Hideki Aita

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.

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.

Harder and Stiffer Bone Osseointegrated to Roughened Titanium

Mechanisms underlying the beneficial anchorage of roughened titanium implants have not been identified. We hypothesized that the implant surface roughness alters intrinsic biomechanical properties of bone integrated to titanium. Nano-indentation performed on two- and four-week post-implantation bone specimens of rats revealed that bone integrated to acid-etched titanium was approximately 3 times harder than that integrated to the machined titanium, both at the osseointegration interface and at the inner area of the peri-implant bone. The hardness of the acid-etched surface-associated bone was equivalent to that of untreated cortical bone at week 4, while the bone hardness around the machined surface was equivalent to that of the untreated trabecular bone. The elastic modulus of the integrated bone was 1.5 to 2.5 times greater around the acid-etched surface than around the machined surface. Analysis of the data suggests that the implant surface roughness affects the biomechanical quality of osseo-integrated bone, and that the bone integrated to the acid-etched surface is harder and stiffer than the bone integrated to the machined surface.

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.

Osteoblasts generate harder, stiffer, and more delamination-resistant mineralized tissue on titanium than on polystyrene, associated with distinct tissue micro- and ultrastructure

This study revealed that osteoblasts generate harder, stiffer, and more delamination‐resistant mineralized tissue on titanium than on the tissue culture polystyrene, associated with modulated gene expression, uniform mineralization, well‐crystallized interfacial calcium‐phosphate layer, and intensive collagen deposition. Knowledge of this titanium‐induced alteration of osteogenic potential leading to enhanced intrinsic biomechanical properties of mineralized tissue provides novel opportunities and implications for understanding and improving bone‐titanium integration and engineering physiomechanically tolerant bone.

Ti Nano-nodular Structuring for Bone Integration and Regeneration

Nanostructuring technology has been proven to create unique biological properties in various biomaterials. Here we present a discovered phenomenon of titanium nano-nodular self-assembly that occurs during physical vapor depositions of titanium (Ti) onto specifically conditioned micro-textured titanium surfaces, and test a hypothesis that the Ti nanostructure has the potential to enhance bone-titanium integration. The nanostructure creation effectively provided geometrical undercut and increased the surface area by up to 40% compared with the acid-etched surface with microtopography. Depending on the size control, the nano-nodules can be added without smearing the existing micro-texture, creating a nano-micro-hybrid architecture. Titanium implants with 560-nm nano-nodules produced 3.1 times greater strength of osseointegration than those with an acid-etched surface in a rat femur model. The discovered titanium nano-nodular self-structuring has been proven feasible on biocompatible materials other than titanium, offering new avenues for the development of implant surfaces and other implantable materials for better bone-generative and -regenerative potential.

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.

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.

N-acetyl Cysteine (NAC)-assisted Detoxification of PMMA Resin:

Despite its proven cytotoxicity, poly-methyl methacrylate (PMMA) resin is one of the most frequently and extensively used materials in dental practice. This study hypothesized that an anti-oxidant amino acid, N-acetyl cysteine (NAC), has the potential to detoxify this material. Ten percent of the rat dental pulp cells were viable when cultured on the PMMA resin for 24 hours, while over 70% of the cells were viable on the NAC-added resin. Nearly all suppressed alkaline phosphatase activity, matrix mineralizing capability, and odontoblastic gene expression, such as dentin sialoprotein, on the untreated control resin was recovered by NAC in a concentration-dependent manner. A Ca/P ratio of 1.65 was found in the extracellular matrix of cultures on NAC-added resin, while that in the untreated resin culture was 0.70. The addition of NAC to PMMA resin significantly ameliorated its cytotoxicity to the dental pulp cells and restored their odontoblast-like cell phenotype to a biologically significant degree.

Molecular and Biomechanical Characterization of Mineralized Tissue by Dental Pulp Cells on Titanium

The application of implant therapy is still limited, because of various risk factors and the long healing time required for bone-titanium integration. This study explores the potential for osseointegration engineering with dental pulp cells (DPCs) by testing a hypothesis that DPCs generate mineralized tissue on titanium. DPCs extracted from rat incisors positive for CD44, alkaline phosphatase activity, and mineralizing capability were cultured on polystyrene and on machined and dual-acid-etched (DAE) titanium. Tissue cultured on titanium with a Ca/P ratio of 1.4 exhibited plate-like morphology, while that on the polystyrene exhibited fibrous and punctate structures. Tissues cultured on titanium were harder than those on polystyrene, 1.5 times on the machined and 3 times on the DAE. Collagen I, osteopontin, and osteocalcin genes were up-regulated on titanium, especially the DAE surface. In conclusion, DPCs showing some characteristics of the previously identified dental pulp stem cells can generate mineralized tissue on titanium via the osteoblastic phenotype, which can be enhanced by titanium surface roughness.