Nanometer-thin TiO2 enhances skeletal muscle cell phenotype and behavior

Ken Ishizaki*, Yoshihiko Sugita*, Fuminori Iwasa, Hajime Minamikawa, Takeshi Ueno, Masahiro Yamada, Takeo Suzuki, Takahiro OgawaLaboratory for Bone and Implant Sciences, The Jane and Jerry Weintraub Center for Reconstructive Biotechnology, Division of Advanced Prosthodontics, Biomaterials and Hospital Dentistry, UCLA School of Dentistry, Los Angeles, CA, USA*Authors contributed equally to this workBackground: 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.Keywords: nanotechnology, orthopedic implants, molten TiO2 nanoparticles, surface chemistr

Dual-optical-comb spectroscopic ellipsometry

Spectroscopic ellipsometry is a means to investigate optical and dielectric material responses. Conventional spectroscopic ellipsometry has trade-offs between spectral accuracy, resolution, and measurement time. Polarization modulation has afforded poor performance due to its sensitivity to mechanical vibrational noise, thermal instability, and polarization wavelength dependency. We equip a spectroscopic ellipsometer with dual-optical-comb spectroscopy, viz. dual-optical-comb spectroscopic ellipsometry (DCSE). The DCSE directly and simultaneously obtains amplitude and phase information with ultra-high spectral precision that is beyond the conventional limit. This precision is due to the automatic time-sweeping acquisition of the interferogram using Fourier transform spectroscopy and optical combs with well-defined frequency. Ellipsometric evaluation without polarization modulation also enhances the stability and robustness of the system. In this study, we evaluate the DCSE of birefringent materials and thin films, which showed improved spectral accuracy and a resolution of up to 1.2x10-5 nm across a 5-10 THz spectral bandwidth without any mechanical movement.Comment: 30 pages, 4 figure

Dual-comb spectroscopic ellipsometry

Spectroscopic ellipsometry is a means of investigating optical and dielectric material responses. Conventional spectroscopic ellipsometry is subject to trade-offs between spectral accuracy, resolution, and measurement time. Polarization modulation has afforded poor performance because of its sensitivity to mechanical vibrational noise, thermal instability, and polarization-wavelength dependency. We combine spectroscopic ellipsometry with dual-comb spectroscopy, namely, dual-comb spectroscopic ellipsometry. Dual-comb spectroscopic ellipsometry (DCSE). DCSE directly and simultaneously obtains the ellipsometric parameters of the amplitude ratio and phase difference between s-polarized and p-polarized light signals with ultra-high spectral resolution and no polarization modulation, beyond the conventional limit. Ellipsometric evaluation without polarization modulation also enhances the stability and robustness of the system. In this study, we construct a polarization-modulation-free DCSE system with a spectral resolution of up to 1.2 × 10−5 nm throughout the spectral range of 1514–1595 nm and achieved an accuracy of 38.4 nm and a precision of 3.3 nm in the measurement of thin-film samples

Long-Term Progressive Degradation of the Biological Capability of Titanium

Titanium undergoes time-dependent degradation in biological capability, or “biological aging”. It is unknown whether the biological aging of titanium occurs beyond four weeks and whether age-related changes are definitely associated with surface hydrophilicity. We therefore measured multiple biological parameters of bone marrow-derived osteoblasts cultured on newly prepared, one-month-old, three-month-old, and six-month-old acid-etched titanium surfaces, as well as the hydrophilicity of these surfaces. New surfaces were superhydrophilic with a contact angle of ddH2O of 0°, whereas old surfaces were all hydrophobic with the contact angle of around 90°. Cell attachment, cell spread, cell density, and alkaline phosphatase activity were highest on new surfaces and decreased in a time-dependent manner. These decreases persisted and remained significant for most of the biological parameters up to six-months. While the number of attached cells was negatively correlated with hydrophilicity, the other measured parameters were not. The biological capability of titanium continues to degrade up to six months of aging, but these effects are not directly associated with time-dependent reductions in hydrophilicity. A full understanding of the biological aging will help guide regulatory improvements in implant device manufacturing and develop countermeasures against this phenomenon in order to improve clinical outcomes

Na+/K+-ATPase as a target for cardiotonic steroids and cisplatin

The sodium (Na+)/potassium (K+)-ATPase is an ion pump located on the surface of all animal cells. It pumps three sodium ions out of the cell while pumping two potassium ions into the cell, hydrolyzing one ATP molecule as the driving force for the reaction. Na+/K+-ATPase forms and maintains the electrochemical gradient in cells, which provides the basis for the excitability of nerve and muscle tissues and contributes to the osmotic regulation of cell volume. In addition, the electrochemical Na+ gradient is the driving force for the secondary transport of nutrients such as amino acids, sugars, and drugs. Recently, the Na+/K+-ATPase has been studied as an important target for cancer treatment, as it has been implicated in the development and progression of many cancers. Na+/K+-ATPase forms a phosphoenzyme intermediate (EP) during ATP hydrolysis.　Cardiotonic steroids have been used to treat congestive heart failure and arrhythmias, and recently their anti-cancer activities have been reported. Ouabain, a specific inhibitor of Na+/K+-ATPase, is a cardiotonic steroid that binds to EP, inhibiting its dephosphorylation and the release of inorganic phosphate. Cisplatin is one of the most potent anti-tumor agents. Many studies have examined the relationship between cisplatin and Na+/K+-ATPase from the viewpoint of cisplatin accumulation and the prevention of nephrotoxicity. It has been suggested that the transport of cisplatin into cells is mediated by the Na+/K+-ATPase and that Na+/K+-ATPase activity is inhibited by cisplatin, although the underlying mechanism remains unclear.　In this review, we evaluate the mechanisms underlying inhibition of Na+/K+-ATPase by cisplatin. We also summarize the structure, function, and enzymatic reaction of Na+/K+-ATPase, as well as the potential for the pump to serve as a target for ouabain and cisplatin. Finally, we will describe experiments conducted by our group showing the mechanism of Na+/K+-ATPase inhibition by cisplatin, and the combined effects of ouabain and cisplatin on cancer cell viability

Calcium Charge and Release of Conventional Glass-Ionomer Cement Containing Nanoporous Silica

The aim of this study was to evaluate calcium charge and release of conventional glass-ionomer cement (GIC) containing nanoporous silica (NPS). Experimental specimens were divided into two groups: the control (GIC containing no NPS) and GIC-NPS (GIC containing 10 wt % NPS). The specimens were immersed in calcium chloride solutions of 5 wt % calcium concentration for 24 h at 37 degrees C, whereupon the calcium ion release of the specimens was measured. The calcium ion release behavior of GIC-NPS after immersion in the calcium solution was significantly greater than that of the control. Scanning electron microscopy and electron-dispersive X-ray spectroscopy results indicated that calcium penetrated inside the GIC-NPS specimen, while the calcium was primarily localized on the surface of the control specimen. It was demonstrated that NPS markedly improved the calcium charge and release property of GIC

Suppression of osteoclastogenesis by lactoferrin

Recent research has shown that lactoferrin indirectly suppresses osteoclastogenesis by affecting osteoblasts and periodontal ligament fibroblasts. However, the mechanism by which lactoferrin directly affects osteoclastogenesis is yet to be reported. Therefore, this study examined the direct effects of lactoferrin on RANKL-induced osteoclast differentiation of murine osteoclastic RAW 264.7 cells. The number of osteoclasts was determined by counting the number of cells positive for tartrate-resistant acid phosphatase staining. The effect of lactoferrin on the number of osteoclasts was measured, and the effect on the mRNA expression of osteoclast differentiation markers was assayed using real-time PCR. Lactoferrin decreased the number of osteoclasts (_2 nuclei) and large osteoclasts (_8 nuclei) in a dose-dependent manner without affecting the viability of RAW 264.7 cells. Additionally, it only mediated these effects within 48 h of culturing the RAW 264.7 cells with RANKL. Lactoferrin also significantly inhibited RANKL-induced mRNA expressions of osteoclastic differentiation genes, such as NFATc1, RANK, DC-STAMP, and OC-STAMP. Thus, these findings suggest that lactoferrin directly suppresses osteoclastogenesis within 48 h of culturing the RAW 264.7 cells with RANKL. Therefore, lactoferrin may be a novel and innovative therapy for treating bone diseases

Cytotoxicity of Strengthened Glass-ionomer Cement by Compounding Short Fibers with CaO-P2O5-SiO2-Al2O3 Glass.

The purpose of this study was to clarify whether the short glass fibers of CaO-P2O5-SiO2-Al2O3 (CPSA) glass possessed the ability to reinforce conventional glass-ionomer cement (GIC). Biocompatibility of the set GIC mixed with short CPSA glass fibers was evaluated in a cell culture cytotoxicity test. Moreover, the rate of release fluoride ions from GIC mixed with short glass fibers was measured. The powder of a conventional GIC was mixed with short CPSA glass fibers (diameter, 9.7±2.1 μm; aspect ratio, 5.0±0.9) before mixing with the liquid of the GIC. Set cements of 40 mass% short CPSA glass fibers mixed with GIC powders showed maximum values of 18 MPa in diametral tensile strength (DTS) after aging for 24 hours due to the effects of specific shape of short glass fibers and reactivity between the mixing liquid and short glass fibers. The cytotoxicity of these cements to rat pulpal cells tested by cellular activity showed that the set GIC disks (13 mm in dia. × 1 mm in thickness) with 40 mass% short CPSA glass fibers had cell activity as that of the set GIC or a cell culture coverslip used as control. Moreover, the addition of short glass fibers to GIC did not disturb the release of fluoride from the specimens

Mechanical Properties and Ions Release of S-PRG Filler-containing Pit and Fissure Sealant

The purpose of this study was to clarify the properties of functional filler-containing pit and fissure sealants (FS). Seventy-two specimens were prepared and divided into three groups of three resin sealants ( S-PRG filler-containing FS, DELTON and Teethmate F1-2.0) and one glass-ionomer sealant (Fuji III LC). Each of six discs (6 mm in diameter ×3 mm in thickness) was used for 24-h, 4-week and 12-week experiments. Diametral tensile strength (DTS) and ion release were measured. S-PRG FS and Delton showed high values of DTS (23.2 MPa and 23.5 MPa, respectively) after 24 hours of storage. The DTS values of each sealant remained relatively constant. A large amount of fluoride was initially released from the sealants. However, fluoride release did not influence on DTS. S-PRG filler-containing FS released large amounts of strontium, boron and fluoride ions. Filler-containing sealants release large amounts of ions, contributing to antibacterial effects