Norio Hori

The effect of ultraviolet functionalization of titanium on integration with bone.

Titanium implants are used as a reconstructive anchor in orthopedic and dental diseases and problems. Recently, ultraviolet (UV) light-induced photocatalytic activity of titanium has earned considerable and broad interest in environmental and clean-energy sciences. This study determines whether UV treatment of titanium enhances its osteoconductive capacity. Machined and acid-etched titanium samples were treated with UV for various time periods up to 48h. For both surfaces, UV treatment increased the rates of attachment, spread, proliferation and differentiation of rat bone marrow-derived osteoblasts, as well as the capacity of protein adsorption, by up to threefold. In vivo histomorphometry in the rat model revealed that new bone formation occurred extensively on UV-treated implants with virtually no intervention by soft tissue, maximizing bone-implant contact up to nearly 100% at week 4 of healing. An implant biomechanical test revealed that UV treatment accelerated the establishment of implant fixation 4 times. The rates of protein adsorption and cell attachment strongly correlated with the UV dose-responsive atomic percentage of carbon on TiO2, but not with the hydrophilic status. The data indicated that UV light pretreatment of titanium substantially enhances its osteoconductive capacity, in association with UV-catalytic progressive removal of hydrocarbons from the TiO2 surface, suggesting a photofunctionalization of titanium enabling more rapid and complete establishment of bone-titanium integration.

Time-dependent degradation of titanium osteoconductivity: an implication of biological aging of implant materials.

The shelf life of implantable materials has rarely been addressed. We determined whether osteoconductivity of titanium is stable over time. Rat bone marrow-derived osteoblasts were cultured on new titanium disks (immediately after acid-etching), 3-day-old (stored after acid-etching for 3 days in dark ambient conditions), 2-week-old, and 4-week-old disks. Protein adsorption capacity, and osteoblast migration, attachment, spread, proliferation and mineralization decreased substantially on old titanium surfaces in an age-dependent manner. When the 4-week-old implants were placed into rat femurs, the biomechanical strength of bone-titanium integration was less than half that for newly processed implants at the early healing stage. More than 90% of the new implant surface was covered by newly generated bone compared to 58% for 4-week-old implants. This time-dependent biological degradation was also found for machined and sandblasted titanium surfaces and was associated with progressive accumulation of hydrocarbon on titanium surfaces. The new surface could attract osteoblasts even under a protein-free condition, but its high bioactivity was abrogated by masking the surface with anions. These results uncover an aging-like time-dependent biological degradation of titanium surfaces from bioactive to bioinert. We also suggest possible underlying mechanisms for this biological degradation that provide new insights into how we could inadvertently lose, and conversely, maximize the osteoconductivity of titanium-based implant materials.

The effect of UV-photofunctionalization on the time-related bioactivity of titanium and chromium-cobalt alloys.

This study examined the possible changes in the bioactivity of titanium surfaces during their aging and investigated the effect of ultraviolet (UV) light treatment during the age-related change of titanium bioactivity. Rat bone marrow-derived osteoblastic cells were cultured on new titanium disks (immediately after either acid-etching, machining, or sandblasting), 4-week-old disks (stored after processing for 4 weeks in dark ambient conditions), and 4-week-old disks treated with UVA (peak wavelength of 365 nm) or UVC (peak wavelength of 250 nm). During incubation for 24 h, only 50% of the cells were attached to the 4-week-old surfaces as compared to the new surface. UVC treatment of the aged surface increased its cell attachment capacity to a level 50% higher than the new surfaces, whereas UVA treatment had no effect. Proliferation, alkaline phosphatase activity, and mineralization of cells were substantially lower on the 4-week-old surfaces than on the new surfaces, while they were higher on the UVC-treated 4-week-old surfaces as compared to the new surfaces. The age-related impaired bioactivity was found on all titanium topographies as well as on a chromium-cobalt alloy, and was associated with an increased percentage of surface carbon. Although both UVA and UVC treatment converted the 4-week-old titanium surfaces from hydrophobic to superhydrophilic, only UVC treatment effectively reduced the surface carbon to a level equivalent to the new surface. Thus, this study uncovered a time-dependent biological degradation of titanium and chromium-cobalt alloy, and its restoration enabled by UVC phototreatment, which surmounts the innate bioactivity of new surfaces, which is more closely linked to hydrocarbon removal than the induced superhydrophilicity.

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.

Ultraviolet light-mediated photofunctionalization of titanium to promote human mesenchymal stem cell migration, attachment, proliferation and differentiation

Improving the osteoconductive potential of titanium implants has been of continuing interest in the fields of dentistry and orthopedic surgery. This study determined the bioactivity of ultraviolet (UV) light-treated titanium. Human mesenchymal stem cells (MSCs) were cultured on acid-etched microtopographical titanium surfaces with and without 48h pretreatment with UVA (peak wavelength of 360n m) or UVC (peak wavelength of 250 nm). The number of cells that migrated to the UVC-treated surface during the first 3h of incubation was eight times higher than those that migrated to the untreated surface. After 24h of incubation, the number of cells attached to the UVC-treated surface was over three times more than those attached to the untreated surface. On the UVC-treated surface, the cellular spread was expedited with an extensive and intensive expression of the focal adhesion protein vinculin. The cells on the UVC-treated surface exhibited a threefold higher bromodeoxyuridine incorporation, a doubling of the alkaline phosphatase-positive area and the up-regulated expression of bone-related genes, indicating the accelerated proliferation and differentiation. The UVC-treated surface did not adversely affect the viability of the cells. These biological effects were not seen after UVA treatment, despite the generation of superhydrophilicity. Thus, we discovered a novel photofunctionalization of titanium dioxide that substantially enhances its bioactivity in human MSCs. Further studies are required to investigate the universal effectiveness of this surface modification for different titanium-containing materials, with varying chemistries and textures, as well as to understand its significance in enhancing in vivo osteoconductivity.

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.

Age-dependent Degradation of the Protein Adsorption Capacity of Titanium:

Reported bone-implant contact percentages are far below the ideal 100%. We tested a hypothesis that the protein adsorption capability of titanium, which is critical to the process of osseointegration, changes over time before its use. Machined, acid-etched, and sandblasted surfaces were prepared and stored under dark ambient conditions for 3 days, 1 week, or 4 weeks. For all surfaces, protein adsorption decreased as the storage time increased, and their decreasing rates were dependent on titanium topography. After 4 weeks, the amounts of albumin and fibronectin adsorbed by the acid-etched surface were only 20% and 35%, respectively, of that adsorbed by the fresh surface after 2 hours of incubation, and remained substantially low even after 24 hours. This time-dependent degradation in protein adsorption of titanium correlated with its naturally decreasing hydrophilicity, which was not observed for the nickel and chromium surfaces, indicating a titanium-specific biological aging.

N-acetyl cysteine (NAC)-mediated detoxification and functionalization of poly(methyl methacrylate) bone cement.

Currently used poly(methyl methacrylate) (PMMA)-based bone cement lacks osteoconductivity and induces osteolysis and implant loosening due to its cellular and tissue-toxicity. A high percentage of revision surgery following the use of bone cement has become a significant universal problem. This study determined whether incorporation of the amino acid derivative N-acetyl cysteine (NAC) in bone cement reduces its cytotoxicity and adds osteoconductivity to the material. Biocompatibility and bioactivity of PMMA-based bone cement with or without 25mm NAC incorporation was examined using rat bone marrow-derived osteoblastic cells. Osteoconductive potential of NAC-incorporated bone cement was determined by microCT bone morphometry and implant biomechanical test in the rat model. Generation of free radicals within the polymerizing bone cement was examined using electron spin resonance spectroscopy. Severely compromised viability and completely suppressed phenotypes of osteoblasts on untreated bone cement were restored to the normal level by NAC incorporation. Bone volume formed around 25mm NAC-incorporated bone cement was threefold greater than that around control bone cement. The strength of bone-bone cement integration was 2.2 times greater for NAC-incorporated bone cement. For NAC-incorporated bone cement, the spike of free radical generation ended within 12h, whereas for control bone cement, a peak level lasted for 6 days and a level greater than half the level of the peak was sustained for 20 days. NAC also increased the level of antioxidant glutathione in osteoblasts. These results suggest that incorporation of NAC in PMMA bone cement detoxifies the material by immediate and effective in situ scavenging of free radicals and increasing intracellular antioxidant reserves, and consequently adds osteoconductivity to the material.

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.

Selective cell affinity of biomimetic micro-nano-hybrid structured TiO2 overcomes the biological dilemma of osteoblasts.

OBJECTIVE There is a great demand for dental implant surfaces to accelerate the process of peri-implant bone generation to reduce its healing time and enable early loading. To this end, an inverse correlation between the proliferation and functional maturation (differentiation) in osteoblasts presents a challenge for the rapid generation of greater amounts of bone. For instance, osteoblasts exhibit faster differentiation but slower proliferation on micro-roughened titanium surfaces. Using a unique micro-nano-hierarchical topography of TiO(2) that mimics biomineralized matrices, this study demonstrates that this challenge can be overcome without the use of biological agents. METHODS Titanium disks of grade 2 commercially pure titanium were prepared by machining (smooth surface). To create a microtexture with peaks and valleys (micropit surface), titanium disks were acid-etched. To create 200-nm TiO(2) nanonodules within the micropits (nanonodule-in-micropit surface), TiO(2) was sputter-deposited onto the acid-etched surface. Rat bone marrow-derived osteoblasts and NIH3T3 fibroblasts were cultured on machined smooth, micropit, and nanonodule-in-micropit surfaces. RESULTS Despite the substantially increased surface roughness, the addition of 200-nm nanonodules to micropits increased osteoblast proliferation while enhancing their functional differentiation. In contrast, this nanonodule-in-micropit surface decreased proliferation and function in fibroblasts. SIGNIFICANCE The data suggest the establishment of cell-selectively functionalized nano-in-micro smart titanium surfaces that involve a regulatory effect on osteoblast proliferation, abrogating the inhibitory mechanism on the micropitted surface, while enhancing their functional differentiation. Biomimetic and controllable nature of this nanonodules-in-micropits surface may offer a novel micro-to-nanoscale hierarchical platform to biologically optimize nanofeatures of biomaterials. Particularly, this micro-nano-hybrid surface may be an effective approach to improve current dental implant surfaces for accelerated bone integration.