Hajime Minamikawa

Enhancement of osteoblast adhesion to UV-photofunctionalized titanium via an electrostatic mechanism.

The mechanism underlying the recently found photofunctionalization of titanium is unknown. We focused on how the initial interaction between the cells and photofunctionalized titanium is enhanced at a molecular-level and the role played by the electrostatic status of the titanium surfaces in the possible regulatory mechanism for determining their bioactivity. Rat bone marrow-derived osteoblasts were cultured on untreated and ultraviolet (UV)-treated titanium surfaces. UV treatment converted the titanium surfaces from hydrophobic to superhydrophilic. The number of osteoblasts attached to UV-treated titanium surfaces was substantially greater than that attached to untreated surfaces (5-fold and 2-fold after 3 and 24 h of incubation, respectively). Osteoblasts cultured for 3 and 24 h on these titanium surfaces were detached mechanically by vibrational force and enzymatically by trypsin treatment. Cell adhesion evaluated by the percentage of remaining cells after these detachments was substantially greater for cells on UV-treated titanium surfaces compared to untreated titanium surfaces (110-120% greater for cells incubated for 3 h and 50-60% greater for cells incubated for 24 h). Osteoblasts on UV-treated surfaces expressed more vinculin. UV-enhancing effect in cell adhesion was also demonstrated under a serum-free condition. UV-enhanced cell adhesion was abrogated when the UV-treated titanium surfaces were electrostatically neutralized by either removing the electric charge or masking with monovalent anions, while the surfaces maintained superhydrophilicity. In conclusion, the establishment of osteoblast adhesion is accelerated and augmented remarkably on UV-treated titanium surfaces, associated with upregulated expression of vinculin. This study has identified an electrostatic property of UV-treated titanium surfaces playing a regulatory role in determining their bioactivity, superseding the effect of the hydrophilic nature of these surfaces. A mechanism underlying the UV-induced conversion of titanium from bioinert to bioactive, in which direct cell-titanium interaction is exclusively enabled, is proposed.

Enhancement of bone–titanium integration profile with UV-photofunctionalized titanium in a gap healing model

In this study, we tested the potential of UV-photofunctionalized titanium surfaces to overcome compromised bone-titanium integration in a gap healing model. Titanium in rod and disk forms was acid etched and then stored for 4 weeks under dark ambient conditions. Titanium rods with and without UV pretreatment were placed into a rat femur with (contact healing) or without (gap healing) contact with the innate cortical bone. The titanium implants were subjected to a biomechanical push-in test, micro-CT bone morphometry, and surface elemental analysis after 2 weeks of healing. The strength of bone-titanium integration in the gap healing model was one-third of that in the contact healing model. However, UV-treated implants in the gap healing condition produced a strength of bone-titanium integration equivalent to that of untreated implants in the contact healing condition. Bone volume around UV-treated implants was 2- to 3-fold greater than that around the untreated implants in the gap healing model. A bone generation profile drawn along the long axis of the implant exhibited greater contrast between the untreated and UV-treated surfaces in the cortical area than in the bone marrow area. The bone tissue formed on UV-treated implants showed a higher Ca/P ratio than that formed on untreated titanium. The rate of cell proliferation, alkaline phosphatase activity, and calcium deposition in femoral periosteal cells and in bone marrow-derived osteoblasts were greater in cultures on UV-treated titanium disks than in cultures on untreated disks. The UV-enhanced function in periosteal cells was more pronounced when they were co-cultured with bone marrow-derived osteoblasts, indicating a synergistic effect of UV-treated titanium with biological signals from bone marrow-derived osteoblasts. Within the limitation of the model used in this study, UV-photofunctionalized titanium surfaces may overcome the challenging condition of bone-titanium integration without cortical bone support. UV treatment of implants induced marked improvements in the behavior of bone formation and quantity and quality of bone tissue around the implants. These effects may be related to the promoted function of both periosteum- and bone marrow-derived osteogenic cells at the local level around UV-treated titanium surfaces.

Electrostatic control of protein adsorption on UV-photofunctionalized titanium.

Ultraviolet (UV)-photofunctionalization of titanium to enable the establishment of a nearly complete bone-implant contact was reported recently. However, the underlying mechanism for this is unknown. We hypothesized that UV-treated titanium surfaces acquire distinct electrostatic properties that may play important roles in determining the bioactivity of these surfaces. The objective of this study was to determine the protein adsorption capability of UV-treated titanium surfaces under various electrostatic environments. The amount of albumin adsorbed on UV-treated and untreated titanium disks was evaluated under different pH conditions above and below the isoelectric points of albumin and titanium. The effects of additional treatment with various ionic solutions were also examined. Albumin adsorption on UV-treated surfaces at pH 7.0 was considerably greater (6-fold after 3h of incubation and 2.5-fold after 24h) than that to UV-untreated surfaces. UV-enhanced albumin adsorption was abrogated at pH 3.0 or when these titanium surfaces were treated with anions, while maintaining UV-induced superhydrophilicity. Albumin adsorption on UV-untreated titanium surfaces increased after treating these surfaces with divalent cations but not after treating them with monovalent cations. These results indicated that UV-treated titanium surfaces are electropositively charged as opposed to electronegatively charged UV-untreated titanium surfaces. This distinct UV-induced electrostatic property predominantly regulates the protein adsorption capability of titanium, superseding the effect of hydrophilic status, and converts titanium surfaces from bioinert to bioactive. As a result, direct titanium-protein interactions take place exclusively on UV-treated titanium surfaces without the aid of bridging ions.

Synergistic effects of UV photofunctionalization and micro-nano hybrid topography on the biological properties of titanium

Titanium surfaces with micro-nano hybrid topography (nanoscale nodules in microscale pits) have been recently demonstrated to show higher biological capability than those with microtopography alone. On the other hand, UV treatment of titanium surfaces, which is called UV photofunctionalization, has recently been introduced to substantially increase the biological capability and osteoconductivity of titanium surfaces. However, synergistic effects of these two advanced surface modification technologies and regulatory factors to potentially modulate the mutual effects have never been addressed. In this study, utilization of a recently discovered controllable self-assembly of TiO(2) nanonodules has enabled the exploration of the relative contribution of different sizes of nanostructures to determine the biological capability of titanium surfaces and their relative responsiveness to UV photofunctionalization. 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, with or without UV treatment. Although UV treatment increased the attachment, spread, proliferation, and mineralization of these cells on all titanium surfaces, these effects were more accentuated (3-5 times) on nanonodular surfaces than on surfaces with micropits alone and were disproportionate depending on nanonodule sizes. For instance, on UV-treated micro-nano hybrid surfaces, cell attachment correlated with nanonodule sizes in a quadratic approximation with its peak for 300-nm nodules followed by a decline for 500-nm nodules, while cell attachment exponentially correlated with surface roughness with its plateau achieved for 300-nm nodules without a subsequent decline. Moreover, cell attachment increased in a linear correlation with the surface area, while no significant effect of the inter-irregularities space or degree of hydrophilicity was observed on cell attachment. These results suggest that the effect of UV photofunctionalization can be multiplied on micro-nano hybrid titanium surfaces compared with the surfaces with micropits alone. This multiplication is disproportionately regulated by a selected set of topographical parameters of the titanium surfaces. Among the nanonodules tested in this study, 300-nm nodules seemed to create the most effective morphological environment for responding to UV photofunctionalization. The data provide a systematic platform to effectively optimize nanostructures on titanium surfaces in order to enhance their biological capability as well as their susceptibility to UV photofunctionalization.

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.

Effect of N-acetylcysteine on Rat Dental Pulp Cells Cultured on Mineral Trioxide Aggregate

INTRODUCTION The purpose of this study was to evaluate the cytotoxicity of mineral trioxide aggregate (MTA) and its potential detoxification by an antioxidant amino acid, N-acetylcysteine (NAC). METHODS Rat dental pulp cells extracted from rat maxillary incisors were directly cultured on MTA with or without NAC in culture medium. The number of cells and their spreading behavior were both assessed 24 hours after seeding. The intracellular levels of reactive oxygen species (ROS) and glutathione (GSH) were also assessed after 24 hours of culture. RESULTS The number of cells attached to MTA was 60% greater when NAC was added to the culture medium. In addition, the area and perimeter of the cells were found to be 2-fold greater in the culture containing NAC. Cells cultured on MTA alone showed large ROS concentrations, which disappeared when the medium was supplemented with NAC. The intracellular GSH level, however, increased 3.5-fold with NAC addition. CONCLUSIONS This study demonstrated that the presence of NAC in environments can substantially improve attachment and spreading behaviors of dental pulp cells on MTA. This biological effect was associated with an improvement in the cellular redox system by NAC and warrants further exploration of NAC for determining its therapeutic value in improving the biocompatibility of MTA.

N-acetyl Cysteine Alleviates Cytotoxicity of Bone Substitute

Lack of cytocompatibility in bone substitutes impairs healing in surrounding bone. Adverse biological events around biomaterials may be associated with oxidative stress. We hypothesized that a clinically used inorganic bone substitute is cytotoxic to osteoblasts due to oxidative stress and that N-acetyl cysteine (NAC), an antioxidant amino acid derivative, would detoxify such material. Only 20% of rat calvaria osteoblasts were viable when cultured on commercial deproteinized bovine bone particles for 24 hr, whereas this percentage doubled on bone substitute containing NAC. Intracellular ROS levels markedly increased on and under bone substitutes, which were reduced by prior addition of NAC to materials. NAC restored suppressed alkaline phosphatase activity in the bone substitute. Proinflammatory cytokine levels from human osteoblasts on the bone substitute decreased by one-third or more with addition of NAC. NAC alleviated cytotoxicity of the bone substitute to osteoblastic viability and function, implying enhanced bone regeneration around NAC-treated inorganic biomaterials.

Photofunctionalization increases the bioactivity and osteoconductivity of the titanium alloy Ti6Al4V.

This study examined the effect of photofunctionalization on bioactivity and osteoconductivity of titanium alloy Ti6Al4V. We also tested a hypothesis that the effect of photofunctionalization is as substantial as the one of surface roughening. Two different surface morphology, a roughened surface (sandblasted and acid-etched surface) and relatively smooth surface (machined surface), was tested. Ti6Al4V samples were photofunctionalized with UV light for 15 min using a photo device. Photofunctionalization converted Ti6Al4V surfaces from hydrophobic to superhydrophilic. The attachment, spread, proliferation, and the expression of functional phenotype of bone marrow-derived osteoblasts were promoted on photofunctionalized Ti6Al4V surfaces. The strength of bone-implant integration examined using a biomechanical push-in test in a rat femur model was at least 100% greater for photofunctionalized implants than for untreated implants. These effects were seen on both surface types. The strength of bone-implant integration for photofunctionalized machined implants was greater than that for untreated roughened implants, indicating that the impact of photofunctionalization may be greater than that of surface roughening. Newly prepared Ti alloy was hydrophilic, whereas the hydrophilic status degraded with time and was converted to hydrophobic in 4 weeks. This finding uncovered biological aging of Ti alloy and allowed us to consider photofunctionalization as a countermeasure for aging. These results suggest that photofunctionalization accelerates and enhances bone-implant integration of Ti6Al4V regardless of smooth and roughened surface features, supporting photofunctionalization as an effective and viable measure for improving efficacy of a wide range of Ti6Al4V-based materials used in dental and orthopedic medicine.

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.