The time required to sense a nylon thread embedded in test food was measured. The test was performed with 3 types of experimental palatal form: no-opening; small-opening, where the opening was located in the anterior region of the palatal base; and large-opening, where the opening was located between the incisive papilla and the third palatal rugae. No palatal coverage was used as a control. Retention force was measured by pulling and loading tests. In the pulling test, pulling force was applied to 2 points on the experimental denture base, and in the loading test, loading force was applied to 6 points. Response times (sec) and retention forces (N) were analyzed using a 1-way ANOVA for repeated measurements followed by the Bonferroni test for multiple comparisons (α=.05). RESULTS A significant difference was observed in response time in fiber perception between bases with or without a small opening (P =.017). In the pulling test, retention force with the small-opening type was 4% lower at the molar region, and 35% lower at the center of gravity than in the no-opening type. Retention force in the large-opening type was lower than in the small-opening type. In the loading test, retention force was 13 N or higher in all 3 types. CONCLUSIONS The small-opening type of palatal coverage improved oral perception and demonstrated sufficient retention force.

Influence of palatal surface shape of dentures on food perception.

The purpose of this study was to clarify the influence of the palatal surface shape of dentures on food perception. Eighteen healthy dentulous subjects (mean age, 24 years) were investigated. Four types of experimental plate were used: (i) a tailor-made plate, (ii) an average-model plate, (iii) a smooth plate, and (iv) a wrinkle plate. Test foods consisted of Bavarian cream cubes containing one to three mustard seeds and six raw carrot pieces of different shapes. Bavarian cream cubes with three seeds were used for analysis. Other foods were used as dummy foods. Subjects were required to wear experimental plates and press test foods placed on the anterior area of the tongue against the experimental plates. We measured time required to perceive number of spherical bodies, rate of correct answers, and level of perception with each type of experimental plate using a 100-mm visual analogue scale. The results showed a significant difference in response time between the average-model plate and the other experimental plates, with response time longest for the average-model plate. On the other hand, no significant differences in rate of correct answers regarding number of spherical bodies or level of perception were found among the experimental plates. When incisive papilla, palatine suture and palatal rugae based on the standard Japanese shape were replicated on the palatal surface of the plates, the time required for food perception during ingestion was prolonged in comparison to plates with other palatal surface shapes.

Ultraviolet-C irradiation to titanium implants increases peri-implant bone formation without impeding mineralization in a rabbit femur model.

Abstract Objectives. Volume and bone quality of peri-implant supporting bone, in particular, at implant neck region, as well as bone–implant contact ratio, is important for long-term stability of implants. Ultraviolet-C (UVC) irradiation is known to enhance the osseointegration capability of titanium implants. However, the histological determination was performed only on a rat model, but not pre-clinical animal model such as a rabbit model. The purpose of this study was to determine the effects of UVC irradiation on titanium implants on the volume and mineral density of peri-implant supporting bone formation in a rabbit femur model. Materials and methods. Acid-etched pure titanium screw implants with or without 3 mW/cm2 UVC irradiation for 48 h were placed in rabbit femur diaphyses. Peri-implant bone tissue formation was analyzed at 3 and 8 weeks post-operatively by histology and micro-CT-based bone morphometry after calibration with hydroxyl apatite phantoms. Results. UVC pre-irradiated implants accumulated a higher density of cells and thicker and longer bone tissue attachments that continued into the inner basic lamellae of the surface of existing cortical bone at 3 and 8 weeks than the implants without irradiation. Although the bone mineral density around both implants was equivalent to that of the existing cortical bone, bone volume was greater with UVC pre-irradiation in two-thirds or more of the apical region throughout the observation period. Conclusions. These results indicate that UVC treatment increased the volume of cortical-like bone tissue in the coronal region of titanium implants without deterioration of bone mineral density.

Anti-infective control in human bronchiolar epithelial cells by mucin phenotypic changes following uptake of N-acetyl-L-cysteine

Purpose. Aspiration pneumonia is infection of the respiratory tract resulting from accumulation of sputum in the larynx. N-acetyl-L-cysteine (NAC) might regulate mucin (MUC) expression and activate inherent anti-infective system in bronchiolar epithelial cells after cellular uptake, and therefore, serve as the preventative agent for chronic lung disease including aspiration pneumonia. The purpose of this in vitro study was to evaluate the effect of uptake of NAC by human bronchiolar epithelial cells on bacterial infection and regulations of mucin expression in association with cellular redox status under co-culture with a representative pathogen for hospital- and community-acquired pneumonia, Streptococcus pneumoniae. Materials and methods. Human bronchiolar epithelial cells preincubated with or without 20 mM NAC for 3 h were co-cultured with or without bacteria for 8 h and evaluated with respect to cellular redox balance, expressions of various types of MUC, proinflammatory cytokines and mediators, and bacterial infection state by biochemical, genetic, and immunofluorescent assays. Results. Markedly increased intracellular reactive oxygen species and oxidized glutathione levels plus increased release and expression of proinflammatory cytokines and mediators were observed in cells co-cultured with bacteria. These bacteria-induced cellular redox disturbance and proinflammatory events were prevented and alleviated by pretreatment with NAC. Cells co-cultured with bacteria did not increase expression of anti-infective membranous MUC4 but exhibited increases in gel-forming MUC5AC expression and bacterial infection. However, NAC-pretreated cells avoided bacterial infection along with enhancement of MUC4, but not MUC5AC, expression. Conclusion. Uptake of NAC by human bronchiolar epithelial cells prevented bacterial infection and upregulated membranous, but not gel-forming, MUC expression along with the increase in intracellular antioxidant level under co-culture conditions with S. pneumoniae.

Maxillofacial prosthetic treatment factors affecting oral health-related quality of life after surgery for patients with oral cancer

Statement of problem: After oral cancer surgery, tissue defects can cause deformity and limited mobility, complicating many essential functions. For patients with mandibular, tongue, and oral floor defects, evidence regarding the effects of maxillofacial prosthetics on their oral health‐related quality of life (OHRQoL) is lacking. Therefore, maxillofacial prosthetic reconstruction has been implemented with no clear treatment goals. Purpose: The purpose of this clinical study was to identify factors affecting the improvement of OHRQoL by using maxillofacial prosthetic treatment after surgery to repair maxillary, mandibular, tongue, and oral floor defects. Material and methods: All individuals who agreed to maxillofacial prosthetics after surgery for oral cancer were enrolled. Oral function and OHRQoL were evaluated before maxillofacial prosthesis placement and 1 month after final adjustments. The oral functions evaluated included masticatory function, swallowing function, and articulatory function. The Oral Health Impact Profile (OHIP‐J54) was used to evaluate OHRQoL. Factors affecting changes in the OHIP‐J54 score for participants’ background and oral functions before and after treatment were analyzed through logistic regression analysis (stepwise method). Results: Participants included 34 men and 16 women with an average age of 72.4 ±8.7 years. “Psychological discomfort” was correlated with the patients sex and masticatory function. “Physical disability” was related to articulatory function. “Handicap” was related to the swallowing function. “Additional Japanese questions” were related to the patients sex. Conclusions: Participants’ sex and their oral functions, including masticatory, swallowing, and articulatory functions, were associated with improved OHRQoL because of maxillofacial prosthetics after surgery for oral cancer.